Aav vectors for retinal and cns gene therapy.

(AAV vectors for AKII therapy and CNS gene therapy) Full description reference reference for relevant requests

0001] This request is based on precedence over the US provisional serial number 5 request

988,131 / 61, filed May 2, 2014, and US provisional serial number application

, Filed on February 10, 2015, both of which have been incorporated into the present application

For reference in their entirety.

Provide a list of sequences in an ASCII TEXT file

000021 The following trim content has been incorporated into an ASCII text file in the current application for reference in its entirety: 10 Computer Readable Form (CRF) from the sequence list (filename? 159792010440SEQLIST.txt, Date of registration: 29 April 2015, Size: 85 KB).

Technical field

[00003 The present invention relates to glandular viral vectors of recombinant recombinant product (rAAV) for advanced transport to the eye and 045 for example for advanced retinal therapy and advanced genetic therapy 15 CNS.

Disclosure of the invention

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Retinal degenerative diseases represent a promising focus of gene therapy mediated by a gene-linked vector (887). AAV vectors can mediate long-term expression of the gene in the retina and stimulate immune responses that make these vectors an attractive choice for transferring the gene into the eye. The retina is attached to a photosensitive tissue at the back of the eye that is made up of many different types of cells including photoreceptor cells, retinal pigment epithelial cells and retinal ganglion cells. The type of target cell and the vector transport pathway of the AAV gene therapy vector depend on the disease indication; for example a clinical trial in phase I of age-related macular degeneration uses transporter in vitreous to achieve retinal ganglion cell transformation and a recent clinical trial for treating patients with Leber's cyclic latex disease Type II, a form of retinitis pigmentosa, transposition of the RPE65 gene under the retina to transform the retinal pigment epithelial cells.

10005 In light of the aforementioned use, there is a need to develop new agents and methods to improve the conduction of 87 m8 in Al Ain.

[0006] Adenovirus-based vectors (AAV) have also become the preferred vector system for neurogenetic therapy as they have an excellent safety record in multiple clinical trials.

Kaplitt, MGet al. (2OO7) Lancet 369: 2097-2105; Eberling, JLet al. (2OO8) Neurology, 1983-70: 1980) However: Fiandaca, Mset al. (2OO9) Neuroimage.47 Suppl 2: Τ27-35

Effective treatment of neurological disorders has been severely hampered by coupled issues with AAV vector transport to affected cell populations. This transport problem is particularly problematic for disorders involving the central nervous system (CNS) based on it. There is an additional need to enhance the transfer of AAV to the CNS machine. 26

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In some aspects. The invention provides a method for transferring a heterogeneous DNA into an individual's eye that includes the administration of a glandular virus particle of the recombinant product (rAAV) into the individual's sub-retina where the rAAV particle comprises a) rAAV capsid comprising rAAV capsid proteins comprising one or more substitutions The amino acid at one or more of the sites that interacted with a protein glycan of heparin sulfate or at one or more of the 5 sites corresponding to the amino acids 484, 487, 527 and 532, 585 or 588 and the numbering is based on the VP1 numbering of 8872, And b) a rAAV vector comprising a heterozygous DNA and an inverted terminal replication of an 887 vector and at least a solution. In some embodiments the rAAV particle includes a serotype 2 AAV capsid (8872). In some embodiments one or more amino acid substitutions reduce the binding of the rAAV particle to the protein glycan of heparin sulfate. In some 10 embodiments one or more amino acid substitutions reduces the rAAV particle binding to the protein glycan of heparin sulfate by about at least 0%, by at least 25%, At least about 50% and at least about or at least about some models, one or more substitutions of the amino acid increase the effectiveness of infecting by the rAAV particle from the cell in the eye or CNS) in some embodiments, one or more substitutions is more than 15. The efficacy of transmitting infection by the rAAV particle from the cell in the eye or CNS by at least about 10%, about 25% at least, about 50% at least, about 75% or at least about 100%, for example, compared to capsid rAAV reference containing the 887-type capsil protein Untreated. In some embodiments the eye cell is a retinal cell, a photoreceptor cell, or pigment epithelial cells of the retina, bipolar cells, 20 horizontal cells and non-axonal cells, Müller cells, and or ganglion cells.

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Of the CNS is an oligodendrocyte, astrocyte, nerve or parenchymal cell, microglial cell, and epidermoid cell / Prokinesse cell

10008 In some embodiments the AAV particles from the invention comprise one or 1 capsid of the amino acid substitutions at position 484, 487,527, 585 or

The numbering is used with the numbering VP1 to 8872 in some embodiments. VP1 for AAV2 comprises the amino acid sequence 5 of the sequence ID: 1 in some embodiments. Substitutions of one or more amphic acid involve the substitution of an amino acid building unit with a positive charge with a metacyltic unit of an amphyoid acid that is not of a positive charge. In some embodiments the positively charged amino acid building unit is replaced by the hydrophobic amino acid building unit. In other embodiments, one or more amino acid substitutions comprise the substitution of the arginine or lysine building unit; in other embodiments as well 10 one or more amino acid substitutions comprise the substitution of the arginine or lysine subunit of the alanine subunit. In some embodiments one or more amphic acid substitutions comprise a substitution at the position R585, 532, R487, R484 Wa or R588, with a numbering based on

Vp1 for 8872 In some embodiments, the rAAV particle includes one or more proteins

RAAV capsid with at least 90%, at least 91%, at least 92%, 15 at least 93%, at least 94%, at least 6%, at least 97/5, at least 98%, at least 99%, or at least ο / 100, of the sequence identity with respect to series No. 2, 4, or 6. In some embodiments, one or more amino acid substitutions involve substitution at the rAAV position. In one embodiment one or more substitutions includes substitutions at the R532 position. In some embodiments one or more of 20 amy acid substitutions include substitutions at the R484 and R487 inhibitors or at the R585 inhibitors.

sucking.

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^R584b <is numbered based on VP1 to 8872. In other embodiments, one or more of the amphic acid substitutions includes R484A and R487A substitutions or the R588A.J R585A substitutions, yna evaluates to VP1 for AAV2. (Other embodiments, the AAV capsid includes the amino acid substitutions R588A, R585A, numbered based on VP1 for 2-AAV. In some embodiments, the rAAV particle includes AAVl capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 5 capsid, AAVrh8R capsid, AAV9 capsid or AAVrhlO ka.

[0009] In some embodiments, the AAV particles of the invention comprise one or more capsids of the amino acid substitutions at position 485, 488, 528, 586 or 589,

In some embodiments, the AAV particle comprises a capsid with one or more of the amino acid deposits of 485, 488, 528 or 533, 10 with a numbering based on VP1 for the numbering ^ 71 ^ In some embodiments the numbering is dependent on VP1 of AAVrhSR. It includes the amphic acid sequence number 9 in some embodiments. Substitutions of one or more of an amino acid involve the substitution of an amino acid residue with a positive charge with an amino acid residue that is not of a positive charge. Some models. The hydrophobic amino acid building unit is substituted for the hydrophobic amino acid building unit. In other 15 embodiments, one or more amino acid substitutions comprise a substitutions of the lignin or arginine unit. In other embodiments also one or 1 more amino acid substitutions include the substitution of the arginine or lysine building unit of the alanine building unit. In other embodiments, one or more amino acid substitutions involve the substitution of the building block of an uncharged positively charged amino acid with the building unit of a positively charged amino acid. Some models. The hydrophilic non-hydrophobic 20-amphic building unit is replaced by the positively charged amino acid building unit. In models

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Additional, one or more amino acid substitutions includes an alanine building block substitution. In additional embodiments, one or more of the amino acid replacements do not include the substitution of an arginine or lysine building block with an alanine building block. In some embodiments the amino acid substitution is at position 488 528, 533, or 589, with a numbering based on the numbering AAVrh8R ^ In some embodiments, the VP1-based notation for AAVrhSR includes the amino acid sequence of the sequence ID: 9. In some embodiments, the amino acid substitution includes the substitution of the position R533, R488, R485, or 589, with a numbering based on the VP1 numbering of

AAVrhSR. In some embodiments, the rAAV particle includes a rAAV capsid of a protein with sequence ID number: 11. In some embodiments, the rAAV particle includes one or more rAAV capsid proteins that have at least 90% or at least 91% or at least 92% or more. At least 93% or at least 94% or at least 95% or at least 97% or at least 98% or at least 95% or at least 99% or at least 100% of the sequence identity of the sequence with ID number 11. In some models The amino acid substitution includes the replacement of R533A with a numbering

It is based on VP1 from AAVrhSR.

In some embodiments, the rAAV particle comprises the capsid 8877, AAVl or AAV9 and where there are one or more amino acid substitutions at position 485, 488, 528, 533, or 589, with a numbering based on the VP1 numbering of AAVl, AAV6, or AAV9 and / or Whereas, the rAAV particle comprises the AAV8 or AAVrhlO capsid and Whereas one or more amino acid substitutions are at position 487, 490, 535, 588 and / or 591 with an opaque VP1 numbering from AAV8 or AAVrhlO.

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In some embodiments the AAV particles of the invention comprise a capsid containing one or more amino acid substitutions that alter the binding to HSPG (for example, reduce or de-link to HSPG) or one or more of the amino acid homologues 484, 487, 527. , 532, or 585, or 588 numbering based on the VP1 numbering of AAV2 and the rl vector comprising a heterocyclic DNA encoding a therapeutic polypeptide or therapeutic DNA. In some embodiments, the heterogeneous DNA encodes a polypeptide selected from the group that consists of an antioxidant neurotrophic factor, an anti-vasodilating agent, an anti-angiogenic agent and an anti-inflammatory agent; in other embodiments, the heterogeneous DNA encodes a polypeptide selected from the group that composes From: LCA5, GUCY2D, AIPLl, RPE65, Prph2, LRAT, CRBl, IMPDHl, RDH12, ABCA4, Clarin, MYO 7a, CEP290, CRX, GNAT2, CNGB3, CNGA3, RPGR, RP2l, RPGRIP1, MNAT1

NFkB, BCL-Χ, BCL2, EPO, PEDF, FGF2, CNTF, GDNF, indostatin, angiostatin, ILIO, sFlt, sPDGF-R, anti-17, ILl-ra, sIL17R, anti-0, si-, sTNF- RI RII and 1444. In other embodiments, heterocyclic DNA encodes a therapeutic nucleic acid. In other embodiments, the therapeutic nucleic acid is siRNA, shRNA RNAi, miRNA, anti-transcription reaction I, ribozyme or DN Azyme. In some embodiments the rAAV vector is a self-completing rAAV vector.

(10012 In some embodiments, the AAV particles of the invention comprise a capsid that comprises one or more amino acid replacements that alter the binding to HSPG (for example, reduce or de-link to HSPG) or at one or more localities of the amino acid. From 484, 487, 527, 532, 585 the numbering is according to VP1 from the AAV2 nucleic acid numbering.

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Heterozygous encodes a therapeutic polypeptide or therapeutic nucleic acid where the heterogeneous DNA is under the control of a reinforced sequence that is expressed in the retina. In some embodiments, the heterogeneous DNA is functionally bound to a suitable promoter for expression of either a therapeutic polypeptide or a therapeutic nucleic acid in one or more retinal cell types. In some embodiments the reticulocyte is a photoreceptor cell, retinal pigment epithelial cell, bipolar cells, lateral cells, amorphous cells, Müller cells and / or a ganglion cell. In some embodiments, the promoter is the rhodopsin kinase promoter (each) the opsin promoter is the CMV promoter, the chicken beta actin promoter (CBA).

10013 In some embodiments the AAV particles of the invention comprise a capsid comprising one or more amino acid substitutions that alter the binding to HSPG (for example, reduce or de-link to HSPG) at one or more of the amino acid positions of 484, 487, 527, 532, 585 were numbered according to VP1 from AAV2 numbering and heterozygous DNA to transfer the heterozygous DNA to the retina of an individual. In some embodiments, the individual is a human being. In some embodiments, heterogeneous DNA is used to treat a selective ophthalmic disorder consisting of acute autosomal early retinal degeneration (congenital Leber's latent), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, conical malnutrition, retinitis pigmentosa. X-ray retinopathy, Usher's syndrome, age-related macular degeneration, amyotrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), Cystoid macular edema, retinopathy central retinopathy. Intraocular inflammation, glaucoma and posterior uveitis.

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In some respects the invention presents methods for improving the rAAV conversion of cells after subretinal rAAV particle transfer to a single eye compared to the transduction of cells with a rAAV vector comprising an untreated capsid type. The method includes the incorporation of one or more amino acid substitutions into the protein of the AAV transporter capsid at one Or more of the sites that interact with protein glycans of heparin sulfate or at one or more sites of the corresponding amino acid of 484, sites of the 487, 527, 532, 585 or 588 were numbered according to VP1 of AAV2 numbering; Where the rAAV particle includes the rAAV vector capsid protein, the rAAV vector comprises heteronuclear DNA and the tip replication of at least one AAV.

In other aspects the invention presents methods for improving the expression of heterocyclic nucleic acid after the subretinal transfer of rAAV particles to Farder. The method includes the incorporation of one or more amino acid substitutions into a transport capsid protein at one or more sites interacting with a protein glycan or at One or more of the corresponding amino acid positions of 484, 487, 532, 585 or 588 were numbered according to VP1 from AAV2 numbering;

Where the rAAV particle includes the rAAV vector capsid protein, the rAAV vector comprises heteronuclear DNA and the tip replication of at least one AAV.

00016 In some embodiments, rAAV particles with evolved conversion and / or heterologous expression of heteronuclear DNA include an AAV capsid of serotype 2 (AAV2). In some embodiments one or more amino acid precipitations reduce the binding of the rAAV particle to the protein glycan of heparin sulfate. In some embodiments, one or more amino acid substitutions reduce the rAAV particle binding to the heparin sulfate glycan by at least 10%.

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At least 25%, about at least 50%, about at least 75%, or about over

Least 100%

[0017] in some embodiments. RAAV particles with evolved conversion and / or evolved expression of heteronuclear DNA comprise a capsid with one or more amino acid substitutions at position 484, 487, 527, 532, 585 or numbering based on VP1 to 8872. In some embodiments the numbering is dependent The VP1 of AAV2 comprises the right acid sequence of the AAV2 sequence, with ID number: 1. In some embodiments, substitutions of one or more of an amino acid involve the substitution of an amino acid building unit with a positive charge with an amino acid residue that is not of a positive charge. In some embodiments the positively charged amino acid building unit is replaced by the hydrophobic amino acid building unit. In other embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine building block; in other embodiments also one or more amino acid substitutions includes the substitution of the arginine or lysine building unit of the alanine subunit. In some embodiments one or more amino acid substitutions comprise a substitution at the position R585, 532, Κ527, R487 U or R588, with the numbering

Based on VP1 for 88272. In some embodiments, the rAAV particle comprises of at least 90% of the rAAV capsid proteins, at least 91%, at least 92% or at least 93%, at least 94% or at least 95% or at least 96%. %, Or at least 97%, or at least 98%, or at least 99% or 100% of the coincidence of the sequence with ID numbers: 2, 4 and / or 6. In some embodiments one or more of the amino acid substitutions includes substitutions at the position R484 and R487 or at the positions R585 and 588D with numbering based on VP1 to 88722. In other embodiments, one or more amino acid substitutions includes

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Substitutions of R484A and R487A, or substitutions of R585A and R588A, with a numbering based on VPl of 8872. In other embodiments, the AAV capsid includes replacements of the amino acid R585A and R588A, numbering based on VPl for 2-87mm. In some embodiments, the AAV2 particle includes a capsid of AAV2 and a capsid. AAV3, AAV6 capsid, AAV8 capsid, AAVrhSR capsids, AAV9 capsids, or AAVrhlO capsids.

10018 In some embodiments, rAAV particles with evolved conversion and / or evolved expression of heteronuclear DNA comprise a capsid with one or more amino acid replacements at position 485, 488, 528, 586 or 589 numbering based on their numbering

AAVrhSR) Some embodiments incorporate AAV particles of the invention onto a capsid of one or more amino acid substitutions at position 485, 488 and 528 or 533 from a numbering based on the VPl numbering of AAVrhSR. In some embodiments the numbering is dependent on the VPl of AAVrhSR incorporating the amino acid sequence of the sequence ID: 9. In some embodiments one or more amino acid substitutions includes the substitution of the positively charged amino acid building unit with the building block of a positively charged amino acid. In some embodiments. The positively charged amino acid building unit is replaced by the hydrophobic amino acid building unit. In additional embodiments, one or more amino acid substitutions include the substitution of the arginine or lysine building block. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block. In other embodiments one or more amino acid substitutions includes the substitution of the building unit of an uncharged amino acid with the building unit of a positively charged amino acid. In some embodiments, the building unit of an amino acid that is non-hydrophilic is replaced by the building unit of an amino acid.

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Positively charged. In additional embodiments, one or more of the amino acid substitutions includes the substitution of the alanine building block. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an alanine building block with an arginine or lysine building block. In some embodiments the amino acid substitution is at position 485, 488, 528, 533, or 589, numbering based on the VP1 numbering of AAVrhSR. In some embodiments, the AAV particles of the invention comprise a capsid with one or more amino acid replacements that are at the positions Of 485, 488, 528, or 533 numbering based on VP1 numbering for AAVrhSR comprising the AAVrhSR sequence of VP1 in some embodiments the numbering is dependent on.

The amino acid from sequence number 9. In some embodiments, the amino acid substitutions include substitutions at the position R533, R488, R485, or 589 where the numbering is based on the numbering of VP1 from the number sequence. In some embodiments, particle 3887 includes the capsid protein of the number sequence. AAVrhSR

11. In some embodiments, the rAAV particle comprises one or more rAAV capsid proteins that comprise at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 97%. Or at least 98%, or at least 95%, or at least 99% or at least 100% of the sequence identity of the sequence with ID 11. In some embodiments, the amino acid substitution involves replacing R533A with a VP1-based numbering from AAVrhSR.

00019 In some embodiments, rAAV particles with evolved conversion and / or evolved expression of heteronuclear DNA comprise a capsid that includes one or more amino acid substitutions that alter HSPG binding (for example, reduce or eliminate binding to HSPG) or one or more of the locus. The amino acid analogs 484, 487, 527, 532, or 585,

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Or 588 with a numbering based on the VP1 numbering of AAV2 and the rAAV vector includes heterocyclic DNA encoding a therapeutic polypeptide or therapeutic DNA. The rAAV vector includes heterocyclic DNA that encodes a therapeutic polypeptide or therapeutic nucleic acid. In some embodiments, heterochromic DNA encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor. Anti-cytokine agent, anti-angiogenic agent, and anti-inflammatory agent. In additional embodiments, the heteronuclear DNA encodes a polypeptide selected from the group consisting of.

,, Clarin, MYO 7a, CEP290, CRX, LCA5, GUCY2D, AIPLl, RPE65, Prph2, RPGR, MERTK, TULPl, ΝΜΝΑΤ1, LRAT, CRBl, IMPDHl, RDH12, ABCA4, EPO, PEDF, FGFDNTF , CNGB3, CNGA3, RPGRIP, RP2

NFkB, BCL-Χ, BCL2, indostatin, angiostatin, ILIO, sPDGF-R, sFlt, IL17 antagonist, ILl-ra, SIL17R, IL4 antagonist, sTNF-R II, sTNF-RI, TGFp. In the other embodiments, heterozygous nucleic acid encodes the therapeutic DNA. In additional embodiments the therapeutic nucleic acid si A RNAi, miRNA, siRNA, anti-transcriptional RNA (ribozyme or DNAzyme) some embodiments is the rAAV vector, the rAAV self-completing vector.

[10020 In some embodiments, rAAV particles with evolved conversion and / or evolved expression of heteronuclear DNA comprise a capsid that includes one or more amino acid substitutions that alter the binding to HSPG (for example, reduce or eliminate binding to HSPG) or At one or more of the homologues of the amino acids 484, 487, 585, or

588 By numbering based on the VP1 numbering of AAV2 and heterologous DNA encoding a therapeutic polypeptide or therapeutic nucleic acid, the heterozygous DNA is under the control of a reinforced sequence that is expressed in the retina. In some embodiments, the heterogeneous DNA is linked

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Operationally with a suitable enhancer for expression of therapeutic polypeptide or therapeutic DNA in one or more reticulocyte types. In some embodiments, the reticulocyte is a photoreceptor cell, a retinal pigment epithelial cell and / or a ganglion cell. In some embodiments the promoter is rhodopsin kinase (RK), the opsin booster is the CMV promoter, the CBA promoter is chicken beta actin.

10021 In some embodiments, rAAV particles with evolved conversion and / or evolved expression of heteronuclear DNA comprise a capsid that includes one or more amino acid substitutions that alter HSPG binding (for example, reduce or eliminate binding to HSPG) or at one or more loci. The corresponding amino acids 484, 487, 527, 532, 585, or 588 numbering based on the numbering of VP1 from AAV2 and heterozygous DNA to transport the heterozygous DNA into the retina of an individual. In some embodiments, the individual is a human. In some embodiments, heterogeneous DNA is used to treat a select group of V-group ophthalmic disorder consisting of acute autosomal early reticulum (Leber's congenital term), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone malnutrition. Retinitis pigmentosa. X-ray retinal cleavage, Usher's syndrome, age-related macular degeneration, neovascular AMD, macular degeneration, diabetic macular degeneration, proliferative diabetic retinopathy (PDR), cystic macular edema, central serous retinopathy, retinal detachment, intraocular inflammation. Glaucoma and posterior uveitis.

10022 In some embodiments. The invention provides methods for treating an eye disorder in an individual (for example, a human) that involve transferring a combination of rAAV particles to the retina of an individual

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Where rAAV particles include a) rAAV capsid includes the rAAV capsid protein that includes one or more amino acid substitutions at one or more sites that interact with a heparin sulfate protein glycan from a glyc protein at one or more of the homologous amino acid sites 484, 487. , 527, 532, 585, or 588 numbering based on the VP1 numbering and b) the rAAV vector comprising heterozygous DNA and tip replication of at least one AAV. Some models. The rAAV particle includes an AAV capsid of serotype 2 (88272). In some embodiments one or more amino acid substitutions reduces the binding of the rAAV particle to the protein glycan of heparin sulfate. In some embodiments one or more amino acid replacements reduce the rAAV particle binding to the protein glycan of heparin sulfate by at least about at least about 25%, at least 50%, at least 75%, or at least 100%.

[0023] In some embodiments, methods include subretinal transport of rAAV particles comprising an rAAV vector encoding heteronuclear acid used in treating eye disorder in an individual (eg, a human), where the rAAV particles comprise a capsid with one or more acid precursors The amino is at position 484, 487, 527, 532, 585 or 588, with the numbering VP1 based on the numbering 8872 A. In some embodiments the numbering includes depending on the VP1 of AAV2 comprising the amino acid sequence of the sequence with ID number 1. In some embodiments one or more substitutions of an amino acid involve the substitution of the building block of a positively charged amino acid with the structural unit of a positively charged amino acid. In some embodiments, the building block of a positively charged amino acid is substituted for the building unit of an amino acid that is non-hydrophilic. In additional embodiments, one or more of the amino acid replacements include a substitution

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Arginine or lysine. In additional embodiments as well one or more of the amino acid metabolites comprise the substitution of an arginine or lysine building block with an alanine building block. In some embodiments one or more amino acid replacements include substitution in the R585, 532, R487, R484 and R588 locus, and the numbering is based on VP1 of AAV2. In some embodiments, the rAAV particle comprises one or more capsid proteins with at least about 90% or at least 091/0 or at least 92% or at least 93% or at least 94% or at least 95% or at least 97 or To; to less than 5/98 or to to less 5/95 or to to less 5/99 or to fold at least 100% of the identity of a series of 2, 4, and / or 6. In some embodiments, one or more of the amino acid substitutions includes substitutions at the R487j position R484 or at the R585 and R588 positions, numbering based on VP1 to 8872. In additional embodiments one or more amino acid substitutions includes the substitutions of R484A and R487A or the substitutions R585A and R588A. In additional embodiments, the AAV capsid includes R588A, R585A, numbering based on VP1 to 8872. In additional embodiments, the AAV capsid includes the amino acid R588A, R585A, numbering based on VP1 for 2-AAV. In one embodiment, the rAAV particle includes an 8871 capsid, AAV2 capsid, AAV3 capsid, AAV6 capsids, AAV8 capsids, AAVrhSR capsids and AAV9 capsids or AAVrhlO capsids.

10024 In some embodiments, the methods include subretinal transport of rAAV particles comprising a rAAV vector encoding a heteronuclear acid used in the treatment of ophthalmic disorder in an individual (eg, a human) wherein the rAAV particles contain a capsid with one or more amino acid replacements at position 485, 488, 533, 586 or 589, numbering based on AAVhR's VP1 numbering. In some embodiments the AAV particles of the invention comprise a capsid with one or more amino acid substitutions at position 485, 488, 528, or

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533 With VP1-based numbering from AAVrhSR. In some embodiments the numbering is dependent on the VP1 of AAVrhSR which includes the amino acid sequence of sequence No. 9. In some embodiments one or more amino acid substitutions includes the substitution of the positively charged amino acid building block with the positively charged amino acid residue. In some embodiments, the building block of a positively charged amino acid is replaced by the building unit of an amino acid that is non-hydrophilic. In additional embodiments, one or more of the amino acid substitutions includes the substitution of the building block arginine or lysine. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block. In the other embodiments one or more amino acid substitutions comprise the substitution of the building block of a positively charged amino acid with the building unit of a positively charged amino acid. In some embodiments the non-hydrophilic amino acid building unit is replaced by the positively charged amino acid building unit. In additional embodiments, one or more of the amino acid substitutions includes the substitution of the alanine building block. In additional embodiments one or more amino acid substitutions also include the substitution of an alanine building block with an arginine or lysine building unit. In some embodiments, the amino acid substitution is at position 485, 8 48, 528, 533, or 589, with the VP construct numbering of AAVrh8R. In some embodiments, the AAV particles of the invention comprise a capsid with one or more amino acid replacements at position 485, 488 and 428 or 533 with a numbering based on the VP1 numbering of AAVrhSR. In some embodiments, the numbering is based on the VP1 of AAVrhSR comprising the amino acid sequence of the amino acid sequence of the sequence ID: 9. In some embodiments, the amino acid substitution involves the substitution of the position R533, R488, R485, or 589, with the numbering based on the numbering AAVrhSR ^ Some

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Models The rAAV particle includes the rAAV capsid of the sequence ID number: 11. In some embodiments, the rAAV particle includes one or more rAAV capsid proteins that have at least about 90% or at least 91% or at least 92% or at least 93%. Or at least 94% or at least 95% or at least 97% or at least 0/988 or at least 95% or at least 99% or at least 100% of the identity of the sequence from sequence # 11. In some embodiments, substitutions include Substituting at least one amino acid on R533A, with a VPl-based numbering of AAVrh8R.

10025 In some embodiments the methods include subretinal transport of rAAV particles comprising a rAAV vector encoding a heterotypic DNA used in treating an eye disorder in an individual (e.g. a human) and a capsid containing one or more amino acid substitutions that alter the binding to the HSPG (e.g. a human). For example, reduce or eliminate the link to HSPG) or at one or more of the positions 484, 487, 527, 532, 585 or 588, with the numbering VP1 based on the numbering 8872. In some embodiments, heterochromic DNA encodes a polypeptide selected from a group consisting of an antioxidant neurotrophic factor, an anti-cellular fading agent, an anti-angiogenic agent and an anti-inflammatory agent. In additional embodiments, heteronuclear DNA encodes a polypeptide selected from a group consisting of CRX, LCA5, GUCY2D, AIPLl, RPE65, Prph2, 1, LRAT, CRBl, IMPDHl, RDH12, ABCA4, Clarin, MYO 7a, 0131290, GDNF,, CNGB3, c A3, RPGRIP, RP2, RPGR, MERTK, TULPl

NFkB, BCL-Χ, BCL2, EPO, PEDF, FGF2, CNTF, indostatin, angiostatin sFlt, ILIO, sPDGF-R, anti-ILl-ra, SIL17R, IL17, antagonist, sTNF-R II, sTNF-RTGFp IL4. The other heterochromic nucleic acid encodes a therapeutic nucleic acid. In additional forms

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-19 anti-directional RNA acid, siRNA, shRNA RNAi, miRNA being the therapeutic nucleic acid.

Transcription, ribosome or DNAzyme In some embodiments, rAAV is a self-executing rAAV vector.

10026 In some embodiments, methods include subretinal transport of rAAV particles comprising a rAAV vector encoding a heterocyclic DNA used in treating an eye disorder in an individual (for example, a human) and a capsid containing one or more amino acid replacements that alter the binding to HSPG. (Eg, reduce or eliminate binding to HSPG), or at one or more of the positions 484, 487, 527, 532, 585 or with a VPl numbering based on the AAV2 numbering where the heterocyclic DNA is under the control of a reinforced sequence that is expressed in Retina. In some embodiments the heterozygous DNA is functionally bound to a suitable promoter for expression of either a therapeutic polypeptide or a therapeutic DNA in one or more retinal cell types. In some embodiments the reticulocyte is a photoreceptor cell, retinal pigment epithelial cell and / or ganglion cell. In some embodiments, the promoter is rhodopsin kinase (RK), the opsin booster is the CMV promoter, the chicken beta actin booster (CBA).

In some embodiments. Methods include subretinal transport of rAAV particles comprising a rAAV vector encoding a heterocyclic DNA used in treating an eye disorder in an individual (eg, a human) and a capsid comprising one or more amino acid substitutions that alter the binding to HSPG (eg , Reduce or eliminate the association with HSPG), or at one or more positions 484, 487, 527, 532, 585 or 588, with a VPl-based numbering from AAV2 wherein the eye disorder is selected from the group consisting of: Acute autosomal early retinal degeneration (congenital potential of Leber), congenital color blindness is a disease

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Stargardt, Best's disease, Doyne's disease, diuretic corticosteroids, retinitis pigmentosa, X-ray-associated retinopathy. Usher syndrome, age-related macular degeneration, age-related macular degeneration, neovascular AMD, diabetic macular degeneration, proliferative diabetic retinopathy (PDR), cystic macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, and posterior ocular inflammation.

[0028] In some embodiments, the methods include transfer of a subretinal composition comprising rAAV particles, wherein rAAV particles include the rAAV vector encoding a heterogeneous DNA used in the treatment of an eye disorder in an individual (for example, a human) and a capsid comprising one or more The amino acid replacements that alter the binding to the HSPG (for example, reduce or eliminate the binding to the HSPG) or at one or more sites 484, 487, 527, 532, 585 or 588, With the VP1 numbering based on the AAV2 numbering. In some embodiments the carrier concentration in the combination is about 1 107 6 (shower) to about 1 14 10 X? Dimple. In some embodiments: the rAAV particle combination is effective in treating an individual's vision function. In some embodiments it is Visual function assessment by visual field measurement, dark-adapted optometry, visual movement evaluation, visual acuity (ERG), or reading assessment. In some models, the method results in an improvement in an individual's vision function. In some embodiments, the method has the effect of preventing or slowing the progression of impaired vision function in a person as a result of the development of a vision disorder.

(00029 in some aspects the invention provides a system for transporting a subretinal vector to Ein Farder comprising a) a composition comprising an effective amount of rAAV particles, where 1) a capsid protein of rAAV particles comprising one or more amino acid substitutions at one or more positions That interacts with heparin sulfate protein glycans or at one or more sites

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484, 487 527, 532 585 or 588, with VP1 numbering based on AAV2 numbering and 2) the carrier includes heterocyclic nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and a tip replication for at least one AAV: and b) a device for transporting rAAV to the retina. In some Embodiments The device includes a micro-bore cannula and a syringe in which the micro-cavity cannula is on a scale from 27 to 45. In some embodiments, the rAAV particle composition is contained within the syringe. In some embodiments, the cannula is transferred with a syringe. In some embodiments, the concentration of particles in the formula is from about 1 X 10 X m / mL to about 1 X 10 X 14 shower.

10030 In some embodiments the rAAV particles of the system incorporate an AAV2 capsid that includes one or more amino acid substitutions that alter HSPG binding (eg, reduce or inhibit binding). In some embodiments one or more amino acid substitutions reduce the binding of the rAAV particle to the heparin sulfate glycan. In some embodiments, one or more amino acid substitutions reduces the rAAV particle binding to the protein glycan of heparin sulfate by at least 10%, about at least 25%, at least about 50%, &lt;at least 75%, or at least 100%. In some embodiments, one or more amino acid substitutions increase the activity of infecting by the rAAV particle in an eye cell or CNS. In some embodiments, one or more amino acid replacements increase the effectiveness of infecting the amount of rAAV particle from the cell in the eye or CNS by about 10%, at least 25%, at least 50% and at least 75% or about 100%. Less, for example, compared to a reference rAAV capsid comprising an untreated AAV capsid protein. In some embodiments, the eye cell is a reticulocyte, a photoreceptor cell, or phallic cells of a network of binary cells and cells of lower cells.

'Sleep.

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Müller, and / or ganglion cells. In some embodiments, the cell from the CNS is an oligodendrocyte, astrocyte, nerve or parenchymal cell, microglia, skin cell, and / or Prokinesse cell.

[10031 In some embodiments the rAAV particles of the system comprise the AAV2 capsid comprising one or more amino acid substitutions that alter HSPG binding (eg reduce or de-link). In some embodiments one or more of the amino acid substitutions is at position 484, 487, 527, 532, 585 or 588, numbered according to the AAV2 numbering. In some embodiments, the numbering is dependent on VP1 of AAV2 which includes the amino acid sequence of the sequence ID number: 1. In some embodiments, one or more amino acid substitutions includes the substitution of the positively charged amino acid building block with the positively charged amino acid residue. In some embodiments the building block of a positively charged amino acid is replaced by the hydrophilic amino acid building unit; in additional embodiments one or more amino acid substitutions comprise the substitution of an arginine or lysine building unit. In additional embodiments it also includes one or more acid substitutions An amino is to replace the building block arginine or lysine with the alanine building unit. Some models. One or more of the amino acid substitutions includes a substitution in the locus of وضع532, 527, R487, R484, R585 and / or R588, with a numbering based on VP1 for AAV2 (some embodiments include the rAAV particle of one or more capsids of rAAV proteins that have at least one About 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 97% or at least 98% or at least 95% or at least 99%

Or at least 100% of the series ID of series 2, 4 and / or 6. In some embodiments,

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One or more amino acid replacements include substitutions at the R487 and locus or at the R585 and 588D positions with numbering based on VP1 to 00AAV2. Additional embodiments One or more AAV capsid amino acid replacements include R585A and R588A replacements numbering based on VP1 to 8872 In some embodiments , AAV capsid includes AAVl capsid, AAV2 capsid, AAV3 capsids, AAV6 capsids, AAV8 capsids, AAVrh8R capsids, AAV9 capsids or AAVrhlO capsids.

10032 In some embodiments the rAAV particles of the system comprise a 2 capsid that comprises one or more amino acid substitutions that alter HSPG binding (eg lowering or de-binding). In some embodiments one or more amino acid substitutions are at position 485, 488, 528, 533, 586 or 589 with numbering based on AAVrhSR numbering in some embodiments, the numbering is based on AAVrh8R's VP1 on the amino acid sequence of the sequence ID number: 9. In some embodiments, the one or more amino acid substitutions includes the substitution of the building unit of a positively charged amino acid by the building unit of a positively charged amino acid. In some embodiments the building unit of a positively charged amino acid is replaced by the building unit of an amino acid that is not hydrophilic. In additional embodiments, one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block. In the other embodiments, one or more amino acid substitutions comprise the substitution of the building block of a positively charged amino acid with the building unit of a positively charged amino acid. In some embodiments. The non-hydrophilic amino acid building unit is replaced by the positively charged amino acid building unit. In additional forms, it includes one or more

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The amino acid precursors to the substitution of the alanine building block. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an alanine building block with an arginine or lysine building unit; in some embodiments, the amino acid substitution is at position 485, 488, 528; 533, or 589, with a numbering based on the AAVrhSR numbering) Some embodiments VP1 for AAVrhSR includes the amino acid sequence mated to ID number: 9. In some embodiments an amino acid substitution includes a substitution at the position R533, R488, R485, or 589, with a numbering based on In some embodiments, the sequence numbering by rAAV ID on the rAAV protein capsid includes a particle. AA Vrh8R

No .: 11. In some embodiments, the slush amino acid substitution involves substitution of R533A, numbering based on VP1 to 0AVrh8R.

10033 In some embodiments the rAAV particles of the system include an AAV capsid with one or more amino acid substitutions that alter HSPG binding (for example, reduce or inhibit binding) or one or more loci corresponding to 484, 487, 527, 532, 585 or 588, numbering 711 based on AAV2. The rAAV vector comprises heteronuclear DNA. In some embodiments the heterocyclic DNA encodes a therapeutic polypeptide or therapeutic DNA. In some embodiments, the heterogeneous DNA encodes a polypeptide selected from the group consisting of an antioxidant neurotrophic factor, an anti-chelating agent, an anti-angiogenic agent and an anti-inflammatory agent. In other embodiments, the heterocyclic DNA encodes a polypeptide selected from the group consisting of: LCA5, GUCY2D, AIPLl, RPE65, Prph2, CRX; CEP290; LRAT, CRBl, IMPDHl, RDH12, ABCA4, Clarin, MYO 7a, GNAT2, CNGB3, CNGA3, RPGRIP, RP2, RPGR, MERTK, TULPl, 1

NFkB, BCL-, BCL2, EPO, PEDF, FGF2, CNTF, GDNF, Indostatin, Angiostatin,

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ILIO, sPDGF-R, sFlt, anti-17 Tal, sTNF-, sTNF-RI ,, TGFp-۵ ^, ILl-ra, SIL17R RII, and FULL. In other embodiments, the heterocyclic DNA encodes a therapeutic nucleic acid. In other embodiments, the therapeutic nucleic acid is miRNA, RNAi, siRNA, anti-transcription RNA, or Azyme. In some embodiments, the rAAV vector is a self-complementing rAAV vector.

10034 In some embodiments the rAAV particles of the system include on the AAV capsid with one or more amino acid primers that alter HSPG binding (for example, reduce or inhibit binding) or at one or more positions of the corresponding amino acids of 484, 487, 527, 532, 585 or 588 were numbered according to VP1 of the AAV2 numbering, and the rAAV vector includes heterocyclic DNA, wherein the heterozygous DNA is under the control of a reinforced sequence that is expressed in the retina. In some embodiments the heterozygous DNA is functionally bound to a suitable promoter for expression of either a therapeutic polypeptide or a therapeutic DNA in one or more retinal cell types. In some embodiments the reticulocyte is a photoreceptor cell, a retinal pigment epithelial cell, and / or a ganglion cell. Some models. The promoter is the rhodopsin kinase (j) promoter, the opsin promoter is the CMV promoter, the chicken beta actin promoter (CBA).

00035 In some embodiments the rAAV particles of the system include on the AAV capsid with one or more amino acid substitutions that alter HSPG binding (for example, reduce or inhibit binding) or at one or more of the amino acid homologues, 484, 487, 527, 532. And 585 or 588 with a numbering based on the VP1 numbering of AAV2, and the rAAV vector containing heterozygous DNA is used to transfer the heterogeneous DNA into the retina of an individual.

R

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Heterogeneous DNA for the treatment of ophthalmic disorder selected from the group consisting of: acute autosomal early retinal degeneration (congenital Leber's latent), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone malnutrition. Retinitis pigmentosa X-ray retinopathy Usher syndrome, age-related macular degeneration, age-related atrophic macular degeneration, neovascular AMD, diabetic macular disease, proliferative diabetic retinopathy (PDR), macular edema, central serous retinopathy. Retinal detachment, intraocular inflammation, glaucoma, and posterior ocular inflammation.

In some embodiments the invention provides a method for transporting heterologous DNA to the central nervous system (CNS) of an individual that includes the administration of a gland-associated virus particle (rAAV) to the individual's CNS where the rAAV particle comprises a) rAAV capsid containing rAAV proteins It includes one or more amino acid substitutions at one or more sites that interact with protein glycans of heparin sulfate or at one or more of the corresponding amino acid sites of 484, 487, 527, 532, 585 or 588. The numbering is according to VP1 from AAV2 numbering and b) the rAAV vector comprising heteronuclear acid and at least one of the 887 inverted terminal repeats in some embodiments. The invention presents a method for improving the conversion of rAAV of cells in the central nervous system (CNS) of an individual compared to the transduction of cells with rAAV comprising a capsid of the untreated type. The method involves administering a glandular recombinant virus particle (rAAV) to the individual's CNS where the rAAV particle comprises A) The rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid substitutions in one or more sites interacting with heparin sulfate protein glycans or at one or more sites of the acids The corresponding amino from

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-η-

484, 487, 527, 532, 585 or 588 numbering according to VPl of AAV2 numbering, And b) a rAAV vector comprising heterotypic DNA and at least one AAV inverted terminal repeat. In additional aspects the invention presents a method for improving the expression of heterozygous DNA in the central nervous system (CNS) of an individual. The method includes administration of a gland-coupled virus particle of the recombinant product ( rAAV) to a single CNS device where the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid precipitates at one or more sites interacting with a heparin sulfate protein glycan or at one or more The positions of the corresponding amino acids from 484, 487, 527, 532, 585 or 588 were numbered according to the VPl of AAV2 numbering, and b) the rAAV vector comprising heteronuclear acid and at least one inverted AAV terminal repeat. In additional aspects, the invention also provides a method for treating a disorder of the central nervous system (CNS) of an individual that includes administering an effective amount of a combination comprising the rAAV particle to the individual CNS system where the rAAV particle comprises a) rAAV capsid comprising one or more rAAV capsid. Of amino acid substitutions in one or more sites that interact with protein glycans of heparin sulfate or at one or more of the corresponding amino acid sites of 484, 487, 527, 532, 585 or 588 have been numbered according to VPl of AAV2 numbering and b) transporter rAAV comprises heteronuclear DNA upon replication of at least one AAV terminal.

[0037] In some of the preceding aspects and embodiments the heterochromatin is expressed by an increased level of expression compared to the expression level of the rAAV heterozygous nucleic acid containing the rAAV reference capsid. In some embodiments, the expression of DNA increases by about 10%, at least about 25%, about 50% at least and at least about 75% or

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2 AWJ ش 9 26912 2926226 2223 mg 6 990 6

92107 A? 6m2 a mm2 with a cube? AW-I LMT222612032۶ mm; Mm km 3 m 2 m 5 s? 2206 a b m 0 m 2 km 1290? (222220969 m 0665۴ 5 mp 169 m coa f 99 liters) mm 00 m 3 m as a ko 99 as a m 0 m m 22 m 2 AWJ 6 m 866 AW^J 6 m; 230 {? 1 m not 60 m? Yum:,? 562,169 6732 m AWJ m d controlled by 877 g

51 Ka 'm 6 c2 ailam maki 6 makki 6 m 9 ka?) 6 wy 9 y 2 mm 6 m m mm 21,23536 AWJ for no maki 699 jas !? J; MI, with D-m1 ki; Mi (% Haj? M, m, m, m, m, m, m, m, m, b, p, 90,?), And m, m, m, m, m, d?, 1226, p., M, m, m, block, 2, m, 99, p, m, m, 3, m,%, m, m, 9, m, m, m, 09%, mira,%, h.

01 0593 1692۴ D 2 m 20 mm 909 K? (202966 AW)

2 6%, 6 3 2; 99 AM, no? What is m0 m 2 m 6290 f?) Kielm 6 is a tragedy ζ ^ ή A WJ D Books! AWT for dirty? 9,26; 562629, m (92 1290 d?), Or “A'am AVVJ? AVV-for scanner? 226; B a

0 3190 yama m col 2 e 93 mmbj {? 1229 ylm ^ '(7AW) h mm 1 m 2 aa pvv

No AWJ for MSC 66002 0 D% 001 5 D m 2 Mira D% 09 Add Dd% H 9 H% 0 Q 21 A 266 ^ 1 KL? 6 E 23 What is an AVW 8 AVV for dirty:; 6629; 6 ٦ ٦ 2? Do not take my breath {? 9 What is mira dd% 001 mm

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Cell in the eye or CNS of at least about 10%, at least about 25%, at least about 50%, at least about 75, or at least about 100% compared to a reference rAAV capsid that takes on an untreated AAV capsid protein. In some embodiments, the rAAV particle includes AAVl capsid, AAV2 capsid, AAV3 capsid, AAV6 capsids, AAV6 capsids, AAV8 capsids, AAVrhSR capsids and AAV9 capsids, or AAVrhlO capsids.

[0038] In some of the aspects and previous embodiments, administration includes direct injection into the spinal cord and / or intracerebral administration. In some embodiments, intracerebral infusion is at a site selected from the group consisting of the brain, medulla, arch, cerebellum, intracranial cavity, meninges surrounding the brain, dura mater, spider mater, pia mater, cerebrospinal fluid (CSF) of the surrounding subarachnoid space In the brain the nuclei of the cerebellum are deep in the cerebellum, the ventricular system of the brain, the subarachnoid space, the striated node, the cerebral cortex, the nasal septum, the optic thalamus, the hypothalamus, and the visceral tissue of the brain. In some embodiments, administration is by injection into a ventricle within the brain, into at least one lateral cerebral ventricle. In some embodiments, intrathecal injection is given to the cervix, chest, and / or lumbar region. In some embodiments, the administration is by injection into the striatum. In some embodiments, the administration is by injection into the hypothalamus. In some embodiments, the administration is by injection into the parenchyma. In some embodiments, administration includes injection directly into the vocal cord or intracranial and / or administration into the brain. In some embodiments, the rAAV particle is given at a single position.

00039 In some of the earlier and later embodiments, the rAAV particle is transported by stereotaxic transport. In some embodiments the rAAV particle is transported by convection-enhanced transport. In some embodiments the rAAV particle is administered using the CED transport system

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Models The CED Delivery System includes a cannula and / or a pump. In some embodiments, the cannula is a reflux or graduated cannula. In some embodiments the pump is a hand pump. In some embodiments, the pump is an osmotic pump. In some embodiments. The pump is an infusion pump.

In some of the previous aspects and embodiments the heparin sulfate proteoglycans are expressed in one or more CNS cells. (Some embodiments are one or more CNS cells Oligodendrocyte astrocyte neurons, brain parenchyma cells, microglial cell, cell The neuronal endothelium, and / or G cell of some forms.Heparin sulfate proteoglycans are expressed in the neuron.

10041 In some of the previous aspects and embodiments, heterochromic DNA is expressed in one or more of the 45 E0 cells. Some embodiments are one or more CNS cells Oligodendrocyte astrocyte neuronal cell and parenchymal tissue cell microglia, cell Endometriosis and / or Purkinjcell cell (In some embodiments, heterozygous nucleic acid is expressed in a nerve cell. In some embodiments, the heterozygous DNA is exclusively expressed in neurons.

[0042] In some embodiments from the previous aspects and embodiments, one or more amino acid substitutions are at 448, 451, 484, 487, 527, 532, 585 and / or 588, with a numbering based on the numbering 88722. In some embodiments it is one or Most of the amino acid replacements are at position 484, 487, 527, 532, 585 or 588, numbering based on the numbering 8872. In some embodiments, VP1 to AAV2 includes the amino acid sequence mated to ID number: 1. In some embodiments it includes one or a More amino acid substitutions than unit substitution

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For a positively charged amino acid by the structural unit of an uncharged amino acid. In some embodiments, the building block of a positively charged amino acid is replaced by the building unit of an amino acid that is not hydrophilic. In some embodiments one or more of the amino acid substitutions includes the substitution of the building block arginine or lysine. In some embodiments, one or more of the amino acid substitutions includes the substitution of the arginine or lysine building block with the a la nene. In some embodiments one or more of the amino acid substitutions includes substitution in the position R347, R585, 532, 527 'R487, R484, R451, R448, 395, 390, R35O and / or R588,

With a numbering based on VP1 to 8872e, some embodiments include one or more amino acid substitutions in the position R585, 532, K527 R87 and / or R588, with the numbering based on VP 1 to 88272. In some embodiments the rAAV particle includes one or more rAAV capsid proteins that have at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least. 97% or at least 98% or at least 95% or at least 99% or at least 100% of the identity of the series 2, 4 and / or 6. In some embodiments one or more amino acid substitutions include R484A, R451A, R448A, Κ395Α, Κ390Α, R350A, R347A, Κ532Α, Κ527Α, R487A, Replace R585A and / or R588A, Numbered based on VP1 to 8872. In some embodiments one or more of the amino acid substitutions includes substitutions at the R484 and R487 position or at the R585 and R588 positions, numbered based on VP1 to 8872. In some embodiments one or more amino acid substitutions includes the R484A and R487A substitutions or the R585A, R585A and R585A substitutions. With a numbering based on VP1 to 88672. In some embodiments, the AAV capsid includes substitutions for the amino acid R585A and R588A 'with a numbering based on VP1.

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For AAV2 ^ In some embodiments the AAV capsid includes substitutions of the amino acid 532, numbered based on VP1 for AAV2.

[0043] In some embodiments from the previous aspects and embodiments one or more of the amino acid substitutions is at position 485, 488, 528, 533, 586 or numbering based on the AAVrlR numbering. Some embodiments, the numbering is dependent on VP1 of AAVrhSR which includes the acid sequence In some embodiments, one or more amino acid substitutions include the substitution of the positively charged amino acid building block with the building unit of a positively charged amino acid. In some embodiments, the building block of a positively charged amino acid is replaced by the building unit of an amino acid that is non-hydrophilic. In additional embodiments one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block. In additional embodiments, one or more amino acid replacements also include the substitution of an arginine or lysine building block with an alanine residue. In other embodiments one or more amino acid substitutions involve the substitution of the building block of a positively charged amino acid with the building unit of a positively charged amino acid. In some embodiments. The hydrophobic amino acid building unit is replaced by the positively charged amino acid building unit. In additional embodiments, one or more amino acid substitutions include an alanine building block. In additional embodiments one or more amino acid substitutions also include the substitution of an alanine building block. Alanine with the building block arginine or lysine. In some embodiments, the amino acid substitution is at position 485, 488, 528, 533, or 589, with a numbering based on the AAVrhSR numbering. In some embodiments, the numbering is based on the VP1 of AAVrhSR which includes the amino acid sequence of the sequence ID: 9. Some models. Amino acid replacement includes

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It replaces the position R533, R485, R488, or 589, with a numbering based on the AAVrh8R numbering. In some embodiments the rAAV C includes an 887 protein capsid] for the sequence ID: 11. In some embodiments the rAAV particle contains one or more capsid proteins with at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 97% or at least 98%. Or at least 95%, or at least 99%, or at least 100% of the identity of the sequence number 11. In some embodiments, a single amino acid substitution involves the substitution of R533A, numbering based on VP1 of the AAVR.

[0044] In some embodiments of aspects and previous embodiments, heterochromic DNA encodes a therapeutic polypeptide or therapeutic DNA. In some embodiments it encodes a heteronuclear DNA coupled with 5Ta. In some embodiments, a heteronucleic acid encodes a polypeptide selected from the group consisting of an enzyme, a neurotrophic factor polypeptide lacking or a mutagen in an individual with a CNS related disorder, an antioxidant factor Anti-cellulite, anti-angiogenic agent, anti-inflammatory agent, alpha-synuclein, beta-glucosidase (GBA), beta-galactosidase-1 (GLBl), ed-ronate-2-sulfatase (IDS), galactosylceramidase (GALC), mannenosidase, alga -Hmannosidase (ΜΑΝ2Β1), beta-mannosidase (MANBA), Pseudo-aryl sulfates N, (ARSA) A-acetyl-glucosamine-1-phospho-transferase (GNPTAB), acid-sphingomylinase (, (ASM Niemann-Pick protein NPCl) C), alpha-1 4-glucosidase (GAA), beta-hexosaminidase (GAA) subunit E-sulfoglucosamine sulfo-hydrolase (MPS3A), T-alpha-acetylgluco-saminidase (NAGLU), heparin-acetyl-CoA, alpha-gluco-saminidase l-acetyltransferase (MPS3C), non-acetyl-guacomamine-e-sulfatase (GNS) T7-acetylcysteine

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Galactosaminidase (NAGA), beta-glucurinidase (GUSB), hexosaminidase alpha 5 subunit (HEXA huntingtin (HTT), lysosomal acid lipase (LIPA), aspartyl glucosaminidase alpha-jackactosidase A, thio-esterase, a palmitoyl butylpeptidase protein. protein

Trans-membrane-to-particle emulsion cysteine-ceramidase acid-alpha-R-fucosidase acid-cathepsin m-alpha-r] -doronidazer aryl sulfates B, aryl sulfatase A, l-acetyl-galactosamine-6-beta-sulfate-acidic galactosidase or alpha-neuramidase. In some embodiments, it encodes a heterocyclic DNA polypeptide that was selected from the group consisting of the neuronal fade inhibitor protein (NAIP), nerve growth factor (NGF), and glial-derived growth factor.

(GDNF), Brain-Derived Growth Factor (BDNF), Ciliary Neurotrophic Factor (CNTF), Tyrosine Hydroxylase (GTP, (TH)^-Cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antiangiogenic polypeptide antioxidant, antiinflammatory polypeptide and aspartocylase (8508) in some embodiments. The heterozygous nucleic acid encodes the therapeutic DNA. In some embodiments, the therapeutic nucleic acid is RNAi, shRNA, siRNA; some embodiments are subjected to the anti-nucleic acid (DNAzyme), ribozym or miRNA, I.

Heterozygous for enhanced sequence control expressed in one or more CNS cells. Some embodiments heterozygous DNA undergoes a reinforced mitotic control that was selected from the group consisting of a current early promoter of cytomegalovirus (MoMLV LTR, RSV LTR, CMV, phosphorylate). Glycerate kinase-1 (PGK), monkey virus booster 40 (SV40), CK6 booster, transethyrin (TTR) booster, TK booster, tetracycline-responsive (TRE) booster, HBV booster, hAAT booster, LSP booster, chimeric liver booster (LSP) E2F booster, telomerase booster (hTERT), enhanced booster

Cytomegalovirus / chicken beta-actin promoter / 0-rabbit globin (CAG), factor booster

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1-alpha prolongation (EF1-alpha), human 3-glucurinidase booster 0-chicken actin (CBA), LTR booster for Russian retrovirus sarcoma virus (RSV), dihydrofolate reductase booster and 13-actin booster. In some embodiments it is nucleic acid A heterozygote operationally linked to a promoter suitable for expression of a therapeutic polypeptide or therapeutic DNA in one or more CNS cells (some embodiments. One or more CNS cells comprising one or more brain cells.) In some embodiments one or more of the CNS cells is an oligodendrocyte, a papillary cell, a neuron, a brain parenchyma cell, a small glial cell, an endothelial cell and / or a Purkinjcell cell (in some embodiments, a brain cell is a neuron.

In some of the flank and earlier embodiments the rAAV bus is the rAAV self-priming bus. In some embodiments, the vector comprises a first nucleic acid sequence encoding a heteronuclear DNA and a second nucleic acid sequence encoding a complement of nucleic acid where the first nucleic acid sequence can form a base pair in the strand with a second DNA sequence of most or all of its length. In some embodiments, a first nucleic acid sequence and a second nucleic acid sequence are linked by the AAV ITR mutant, wherein the AAV ITR mutant includes a D region deletion and includes mutagenesis of the terminal resolution sequence.

10046 In some of the previous aspects and models the individual is a human being.

[0047] In some embodiments from previous aspects and embodiments, heterochromic DNA encodes a therapeutic polypeptide or therapeutic DNA used in the treatment of CNS disorder (some embodiments. CNS disorder is lysosome storage disease (LSD), Huntington's disease, Parkinson's disease, epilepsy, Alzheimer's disease, stroke, basal cortical degeneration (CBD), basal cortical nodal degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy

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(MSA), progressive supranuclear palsy (PSP) or brain cancer. In some embodiments. The disorder storage disease with the present particles was selected from the group consisting of

Fabry, Aspartylglusoaminuria, Batten disease in infants (CNLl), Batten disease in infants classic late (CNL2), Juvenile Batten disease (CNL3), photo, Batten CNL4, Batten CNL5 picture, Batten CNL6 picture, Batten CNL7 picture, Batten CNL8 photo, Cystinuria 5, Farber, Galactosidosialidosis, Gaucher's disease Type 1, Gaucher disease Type 2, Gaucher disease Type 3, Gangliosidic disease GM1, Hunter's disease, Krabbe disease, 0 Manusoid disease Manusoid disease Maroteaux-Lamy, Metachromatic Leukoplakia disease Morquio A disease, Morquio B disease, Mucous lipodystrophy disease

11/111, Niemann-Pick A disease, Niemann-Pick B disease, Niemann-Pick C disease, 10 Pompe disease, Sandhoff disease, Sanfillipo A disease, Sanfillipo B disease, Sanfillipo C disease, Sanfillipo D disease, Schindler disease, sialidosis disease , Schindler-Kanzaki disease, Sly's disease, Tay-Sachs disease, and Wolman's disease (some instances of a disorder in CNS being Huntington's disease or Parkinson's disease.

10048 In some embodiments. The invention provides a method for treating Huntington's disease in an individual comprising 15

Give an effective amount of a pouch containing a gland-associated virus particle of rAAV recombinant product (rAAV) to the single striated node where the rAAV particle comprises a) rAAV capsid comprising the rAAV capsid comprising one or more amino acid substitutions in one or more solutions. The sites that interact with heparin sulfate protein glycans or at one or more of the homologous sites of the amino acids 484, 487, 527 and 532, 585 or 588 and 20 numbering is based on the VP1 numbering of AAV2, and b) the rAAV vector containing the heteronuclear acid

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Repeat both ends of AAV at least one. In some embodiments. It encodes a heterocyclic polypeptide

Therapeutic or therapeutic DNA. In some embodiments a therapeutic polypeptide is a polypeptide

Huntington or a sliver of it. In some embodiments the huntingtin polypeptide or a fragment thereof is a polypeptide

Huntingtin functional or a functional fragment thereof. In some embodiments, therapeutic DNA includes

RNAi is directed to Huntingtin; in some embodiments RNAi is miRNA.

10049 In some embodiments the invention presents a method for treating Parkinson's disease in an individual that includes administration of an effective amount of a combination comprising a virus particle associated with the gland of the product of recombinant gene recombination into the striatal node of the individual where the rAAV particle comprises a) rAAV capsid comprising one or more rAAV capsid. Of amino acid substitutions in one or more sites that interact with protein glycans of heparin sulfate or at one or more of the corresponding sites of amino acids 484, 487, 527 and 532, 585 or 588 and the numbering is based on the numbering VP1 from AAV2 ,, and b) the rAAV vector comprising heteronuclear acid and replicating at least one AAV terminals. In some embodiments, heterochromic DNA encodes a therapeutic polypeptide or therapeutic DNA. In some embodiments the therapeutic polypeptide is GTPCII, TH, BDNF, GDNF, and / or AADC; Or a sliver of it. In some embodiments. A therapeutic polypeptide of which AADC is a fraction.

[0050] In some of the preceding aspects and embodiments the heteronuclear nucleic acid is expressed at an increased level of expression compared to the expression level of the heteronucleic acid of the rAAV particle comprising the rAAV reference capsid. In some embodiments the rAAV particle causes reduced inflammation compared to the rAAV particle comprising a reference rAAV capsid. In some embodiments the rAAV particle includes an AAV capsid of serotype 2 (8872). In some models, one lowers

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One or more amino acid replacements are rAAV particle binding to heparin sulfate proteoglycans. In some embodiments. One or more amino acid substitutions reduce the binding of the rAAV particle to the heparin sulfate proteoglycans, compared to the binding of the rAAV particle comprising a reference rAAV capsid to the heparin sulfate proteoglycan. Some models. One or more amino acid substitutions reduces the rAAV particle binding to heparin sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75% or at least about 100%. Some embodiments reduce one or Most of the amino acid substitutions is the binding of the rAAV particle to the proteoglycan heparin sulfate by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 6100 ?, compared to the rAAV particle binding of a reference capsid to a proteoglycan Heparin Sulfate. In some embodiments the rAAV reference capsid includes the untreated rAAV capsid or capsid protein in some embodiments. A reference rAAV capsid includes an rAAV capsid or a capsid protein that lacks one or more amino acid substitutions at one or more sites that interact with the heparin sulfate proteoglycans.

[10051 In some embodiments from the aspects and earlier embodiments the rAAV particle is transported by stereotaxic transport. In some embodiments. The rAAV particle is transported by convection-enhanced transport. In some embodiments, the rAAV particle is administered using a 0i transport system. In some embodiments the rAAV particle is an anti-reflux cannula or a gradient cannula. In some embodiments, the CED transport system includes a cannula and / or a pump. In some embodiments the rAAV particle is administered using a CED transmission system; in some embodiments the pump is a hand pump. In some embodiments the pump is an osmotic pump. In some embodiments the pump is an infusion pump.

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[0052] In some of the previous aspects and embodiments the proteoglycans of heparin sulfate are expressed in one or more CNS cells (some embodiments are one or more of the CNS cells Oligodendrocyte, astrocyte, neuron, parenchymal tissue cell in the brain cell) Microglia neuronal endothelium and / or j.Purkinjcell cell, some embodiments of heparin sulfate proteoglycans are expressed in the neuron.

[0053] In some embodiments from previous aspects and embodiments, heteronuclear DNA is expressed in one or more tCNS cells in some embodiments. One or more CNS cells are oligodendrocyte, astrocyte, neuron, parenchymal tissue cell, microglial cell, neuroendothelial cell, and / or Purkinjcell cell. 0 Some embodiments of heterologous DNA are expressed in a nerve cell. In some embodiments, the heterozygous DNA is exclusively expressed in neurons.

10054 In some embodiments of the previous aspects and embodiments one or more substitutions of the amino acid are at 448, 451 484, 487, 527, 532, 585. The VP1 numbering is based on the AAV2 numbering. In some embodiments. The numbering is based on the VP1 numbering of AAV2 which includes the amino acid sequence of the sequence ID number: 1. In some embodiments, one or more amino acid substitutions includes the substitution of the positively charged amino acid building block with the positively charged amino acid residue. In some embodiments. The positively charged amino acid building unit is replaced by the hydrophilic amino acid building unit. In some embodiments one or more of the amino acid substitutions includes the substitution of the building block arginine or lysine. In some embodiments one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block.

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One or more of the amino acid substitutions includes substitutions in the position R35O, R347, R585, 532, 527, R487, R484, R451, R448, 395, 390 Wa or R588, with a numbering based on VP1 for 8872. In some embodiments it includes one or Most of the amino acid substitutions on substitutions in the position R585, 532, 527, R487, R484 and / or R588, By numbering based on VP1 for 88672. In some embodiments, the rAAV particle includes one or more rAAV capsid proteins that have at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% or more. At least 95% or at least 97% or at least 98% or at least 95% or at least 99% or at least 100% of the identity of the sequence number 2, 4 and / or 6. In some embodiments one or more of the amino acid substitutions includes R487A, R484A, R451A, R448A, 395Α, 390Α, R350A, R347A, 532Α, 527Α, numbering based on VP1L 8872. In some embodiments one or more amino acid substitutions include the substitution of R585A and / or R588A at the position R484 and R487 or at the positions R585 and 588 ^ numbered based on VP1 for AAV2. In some embodiments one or more amino acid substitutions include the R484A and R487A substitutions or substitutions. R585A and R588A, numbered based on VP1 to 8872. In some embodiments, the AAV capsid includes the amino acid R585A and R588A replacements, numbered based on VP1 for AAV2.0 Some embodiments The AAV capsid includes the amino acid 532Α capsid. Numbering based on VP1 to 8872-

10055 In some embodiments of the foregoing features and embodiments there is one or more amino acid substitutions at position 485, 488, 528, 533, and 589, which are non-numbering based on the VP1 numbering of AAVrhSR (some embodiments, the particles of the invention include c

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A capsid with one or more amino acid substitutions at positions 485, 488, 528, 533 and the numbering is dependent on the VP1 numbering of AAVrhSR. In some embodiments the numbering is dependent on the VP1 of AAVrhSR comprising the amino acid sequence of sequence No. 9. In some embodiments, one or more amino acid substitutions includes the substitution of the positively charged amino acid building unit with the positively charged amino acid building unit. In some embodiments, the positively charged amino acid building unit is replaced by the hydrophobic non-hydrophilic amino acid building unit. In additional embodiments, one or more of the amino acid substitutions includes the substitution of the building block arginine or lysine. In additional embodiments, one or more amino acid substitutions also include the substitution of an arginine or lysine building block with an alanine residue. In other embodiments, one or more amino acid substitutions includes the substitution of the positively charged amino acid residue with the positively charged amino acid residue. . In some embodiments the non-hydrophilic amino acid building unit is replaced by the positively charged amino acid building unit. In additional embodiments, one or more of the amino acid substitutions comprise the substitution of the alanine building block. In additional embodiments one or more of the amino acid substitutions also includes the substitution of an alanine building block for an arginine or lysine building unit; in some embodiments, the amino acid substitution is at position 485, 488, 528, 533, or 589, numbered according to the AAVrhSR numbering. In some embodiments the numbering is based on the VP1 numbering of AAVrhSR comprising the amino acid sequence of the sequence ID number: 9. In some embodiments, the amino acid substitution includes the substitution of the position R533, R488, R485, or 589, with a numbering based on the AAVrhSR numbering. In some embodiments, the rAAV particle includes the rAAV capsid of the sequence ID: 11. In some embodiments the rAAV particle includes one or more

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Capsid proteins have at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 97% or at least 98% or at least 95% or more At least 99% or at least 100% of the 11th sequence identity. In some embodiments, the amino acid substitution includes a substitution of R533A, Yukon numbering dependent on VP1 from AAVrh8R.5. In some embodiments, the rAAV particle includes AAVl capsid, AAV2 capsid, 8873 capsid, AAV6 capsid, AAV8 capsids, AAVrhSR capsids, AAVrO capsids, AAVrO capsids. In some embodiments, a single amino acid substitution includes a substitution of R533A, with a numbering based on VP1 of the AAVrhSR.

In some embodiments from previous aspects and embodiments, heterozygous DNA undergoes enhanced sequence control that is expressed in one or more tCNS cells. Some embodiments, heterozygous DNA is subject to enhanced sequence control selected from the group consisting of the cytomegalovirus (CMV) promoter. Early-Prompt MoMLVLTR, RSV LTR, Phosphoglycerate Kinase-1 Booster (PGK), Monkey Virus Booster 40 (SV40), CK6 Booster, Transthretin Booster (TTR),

TK booster, tetracycline-responsive (TRE) booster, HBV booster, hAAT booster, LSP booster, chimeric liver booster (LSP), E2F booster, telomerase (hTERT) booster, CMV booster / chicken beta-actin booster / 0-globin rabbit (CAG), α-prolonging factor-1 (EF1— alpha) booster,; human-glucorinidase booster — chicken actin (CBA), Russian retrovirus sarcoma booster (LTR), dihydrofolate reductase booster and actin 13 booster. In some embodiments. . Be a heterotypic nucleic acid operatively attached to a promoter suitable for expression of a therapeutic polypeptide or therapeutic DNA in one or more CNS cells ^ some c

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Models include one or more CNS cells containing one or more brain cells. In some embodiments, one or more of the CNS cells is an oligodendrocyte, an astrocyte, a neuron, a brain parenchyma cell, a small glial cell, an endothelial cell, and / or an 11-cell glial. In some embodiments, a brain cell is a neuron.

In some embodiments of the aspects and earlier embodiments, the rAAV vector is a self-executing rAAV vector; in some embodiments the vector includes a first DNA homolog encoding heterozygous DNA and a second nucleic acid sequence encoding a complementary DNA where the first nucleic acid sequence can form a base pair in the strand with A second nucleic acid sequence of most or all of its length. In some embodiments. A first nucleic acid sequence and a second nucleic acid sequence are linked by the AAV ITR mutant, wherein the AAV ITR mutant includes a D region deletion and includes the mutagenesis of the terminal resolution sequence.

(0058; In some of the preceding aspects and embodiments, the individual is a human being.

In some embodiments, the invention provides a kit for use in any previous embodiments comprising a gland-associated virus particle of the recombinant product (rAAV), Where the rAAV particle comprises a) a rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid substitutions at and one or more sites that interact with heparin sulfate protein glycans or at one or more of the homologous sites of the amino acids 484, 487. 527, 532, 585 or 588 and the numbering is based on AAV2's VP1 numbering; And b) a rAAV vector comprising heteronuclear DNA and at least one-end flip-flop AAV solution. In some embodiments, the invention provides a kit for transporting heterologous DNA into the central nervous system (CNS) of an individual

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It includes a combination comprising a glandular conjugated virus particle of a recombinant product (rAAV), wherein the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid precipitants at one or more sites that interact with the proteoglycans Heparin sulfate, and b) the rAAV vector comprising heteronuclear acid and at least one inverted AAV terminal repeat. In some embodiments, the invention presents a treatment kit for a central nervous system (CNS) 5 disorder in an individual comprising a combination comprising a glandular recombinant virus (rAAV) particle, wherein the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising On one or more amino acid substitutions in one or more sites interacting with heparin sulfate proteoglycans, and b) the rAAV vector comprising heterotypic DNA for treating CNS disruption and at least the inverted AAV terminal replication. 10

[0060] In some embodiments from previous aspects and embodiments it is a disorder of the CNS of Huntington's disease In some embodiments the heterocyclic DNA encodes a therapeutic polypeptide or the therapeutic DNA. In some embodiments, the therapeutic polypeptide is a huntingtin polypeptide or a fragment thereof. In some embodiments, huntingtin polypeptide or a fragment thereof is a functional huntingtin polypeptide or a functional huntingtin fragment thereof. In some embodiments, the therapeutic nucleic acid includes RNAi directed to huntingtin 15. In some embodiments, RNAi is 1 h. In some embodiments, this disorder is Parkinson's disease. In some embodiments, the heterochromic DNA encodes a therapeutic polypeptide or therapeutic DNA. In some embodiments, the therapeutic polypeptide is GDNF, GTPCII, TH, BDNF, and / or AADC; Or a sliver of it. In some embodiments of the therapeutic polypeptide it is AADC or a fragment thereof.

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[0061] In some embodiments, the invention introduces an adenovirus particle of recombinant gene (rAAV) for use in any previous embodiments. In some embodiments the invention introduces a gland-coupled virus particle of a recombinant product (rAAV) to transport heterozygous DNA into the central nervous system (CNS) of the individual where the rAAV particle comprises a) rAAV capsid comprising a rAAV protein capsid comprising one or more substitutes of an acid Amino acids at one or more sites that interact with a protein glycan of heparin sulfate or at one or more of the corresponding sites of amino acids 484, 487, 527 and 532, 585 or 588 and the numbering is dependent on the VP1 numbering of AAV2, and b) the rAAV vector containing the amino acid Nuclear heterozygote and repeat at least one inverted AAV terminal. In some embodiments the invention introduces a gland-associated virus particle of a recombinant product (rAAV) for treating a central nervous system (CNS) disorder of the individual where the rAAV particle comprises a) rAAV capsid comprising the rAAV protein capsid comprising one or more amino acid substitutions at one time. One or more of the sites that interact with heparin sulfate proteoglycans and include one or more homocysteine ​​metabolites at one or more sites that interact with heparin sulfate protein glycans or at one or more of the acid homologues Amino 484, 487, 527 and 532, 585 or 588 and the numbering is dependent on the VP1 numbering of AAV2 and b) the rAAV vector comprising heterocyclic DNA at at least one terminal repeat unit of AAV. In some embodiments the invention provides a glandular recombinant virus (rAAV) particle for treating Huntington's disease in an individual, wherein the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid substitutions in one or more loci. That interacts with heparin sulfate proteoglycans or one or more sites

<img file="MA39439B2_D0003.tif" />

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The corresponding amino acids 484, 487, 527 and 532, 585 or 588 and the numbering is based on the VPl numbering of AAV2, and b) the rAAV vector comprising the heteronuclear acid and at least one from a repeat of at least one AAV terminus, wherein the rAAV particle is formulated for delivery to the layers. Some embodiments The invention provides a gland-associated virus particle of a recombinant product (rAAV) for treating Parkinson's disease in an individual, wherein the particle includes rAAV a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid substitutions in one or more of the The sites that interact with heparin sulfate proteoglycans or one or more of the corresponding amino acid sites 484, 487, 527 and 532, 585 or 588 and the numbering is based on the VPl numbering of AAV2, and b) the rAAV vector comprising the heteronuclear acid and at least one of the replicates At least one AAV ends, as the rAAV particle is formulated for conduction to the layers. In one aspect, in some embodiments the invention provides a glandular recombinant virus (rAAV) particle for treating Huntington's disease in an individual, wherein the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more amino acid substitutions in One or more of the sites that interact with the proteoglycan heparin sulfate or one or more of the homologous sites of the amino acid 484, 487, 527 and 532, 585 or 588 and the numbering is based on the VPl numbering of AAV2, and b) the rAAV vector comprising one heteronuclear acid and one From at least one AAV end replication, where the rAAV particle is formulated to conduct into a single locus (eg, CNS in an individual). In some embodiments the invention provides a glandular recombinant virus (rAAV) particle for treating Parkinson's disease in an individual, wherein the rAAV particle comprises a) rAAV capsid comprising a capsid of rAAV proteins comprising one or more substitutions

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The amino acid in one or more of the sites that interact with the proteoglycan heparin sulfate or one or more of the homologous sites of the amino acid 484, 487, 527 and 532, 585 or 588 and the numbering is dependent on the VP1 numbering of AAV2, and b) b) the rAAV vector comprising Nucleic acid heterozygous and at least one of at least one AAV terminal replication, wherein the rAAV particle is formulated for delivery to a single locus (for example, to the CNS of the individual)

[0062] In some of the preceding aspects and embodiments, the heteronuclear nucleic acid is expressed at an increased level of expression compared to the expression level of the heteronuclear rAAV particle comprising the rAAV reference capsid. In some embodiments, the rAAV particle causes reduced inflammation compared to the rAAV particle comprising the rAAV capsid comprising a reference capsid. In some embodiments the rAAV particle includes an AAV capsid of serum type 2 (AAV2). 5. Some embodiments reduce one or more amino acid precipitations of the rAAV particle binding to the heparin sulfate proteoglycan. In some embodiments one or more amino acid substitutions reduces the binding of the rAAV particle to the heparin sulfate proteoglycan compared to the rAAV particle binding of a reference rAAV capsid to the heparin sulfate proteoglycan. In some embodiments one or more amino acid replacements reduce the rAAV particle binding to heparin sulfate proteoglycans by at least 10%, at least 25%, at least 50%, at least 75% or at least 100% in some embodiments. , One or more amino acid substitutions reduce the rAAV particle binding to heparin sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, Or at least about 100% of the rAAV particle binding to a reference capsid with heparin sulfate proteoglycans. In some embodiments, the rAAV reference capsid includes a capsid

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rAAV is an untreated type or a capsid protein. In some embodiments, the rAAV reference capsid includes an rAAV capsid or a capsid protein that lacks one or more homocysteine ​​proteins in one or more sites that interact with the heparin sulfate proteoglycan.

10063 In some of the previous aspects and embodiments, heparin sulfate proteoglycans are expressed in one or more CNS cells. In some embodiments one or more 5 CNS cells are oligodendrocyte, astrocyte, neuron, parenchymal tissue cell in the brain cell Microglia neuronal endothelium and / or Purkinjcell cell (Some embodiments. Heparin sulfate proteoglycans are expressed in the neuron.

[0064] In some embodiments of the preceding aspects and embodiments, heterozygous DNA is expressed

In one or more of the 5 L cells, in some embodiments one or more of the CNS 10 cells is oligodendrocyte, astrocyte, neuron, parenchymal cell, microglial cell, neuroendothelial cell and / or Purkinjcell cell (some embodiments In a nerve cell, heterozygous DNA is expressed, and in some embodiments, the heteronuclear expression is exclusively expressed in the neuron.

In some embodiments of the previous aspects and embodiments one or more of the 15 amino acid substitutions is at 484, 487, 527, 532, 585 and / or 588, numbering based on the numbering 88722. In some embodiments one or more amino acid substitutions are in situ.

484, 487 532, 585 or 588, numbering based on the numbering 88722. Some models

One or more amino acid substitutions includes the building block substitution of an amino acid

The positively charged by the structural unit of an uncharged amino acid. In some embodiments, Module 20

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The structural unit of a positively charged amino acid is replaced by the structural unit of an amino acid that is non-hydrophilic. In some embodiments one or more of the amino acid substitutions includes the substitution of an arginine or lysine building unit; in some embodiments one or more amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine subunit. In some embodiments. One or more amino acid substitutions includes the substitution in the position R35O, R347, R585, 532, 527, R487, R484, R451, R448, 395, 390 and / or R588, with the numbering

Based on VP1 to 8872, in some embodiments one or more amino acid substitutions includes substitutions in the R585, 532, 527 and / or R588 sites, with a construct number

On VP1 to AAV2 ^ some embodiments, the rAAV particle includes the rAAV particle comprising one or more rAAV capsid proteins in which it is at least about 90% or at least 91% or at least 92% or at least 93% or at least 94% Or at least 95/5, fold '97/5, fold 98 / e, or fold 95/5, or fold 99/5, or at least 100% of the identity of the sequence No. 2, 4, and / or 6. In In some embodiments, one or more amino acid substitutions include 395Α, Κ390Α, R350A, a R347A, 532Α, 527Α, R487A, R484A, R451A, R448A, Replace R585A and / or R588A,

With numbering based on VP1 for AAV2 ^ some embodiments, one or more amino acid substitutions include substitutions at position R484 and R487 or at positions R585 and R588, numbering based on VP1 for AAV2. Some embodiments include one or more amino acid substitutions on R484A substitutions. And R487A or substitutions R585A and R588A, with a numbering based on VP1 to AAV2. 5 Some embodiments The AAV capsid includes the amino acid R585A and R588A replacements, numbered based on VP1 for 88722. In some embodiments the AAV capsid includes

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The amino acid substitutions Α532Α, numbering based on VP1 to 8872. In some embodiments, the 48827 particle includes AAVl capsid, AAV2 capsid, AAV3 capsids, AAV6 capsids, AAV8 capsids, AAVrhSR capsids, AAV9 capsids or AAVrhlO capsids.

[0066] In some embodiments from the aspects and previous embodiments one or more of the amino acid substitutions is at position 485, 488, 528, 533, 586 or 589, numbering according to the numbering] 887118. In some embodiments the numbering is dependent on the VP1 of AAVrhSR which includes the amino acid sequence of the sequence ID number: 9. In some embodiments, one or more amino acid substitutions includes the substitution of the positively charged amino acid building block with the positively charged amino acid residue. In some embodiments the positively charged amino acid building unit is replaced by the hydrophobic amino acid building unit. In additional embodiments one or more amino acid substitutions include the substitution of the arginine or lysine building block. In additional embodiments also one or more of the amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block. In the other embodiments one or more amino acid substitutions comprise the substitution of the building block of a positively charged amino acid with the building unit of a positively charged amino acid. In some embodiments the non-opaque amino acid building block of water is substituted for the positively charged amino acid building unit. In additional embodiments one or more amino acid substitutions include the substitution of the alanine residue. In additional embodiments also one or more amino acid replacements include the substitution of an arginine or lysine building block with an alanine building block. In some embodiments, the amino acid substitution is at position 485, 488, 528, 533, or 589 with a numbering based on the numbering.

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-K-

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A lectin. In some embodiments, the heteronuclear DNA is operationally attached to a promoter suitable for expression of either the therapeutic polypeptide or the therapeutic DNA in one or more of the 5LE cells of some embodiments. One or more CNS cells contain one or more brain cells. In some embodiments. One or more of the CNS cells are oligodendrocyte, astrocyte, neuron, parenchymal tissue cell. Small glial cell neuroendothelial cell wa or 5 Purkinjcell cell.

In some of the lateral and earlier embodiments the rAAV vector is a rAAV autotransporter. The carrier comprises a first nucleic acid sequence encoding a heteronuclear DNA and a second nucleic acid sequence encoding a complementary DNA, where the first nucleic acid sequence can form a base pair in the strand with a second nucleic acid sequence of most or all of its length. A first nucleic acid sequence and a second nucleic acid sequence are linked by the AAV ITR mutant, wherein the AAV ITR mutant includes a D region deletion and includes mutagenesis of the terminal resolution sequence.

[0069] In some of the previous aspects and embodiments, the individual is a human being.

In some of the preceding aspects and embodiments the rAAV particles are in a combination. J15 Some embodiments of Turkish include a blocking buffer solution and / or pharmaceutically acceptable excipients. In some embodiments the kit or rAAV particle further includes instructions for transferring the rAAV particle composition to the CNS device. In some embodiments the kit or rAAV particle further includes instructions for transferring the rAAV particle composition to the stripped node.

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In some embodiments, the invention introduces a rAAV particle comprising AAVrhSR capsid protein, Where the AAVrhSR capsid protein comprises one or more amino acid substitutions where one or more amino acid substitutions increase the binding of the rAAV particle to the heparin sulfate proteinoglycan compared to the AAV particle comprising the untreated AAVrhSR capsid protein or where one or more substitutions are At one or more 5 positions to correspond to the amino acids of 484, 487, 527, 532, 585 or 588 with VP1-based numbering for AAV2- in some embodiments. One or more amino acid substitutions increase the rAAV particle attachment to heparin sulfate proteoglycans by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% in some embodiments. . The amino acid substitution is at position 10, 586

With a VP1-based numbering from AAVrhSR, in some embodiments the numbering is based on VP1 for AAVrhSR that includes the amino acid sequence of the AAVrhSR sequence. In some embodiments, the amino acid substitution includes a substitution at the 586 position, with a VP1 based numbering from the AAVrhSR numbering. In some embodiments, the amino acid substitution includes the replacement of A586R or the substitution of 586Κ with a VP1-based numbering from the AAVrh8R ^ in some embodiments a 15 rAAV particle comprising the rAAV protein capsid of the sequence ID: 10.

10072 In some embodiments, the invention presents a method for increasing the binding of a rAAV particle comprising the AAVrh8R capsid protein to the heparan sulfate proteoglycans, comprising one or more advances of an amino acid to a capsid protein, in which one or more amino acid substitutions increase the binding of the rAAV particle to the heparan sulfate proteoglycan. Compared to AAV particle 20 comprising an untreated type AAVrhSR capsid protein. In some embodiments, more than one or more than one

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Amino acid substitutions from rAAV particle binding to heparan sulfate proteoglycan by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the amino acid substitution is in Position 586, numbering based on AAVrh8R. In some embodiments, the numbering is based on VPlg AAVrh8R and includes the amino acid sequence of the sequence ID number: 9. In some embodiments, the amino acid substitution includes the substitution at the 586 locus, with a numbering based on the AAVrhSR numbering. In some embodiments, the amino acid substitution includes the replacement of A586R or 586, with a numbering based on the AAVrhSR numbering. In some embodiments, the rAAV particle includes RAAV capsid protein for sequence ID number: 10.

In some aspects, the invention presents a method for delivering heterogeneous DNA into an individual's retina that includes the administration of within the vitreous a virus particle associated with an adenoma of a recombinant product (rAAV) to the individual, wherein the rAAV particle comprises a) rAAV capsid comprising rAAV capsid proteins It includes one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycans or at one or more of the homologues of the amino acids 484, 487, 527, 532, 585 or 588, numbering based on the VPl AAV2 numbering, And b) The rAAV vector comprising heterocyclic DNA and replicating at least one inverted AAV tip. In some respects, the invention presents a method for improving the rAAV conversion of cells after intravitreal delivery of the rAAV particle to a single eye compared to the conversion of cells with rAAV involving an untreated capsid. The method includes the inclusion of one or more amino acid substitutions in the AAV capsid protein when One or more sites that interact with heparan sulfate proteoglycans or at one or more of the corresponding sites

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For amino acids 484, 487, 527, 532, 585 or 588, numbering based on VPI AAV2 numbering; Where the rAAV particle comprises the rAAV capsid protein, the rAAV vector comprises heteronuclear DNA and replicates at least one AAV terminal. In some aspects, the invention presents a method for improving the expression of heterozygous nucleic acid after the intravitreal delivery of rAAV particles to the eye of an individual. The method includes the inclusion of one or more amino acid precursors into the AAV capsid protein at one or more sites where they interact. With heparan sulfate proteoglycans or at one or more of the corresponding positions of amino acids 484, 487, 527, 532, 585 or 588, numbering according to the numbering 711 AAV2; Where the rAAV particle comprises the rAAV capsid protein, and the rAAV vector comprises heteronuclear DNA. Repeat at least one AAV tip. In some respects, the invention presents a method for treating an eye-to-eye disorder involving the delivery in the vitreous of a combination that includes rAAV particles to an individual's retina, wherein rAAV particles comprise a) rAAV capsid comprising a rAAV capsid protein comprising one or more amino acid antigens At one or more sites that interact with heparan sulfate proteoglycans or at one or more of the homologous sites of the amino acid 484, 487, 527, 532, 585 or 588, numbering based on the numbering VP1 AAV2, and b) the rAAV vector comprising acid Nuclear heterogeneous and repeatable At least 1 AAV tip. In some respects, the invention introduces a system for the delivery within the vitreous of a transporter to the eye of an individual, comprising a) a composition comprising an effective amount of rAAV particles, where i) a protein capsid of rAAV particles comprising one or more amino acid substitutions of one or more More than the sites that interact with heparan sulfate proteoglycans or at one or more of the sites corresponding to the amino acid 484, 487, 527, 532, 585 or 588, Wa)

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A vector comprising a heterocyclic nucleic acid encoding a therapeutic polypeptide or RNA and a replication of at least one AAV tip; And b) a device for delivering rAVVV into the vitreous. In some embodiments, the rAAV particle includes capsid of serotype 76, 4871, AAVrh8R e8, AAV9, AAV8, or AAVrhlO capsid of serotype. In some aspects, the invention presents a set of treatment for an eye disorder comprising a) a combination that includes rAAV particles, where the rAAV particle comprises i) a rAAV capsid comprising rAAV capsid proteins comprising one or more amino acid substitutions at one or more of the sites that interact With heparan sulfate proteoglycans or at one or more of the corresponding positions of amino acids 484, 487, 527, 532, 585 or 588, numbering based on VPl AAV2 numbering, and Ü) the rAAV vector comprising heterocyclic DNA for treating ophthalmic disturbance and AAV tip replication At least one inverted; And b) pharmaceutical excipients suitable for vitreous administration. In some embodiments, the rAAV particle includes a capsid AAV9, AAV8, AAV6, AAVl, AAVR, or AAVrhlO. In some respects, the invention introduces an rAAV capsid comprising the AAVl capsid protein, wherein the AAVl capsid protein includes one or more amino acid substitutions, wherein One or more amino acid substitutions increases the efficacy of converting the rAAV capsid into a cell in the eye compared to an AAV particle comprising the 1 887 capsid protein of the untreated type, or where one or more amino acid substitutions are at one or more of the 484 amino acid homologues. , 487, 527, 532, 585 or 588, numbering based on AAV2 711. In some respects, the invention introduces an rAAV particle comprising the AAV6 capsid protein, wherein AAV6 capsid protein comprises one or more amino acid substitutions, denoting one or more substitutions. The amino acid efficacy of converting rAAV capsid into a cell in the eye compared to a particle

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AAV includes a capsid protein of the untreated type AAV6, or where there is one or more amino acid substitutions at one or more of the homologous sites of the amino acids 484, 487, 527, 532, 585 or 588, a numbering AAV2 711. In some aspects, the invention introduces a rAAV particle comprising the AAV8 capsid protein, wherein AAV8 capsid protein comprises one or more amino acid substitutions, where one or more amino acid substitutions increase the efficacy of converting the rAAV capsid into a cell in the eye compared to the AAV particle. It includes protein A capsid of the untreated type AAV8, or where one or more amino acid substitutions are at one or more sites corresponding to the amino acids 484, 487, 527, 532, 585 or 588, numbering based on AAV2 711. The invention introduces a rAAV particle comprising the AAV9 capsid protein, in which the AAV9 capsid protein comprises one or more amino acid substitutions, in which one or more amino acid substitutions increase the efficacy of converting the rAAV capsid into a cell in the eye compared to the AAV particle comprising the capsid protein of Untreated type AAV9, or where one or more amino acid substitutions are at one or more of the corresponding positions of the amino acids 484, 487, 527, 532, 585 or 588, numbering based on AAV2 711. In some respects, the invention introduces a rAAV capsid that includes AAVrhlO capsid protein, in which AAVrhlO capsid protein includes one or more amino acid replacements, where one or more amino acid substitutions increase the efficacy of converting rAAV capsid into a cell in the eye compared to an AAV particle comprising the untreated AAVrhlO capsid protein, or Wherever it is One or more substitutions of an amino acid at one or more of the corresponding positions of the amino acids 484, 487, 527, 532, 585 or 588, numbering based on AAV2 6711.

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The invention introduces the rAAV capsid comprising the AAV3 capsid protein, wherein the AAV3 capsid protein comprises one or more amino acid substitutions at one or more homologous sites of the amino acids 484, 487, 527, 532, 585 or 588, numbering according to the AAV2 numbering 711. In some embodiments, the rAAV particle includes AAVl capsid, AAV2 capsid, AAV3 capsid, AAV6 capsids, AAV8 capsids, AAVrhSR capsids, AAV9 capsids, or AAVrhlO capsids (in some embodiments, the transfer efficiency is increased by at least about 10%, upon At least about 25%, at least about 50%, on The lowest is about 75%, or at least about 100%.

In some embodiments, one or more proteins of the amino acid that binds the capsid rAAV to the heparin sulfate proteoglycans increase. In some embodiments, one or more amino acid substitutions that bind the capsid rAAV to the heparin sulfate proteoglycans increase by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about In some embodiments, one or more amino acid substitutions increases the efficiency of rAAV capsid conversion to a cell in the eye or central nervous system, compared to an AAV particle comprising a non-treated AAVrhSR capsid protein, by at least about 10%, respectively. The least is about 25%, at least About 50%, at least about 75%, or at least about 100%. In some embodiments, the eye cell is a reticulocyte, photoreceptor cell, retinal pigment epithelial cells, bipolar cells, lateral cells, parenchyma cells, Müller cells and / Or ganglion cells. In some embodiments, one or more amino acid substitutions involve the substitution of a single amino acid building block that is not positively charged with a positively charged amino acid building block. In some embodiments, unity

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The positively charged amino acid structure replaces the hydrophobic amino acid building block. In some embodiments, one or more of the amino acid substitutions includes the substitution with an arginine or lysine building block. In some embodiments, one or more amino acid substitutions include substituting alanine, serine, glutamine, or threonine with an arginine or lysine building block. In some embodiments, the rAAV particle comprises the AAV capsid of the rh8R serotype (AAVrh8R). 5 In some embodiments, one or more amino acid substitutions are at positions 586 and / or 589, numbering based on AAVrh8R 711. In some embodiments, the numbering is MBE on VPl g AAVrh8R includes the amino acid sequence of homozygous No .: 9. In some embodiments, one or more amino acid substitutions comprise a substitution at the Α586 and / or 589 position, numbering based on the AAVrhSR 711 numbering. In some embodiments, it includes one or more amino acid substitutions. One or more of the amino acid replacements Substituting A586R or 586Κ, numbering based on VP1 for AAVrh8R. In some embodiments, one or more amino acid substitutions include substituting T589R or 589Κ, numbering based on VP1 for AAVrhSR. In some embodiments, the rAAV particle includes the AAV serotype for capsid 1 (AAV1) · In some embodiments, one or more amino acid substitutions are at positions 586 and / or 589, numbering based on AAV1 VP1. In some embodiments, VP1 for AAVl includes the amino acid sequence of the homozygous sequence number: 12 In some embodiments, one or more amino acid substitutions include substitution at the S586 and / or 589 position, numbering based on the AAVl 711 numbering. In some embodiments, one or more amino acid substitutions include the S586R or S586K substitution, the numbering Based on VP1 for AAV1. In some embodiments, one or more amic acid substitutions comprise a replacement for T589R or 589Κ, numbering based on VP1 for 8871. In some embodiments, a particle is included.

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rAAV is based on serotype AAV capsid 6 (AAV6) (some embodiments have one or more amino acid substitutions at positions 586 and / or 589, numbering based on VPI numbering 88776. In some embodiments, the numbering is based on VP1 for AAV6 containing The amino acid sequence of the sequence ID: 13. In some embodiments, one or more amino acid replacements include substitution at position S586 and / or 589, numbering based on the numbering 711 AAV6. In some embodiments, one or more amino acid substitutions include a substitution of S586R, numbering based on VP1 for AAV6.5. In some embodiments, one or more amino acid substitutions include a substitution of T589R or 589, numbering based on VP1 for 88776. In some embodiments, one or more amino acid substitutions include a substitution of T589R or 589. Embodiments, the rAAV particle includes a capsid of the AAV8 (AAV8) serotype. In some embodiments, one or more amino acid replacements are at positions 588 and / or 591, numbering according to AAV8 711. In some embodiments, VP1 of AAV8 comprises the amino acid sequence of the primordial sequence No. 4: 1. In some embodiments, one or more of the amino acid substitutions includes substitution at the position Q588 and / or 591, numbering according to the numbering AAV8 711. In some embodiments, one or more amino acid substitutions include a Q588R or Q588K substitution, numbering based on VP1 G 88788. In some embodiments, one or more amino acid substitutions includes a replacement for T591R, numbering based on VP1 for 88788. In some embodiments , The rAAV particle includes an AAV capsid of the type Serum 9 (8879). In some embodiments, one or more amino acid substitutions are at positions 586 and / or 589, numbering based on AAV9 numbering 711. In some embodiments, VP1 from AAV9 includes the amino acid sequence of the sequence ID: 15 In some embodiments, one or more amino acid replacements include substitution at the S586 and / or 589 position, numbering based on the VP1 numbering.

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8879. In some embodiments, one or more amino acid substitutions include a substitution of S586R or S586K, numbering based on VPl for AAV9. Some embodiments, one or more amino acid substitutions include substituting A589R or 589, numbering based on VPl for 88799. Models, Particle 3887 includes a capsid of 8827 of the Rhio serotype (AAVrhlO). In some embodiments, one or more amino acid substitutions are at positions 588 and / or 591, numbering based on AAVrhlO 711. In some embodiments, the VPl of AAVrhlO includes the amino acid sequence of the sequence ID: 16. In some embodiments, One or more amino acid substitutions include substitutions at position Q588 and / or 591, numbering based on AAVrhlO 711. In some embodiments, one or more amino acid substitutions includes substitutions of Q588R or Q588K, numbering based on AAVrhlO 1 VPl. In some embodiments, it includes one or more Of amino acid substitutions on the substitution of A591R or 591قيم, numbering based on VPl and AAVrhO ^ In some embodiments, the invention introduces an rAAV particle comprising the AAV3 capsid protein, wherein the capsid protein AAV3 comprises one or more amino acid substitutions at one or more loci The amino acid analog for 484, 487, 527, 532, 585 or 588, numbering based on AAV2 711. In some embodiments, one or more amino acid substitutions increases the efficiency of the rAAV capsid conversion of a cell in the eye or central nervous system, compared to the AAV particle. Includes Contains untreated AAVrhSR capsid protein, in a ratio of at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the rAAV particle includes AAVl capsid, AAV2 capsid, AAV3 capsid, AAV6 capsid, AAV8 capsids, AAVrhSR capsids, AAV9 capsids, or AAVrhlO capsids. In some embodiments,

<img file="MA39439B2_D0004.tif" />

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The heterocyclic amino acid encodes a therapeutic polypeptide or nucleic acid. In some embodiments, the heterocyclic amino acid encodes a polypeptide selected from the group consisting of an antioxidant, a neurotrophic factor, an anti-cytostatic agent, an anti-angiogenic agent, and an anti-inflammatory agent. In other embodiments, the heterocyclic amino acid encodes a polypeptide selected from the group consisting of: CEP290, CRXLCA5, GUCY2D, AIPLl, RPE65._،Prph2 ,, TULPINMNATI, LRAT, CRBl, IMPDHl, RDH12, ABCA4 ,, Clarin, MYO 7a, FGF2, CNTF, GDNF, GNAT2, CNGB3, CNGA3, RPGRIP, RP2, RPGR, MERTK, AngDlt-1110, RPGR, MERTK , Endostatin, NFkB, BCL-X, BCL2, EPO, PEDF

Anti-17, sTNF-R II, sTNF-R I, TGFp۵L- ^, ILl-a, sIL17R, and 4) 1. In other embodiments, the heterocyclic amino acid encodes a therapeutic nucleic acid. In other embodiments, the therapeutic DNA is miRNA, shRNA RNAi, siRNA, anti-transcriptional RNA, ribonim or DNAzyme. (In some embodiments, the rAAV vector is a self-complementing rAAV vector. In some embodiments, the AAV particles of the invention comprise a capsid that comprises one or more amino acid substitutions that alter the binding to HSPG (for example, reduce or eliminate HSPG) or at one or more amino acid homologues 484, 487, 527. , 532, 585 or 588, numbering based on VPl AAV2 numbering and heterocyclic DNA encoding a therapeutic polypeptyl or therapeutic DNA, wherein the heterocyclic amino acid is under the control of a reinforced sequence that is expressed in the retina. In some embodiments, the heterocyclic amino acid is operationally bound to a suitable promoter for expression of a therapeutic polypeptide or therapeutic nucleic acid in one or more retinal cell types. In some embodiments, the retina cell is a photoreceptor cell, retinal pigment epithelial cells, bipolar cells, lateral cells, parenchyma cells, Müller cells and / or

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Ganglion cells. In some embodiments, the promoter is a rhodopsin (RK) booster, an opsin booster, a cytomegalovirus (CMV) booster, a chicken beta actin booster (38 I0). In some embodiments, the individual is a human. In some embodiments, the heterocyclic amino acid is used to treat an eye disorder selected from the group consisting of acute autosomal early retinal degeneration (congenital potential for Leber's), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone malnutrition, inflammation Retinitis pigmentosa, X-ray retinopathy, Usher syndrome, age-related macular degeneration, atrophic macular degeneration, age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), macular edema, central retinopathy For serous, retinal detachment, intraocular inflammation, and posterior anthritis. In some embodiments, the rAAV vector is a self-complementing rAAV vector. In some embodiments, the vector includes a first nucleic acid sequence encoding a heterozygous amino acid and a second nucleic acid sequence encoding a complement of nucleic acid, wherein the first DNA twisting can form base pairs within the strand with the second DNA sequence encoding most or all of its length. Models, the first nucleic acid sequence and the second nucleic acid sequence are linked to AAV ITR mutagenesis, wherein AAV ITR mutagenesis includes D-region deletion and mutagenesis includes terminal degeneration sequence. In some embodiments, the individual is a human being. In some embodiments, one or more amino acid substitutions increase the conversion efficiency of the rAAV capsid to a cell in the eye or central nervous system, compared to an AAV particle containing the untreated AAVrhSR capsid protein, by at least about 10%, at least about 25%. %, At least about 50%, at least about 75%, or at least about 100%.

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[0075] All references cited in the present application, including patent applications and publications, are merged for reference in their entirety.

Brief description of shapes

Figure 1 shows the capsular building blocks involved in the proteoglycan binding of heparan sulfate and the mutations that were introduced to generate the 8872 HBKO mutant. The numbering is based on the amino acid sequence of VP1.

[0077] Figure 2 shows the decrease in conversion of 293 cells in culture observed with AAV2 particles of the AAV2 HBKO CBA-SFLTO2 (HBKO) mutant, compared to untreated AAV2 particles (AAV2 CBA-SFLTO2). Soluble sFLT present in cell culture media 48 hours after injection with AAV2 particles of an untreated type mutant or HBKO mutant carrying vectors that use CBA promoter to guide expression of Fit.

[0078] Figure 3 shows the reduction in the conversion of 293 cells and Hela cells in culture observed with AAV2 particles of the 8872 HBKO CBA-GFP (HBKO) mutant, compared to untreated AAV2 particles (AAV2 CBA-GFP). For cells taken after injection of B 48 with an untreated species or AAV2 particles of the HBKO mutant carrying vectors that use a CBA promoter to direct the expression of EGFP

[0079] Figs 4a and 4b illustrate the conversion observed in vitreous (Fig. 4a) or subretinal injection (Fig. 4b) of an untreated type of AAV2 or AAV2 particles of a mutant 0 ks. The conversion was tested by expression of Soluble Fit (sFLT) after conversion with encoding vectors. Fit shows the number of vector genomes injected per experiment (10 8 or 10 9 vg).

[0080] Figure 5 shows that the AAV2 particles of the HBKO mutant fail to transform the mouse eye after

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Vitreous injection. Mice were given an in vitro injection of untreated (AAV2 CBA-GFP) type or AAV2 particles of an AAV2 HBKO CBA-GFP (HBKO) mutant carrying vectors that used the CBA booster to guide the expression of EGFP, and the sections were imaged with fluorescence.

[0081] Shakzig 6 illustrates that AAV2 swarms of the AAV2 CBA HBKO (HBKO) mutant induce a statistically significant increase in conversion after subretinal injection, compared to untreated type particles (2 CBAd). The conversion was tested by expression of Soluble Fit (sFLT). ) HBL injection with AAV2 particles carrying vectors that use a CBA promoter to direct Fit expression. The number of injected vector genomes (810 or 10 9 vg) is indicated.

[0082] Fig. 7 shows that AAV2 particles of the AAV2 HBKO CBA-GFP (HBKO) mutant exhibit a statistically significant increase in photoreceptor cell conversion (as named) after subretinal injection, compared to untreated AAV2 CBA-GFP particles. Transformation was measured by fluorescence imaging of post-transduction expression using vector-bearing AAV2 particles using a CBA promoter to direct expression of EGFP.

[0083] Figure 8 shows that AAV2 particles of the AAV2RKHBKO mutant (HBKO) induce a statistically significant increase in photoreceptor conversion after subretinal injection, compared to untreated-type particles (8872 RK). The transformation was tested by soluble Fit expression (sFLT). After injection with AAV2 particles carrying vectors that use rhodopsin promoter (RK) to direct expression of Fit. The number of vector genomes injected is indicated (810 or 10 9 (vg)).

[0084] Figures 9A and 9B show the expression of EGFP in the mouse brain 30 days after injection into the striatum of AAV2HBKO-EGFP (Fig. 9A), compared to AAV2-EGFP (Fig.

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9 b) in an untreated mouse. In each plate, the expression of EGFP was guided by a CBA booster and visualized with fluorescence pyrophoresis.

Figures 10a and 10b show the expression of GFP in the mouse brain 30 days after the injection into the striatum of AAV2HBKO-miRNA-Htt-GFP (Fig. 10a), compared to AAVl-miRNA-Htt-GFP (Fig. 10b) in the 80128 HD * mouse. The miRNA-Htt-GFP vectors indicate 5 constructs expressing a synthetic miRNA targeting human Htt and a 1T81 transmitter. In each plate, expression of GFP was directed by a CBA promoter and visualized with a fluorescence microscope at three different magnifications (, 4, and 20 T, according to its label).

[0086] Figure 1A shows qPCR analysis of levels of human HTT mRNA in holes in the mouse brain striatum 30 days after injection of AAVl-miRNAHtt and 10, AAV2HBKO-miRNA-Htt

Compared to the untreated comparison groups.

Figure 11b shows Western blot analysis of human Htt protein levels in holes in the cortex of mouse brain 30 days after injection of AAVl-miRNA-Htt and AAV2HBKO-miRNA Htt, compared to groups for a comparison of untreated.

Figures 12a-12c show the expression of Ibal in the striatal node of a 15 YAC128 mouse 30 days after injection of AAV2HBKO-miRNA-Htt-GFP (Fig.12b) or -AAVl miRNA-Htt-GFP (Fig.12c), compared to the untreated comparison groups (Fig. 12 a).

[0089] Figures 13a-13c show the expression of GFP in the striated node of a YAC128 mouse 30 days after injection of AAV2HBKOmiRNA-Htt-GFP (Fig.13b) or -AAVl miRNA-Htt-GFP (Fig.13c), compared to groups for comparison of untreated (Fig. 13 a). 20

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(0090; Figure 14 compares the building blocks of a capsid involved in the proteoglycan binding of heparan sulfate between the AAVrhSRj AAV2 capsids The numbering is based on the amino acid sequence VP1 · [0091] Figure 15 shows the amino acid alignment of AAV2 and AAVrh8R in the building blocks responsible for binding heparan to the 88672 circuit. About the loci of AAVrhSR-arginine capsid modifications.

[0092] Fig. 16a shows the advanced in vitro conversion of HeLa cells imaged by the AAVrhSR A586R mutant, as compared to the untreated AAVrhSR. Conversion by SFLT02 was monitored in current settings 48 hours after infection with AAVrhSR or AAVrhSR arginine-modified vectors.

[10093 Figure 16b shows the in vitro latent conversion of HeLaRC32 cells imaged by AAVrhSR R533A mutant, compared to untreated AAVrhSR. The conversion was monitored by SFLT02 in the present media 48 hours after infection with AAVrhSR or AAVrhSR arginine-modified vectors.

[0094] Figures 117-17d show the in vitro conversion levels imaged by AAVrhSR A586R and R533A mutants, compared to untreated AAVrhSR mutant type AAVrhSR A586R (Fig.17b) illustrating the increased in vitro conversion of NSI cells, compared to AAVrhSR of Fig.17d) The transformation (AAVrhSR R533A) illustrates the untreated type (Fig.17a)

In vitro reduced HeLa cells, compared to untreated AAVrhSR (Fig. 17c); conversion was monitored by expression of EGFP in cells 48 hours after infection with AAVrhSR or AAVrhSR arginine-modified vectors.

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0095 Figs 18a and 18b show the levels of subretinal conversion in C57B16 mice imaged by AAVrhSR A586R and 125338 mutants. (Fig.18a) The AAVrhSR mutant A586R demonstrates a reduced subretinal transformation, compared to the untreated type AAVrhSR. M also tested AAV2 bus. (Fig.18b) AAVrhSR mutant R533A demonstrates increased subretinal shunt, compared to untreated AAVrhSR and untreated mice. Conversion was monitored by SFLT02 in retinal analysis products of C57B16 mice after 30 days of subretinal administration of AAVrhSR or AAVrhSR arginine-modified vectors.

00096 Figure 19 shows the levels of SFLT02 retinal degradation products in C57B16 mice after administration of AAVrhSR, AAV2 or AAVrh8R-A586R vectors in the vitreous at 30 days 100971 Figure 20 shows the amino acid alignment of the building blocks responsible for binding HRAN to AAV2 vectors with AAV8, 88679, AAV6. , AAVl, AAVrhSR, and AAVrhl0.

Detailed description

According to what has been described in the present application, 1 the inventors surprisingly discovered that modifications in the rAAV particles corresponding to the amino acids 484, 487, 532, 585, and / or 588, numbering based on the VPl AAV2 numbering, exhibit an incremental transformation of cells after administration into an eye. Or the nervous system of my subordinate CNS. Without wishing to be bound by any theory, it is believed that these rAAV particles reduced or removed the attachment to the HSPG or modified the charge on the capsid so that the administration of rAAV particles would result in an increased transformation of cells in the eye or in the CNS of a subject. Therefore, the present invention provides methods for transporting heterogeneous DNA into the eye or the CNS of an individual that include administering a glandular virus particle of the recombinant product (rAAV) to the eye or the CNS of the individual, wherein the rAAV particle comprises a) rAAV capsid comprising proteins

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The rAAV capsid comprises one or more amino acid substitutions at one or more sites that interact with a protein glycan of heparan sulfate, and b) the rAAV vector comprising a heterozygous DNA and an inverted terminal replication of at least one AAV transporter. In some embodiments, the amino acid substitutions reduce or inhibit HSPG binding

00099 In some embodiments, the present invention provides methods for transferring heterologous DNA into the eye of an individual that include administering a gland-associated virus particle of the recombinant product (rAAV) to the sub-retina of an individual, wherein the rAAV particle comprises a) rAAV capsid comprising the rAAV capsid protein. It comprises one or more amino acid substitutions in one or more sites that interact with heparan sulfate proteoglycans, and b) the rAAV vector comprising heteronuclear DNA and at least one inverted AAV terminal replication.

10100 In some respects, the invention presents methods for improving the rAAV conversion of cells after subretinal rAAV particle transfer to a single eye compared to the transduction of cells using an rAAV vector comprising an untreated capsid of the rAAV type. At one or more sites that interact with heparan sulfate protein glycans; Where the rAAV particle comprises the rAAV vector capsid protein, the rAAV vector comprises heteronuclear DNA and the tip replication of at least one AAV. In some embodiments, amino acid substitutions result in low or variable binding to HSPG (some embodiments, rAAV particles include a capsid that includes R588Aj R585A replacements for rAAV2, numbering based on VP1 for AAV2 (sequence ID: 1). AAV particles on a capsid comprising the A586R and / or R533A replacements for AAVrhSR, numbered based on VP1 for AAV2AAVrh8R (sequence ID number: 9).

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[0101] In some respects, the invention presents methods for improving the expression of heterozygous nucleic acid after the subretinal transfer of rAAV particles into an individual eye, the method includes the incorporation of one or more amino acid substitutions into the AAV capsid protein at one or more of the locations where Reacts with heparan sulfate protein glycans; Where the rAAV particle comprises the rAAV vector capsid protein, the rAAV vector comprises heteronuclear DNA and the tip replication of at least one AAV. In some embodiments, the amino acid substitutions result in reduced binding or ablation of HSPG (some embodiments, rAAV particles include a capsid that includes R585A and R588A replacements for rAAV2, numbering based on the VPl of AAV2 (sequence ID: 1). The rAAV particles comprise a capsid containing the A586R and / or R533A replacements for AAVrhSR, with a VPl-based numbering for AAVrhSR (sequence ID number: 9).

[20102 In some embodiments, the invention presents methods for improving the expression of heterozygous DNA following the subretinal transfer of rAAV particles to an individual eye. The method includes the incorporation of one or more amino acid replacements into the AAV capsid protein at one or more sites that interact with the proteoglycans. Heparan sulfate; Where the rAAV particle comprises the rAAV capsid protein, the rAAV vector comprises heteronuclear DNA and replicates at least one AAV terminal. The evolution in transformation is compared to rAAV particles comprising an untreated type capsid. In some embodiments, the amino acid replacements result in a reduced or eradicated binding to HSPG. In some embodiments, rAAV particles comprise a capsid comprising the R585A and R588A replacements for the rAAV2 capsid, numbering based on the VPl of AAV2 (sequence ID: 1). , RAAV particles comprise a capsid containing the A586R and / or R533A replacements for AAVrhSR, numbered based on the VPl of AAVrhSR (sequence ID number: 9).

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In some respects, the invention presents methods for treating an ophthalmic disorder in an individual that involve the transfer of a combination containing an effective amount of rAAV particles to the retina of an individual, wherein it includes rAAV particles a) rAAV capsid containing the rAAV capsid protein that includes one or more amino acid antigen At one or more sites interacting with heparan sulfate protein glycans, and b) the rAAV vector comprising heteronuclear DNA and replicating a tip for at least one AAV. In some embodiments, the amino acid substitutions result in a low or forbidden binding to HSPG (some embodiments, rAAV particles include a capsid that includes the R585A and R588A substitutions for the rAAV2 vector, numbered based on VP1 for AAV2 (sequence ID number 1)). The rAAV particles on a capsid comprising the A586R and / or R533A replacements for AAVrhSR, numbered based on VP1 for AAVrhSR (sequence ID number: 9).

The invention also provides systems for transporting a subretinal vector to an individual eye, comprising a) a combination comprising an effective amount of rAAV particles, where 1) a capsid protein of rAAV particles comprising one or more amino acid substitutions at one or more sites interacting with Protein glycans of heparan sulfate, and 2) the carrier includes heterocyclic nucleic acid encoding a therapeutic polypeptide or therapeutic RNA and tip replication for at least one AAV; And b) a device for transferring rAAV to the retina. In some embodiments, the amino acid substitutions result in low or no-no binding to HSPG (some embodiments, rAAV particles include a capsid that includes R585A and R588A replacements for the rAAV2 vector, numbered based on VP1 for AAV2 (sequence ID: 1). The rAAV particles on a capsid comprising the A586R and / or R533A replacements for AAVrhSR, numbered based on VP1 for AAVrhSR (sequence ID number: 9).

[0105] In some embodiments, the present invention further provides methods for transporting nucleic acid

<img file="MA39439B2_D0005.tif" />

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Heterozygous to the central nervous system (CNS) of an individual comprising the administration of a gland-associated virus particle (rAAV) to the chirp's CNS The rAAV particle comprises (A) rAAV capsid comprising rAAV capsid protein comprising one or more amino acid substitutions In one or more of the loci interacting with proteophilicans as heparan bits, and (b) the rAAV vector comprising heteronuclear DNA and at least one AAV inverted terminal replication. These methods demonstrate advanced expression of heterozygous DNA and / or rAAV transformation of cells following particle delivery. rAAV to For an individual's CNS device, for example, compared to cell transduction with rAAV incorporating a capsid of the untreated type. Moreover, the methods from the present invention are able to infect specific cells (for example, neurons) while still achieving strong and broad-spectrum conversion efficiencies. RAAV particles and the mentioned methods are suitable for use.

In the treatment of CNS disorders, including but not limited to Huntington's disease. In some embodiments, amino acid substitutions result in a reduced or elastomeric binding to 0%. In some embodiments, rAAV particles comprise a capsid comprising R585A and R588A replacements for rAAV2, with a construct numbering On VP1 for AAV2 (sequence ID #: 1). In some embodiments, rAAV particles include a capsid that includes the replacements of A586R and / or R533A for AAVrhSR, numbered based on VP1 for AAVrhSR (sequence ID: 9).

[0106] The present invention also presents sets containing rAAV particles or formulations containing rAAV particles comprising (a) an rAAV capsid comprising the rAAV capsid protein comprising one or more amino acid substitutions at one or more sites that interact with the proteoglycans. Heparan sulfate, and (b) the rAAV vector comprising heteronuclear acid and repeating at least one inverted AAV terminal. These kits are useful for transporting nucleic acids directly

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Heterochromia to the eye or CNS of the individual, as well as for the treatment of eye disorders or CNS disorders in an individual (for example, treatment of retinopathy or Huntington's disease).

1. General techniques

[0107] The techniques and procedures described or referenced in the application will be quite generally understood and commonly used using traditional methodologies by those experienced in the field, for example, the widely used methodologies which are described in 0

Molecular Cloning: A Laboratory Manual (Sambrook et ah 4^th ed., Cold Spring Harbor

Current Protocols in Molecular (2012); Laboratory Press, Cold Spring Harbor, NY the series Methods in Enzymology (Academie); 2003; Biology (FMAusubel, et reds).

Press, Inc.); PCR 2: A Practical Approach (MJMacPherson, B.Ü.Hames and

Antibodiesi 4 Laboratory Manual (Harlow and Lane eds, 1995), G. Raylor eds.

116 The Culture Animal Cells: A Manual 0 Basic Technique and Specialized

Oligonucleotide Synthesis (2010, Applications (R.L Freshney, 6, h ed., J. Wiley and Sons)

QAA.GA is this? Methods in Molecular Biology, Bu Cell Biology: A

NcBmic, Introduction to Cell and £ 1 for Laboratory Notebook Cell and Tissue; 1998), Tissue Culture (JPMather and PERoberts, Plénum Press).

Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DGNewell, eds., J. Wiley Handbook of Experimental Immunology (DMWeïr and 1998-8), and Sons CCBlackwell, eds., Gene Transfer Vectorsfor Mammalian Cells (JMMiller and

PCR: The Polymerase Chain Reactionc (Mullis et ah eds, 1987) .MPCalûs, eds.

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Short Protocols (1991, eds, z. Current Protocols in Immunology (JEColigan et) 1994

Immunobiology; 2002, in Molecular Biology (Ausubel et ait eds., J. Wiley and Sons et 3) 2.1 deck Antibodies 9961 L, Antibodies: A Practical

39 ^ -39 ^ 9) 3 Monoclonal Antibodies: A Practical .; Vress L, C. Guides 1 £ e 2 Approach

Approach (P.Shepherd and C. Dean, eds., Oxford University Press, Using

Antibodies: 4 Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory

The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academie; (1999, Press)

and Cancer: Principles and Practice of Oncology (VTDeVita et al.c); (1995, Publishers)

Eds., JBLippincott Company

2- Definitions 10

[010 8] The expression refers to a vector, according to its use in the present application, to a plasmid or recombinant virus that includes DNA required to be transported in a host cell, either in the laboratory or in the organism.

[0109] The term polynucleotide or nucleic acid as used in the current application refers to a polymeric form of nucleotides of any length, either stripe nucleotides or deoxy-15 bionucleotides. Consequently, this term includes, but is not limited to, DNA or RNA acid, single, double, or multi-strand genomic DNA, cDNA, DNA-RNA hybrids, a polymer comprising purine and pyrimidine bases, or a nucleotide preparation. Other natural, chemically modified or biochemically modified, abnormal, or derivative. The basis of the poly nucleotide may include groups of sugars and phosphates (according to what can typically be found in RNA or DNA, or with modified or substituted sugars and phosphates). Alternatively, a foundation can be included

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The polynucleotide contains a synthetic subunit polymer such as phosphoramides and thus can be a phosphoramide oligonucleoside (Ρ-2) or a phosphoramides-phosphodiester oligomer. In addition to the above, the double-stranded polynucleotide from a single-stranded polynucleotide product can be obtained from chemosynthesis either of the complement strand and annealing of strands under the correct conditions, or by the synthesis of the new complementary strand using a DNA polymerase with the correct primer.

[0110] The terms polypeptide and protein are used interchangeably to refer to the polymer of an amino acid building block, and is not limited to minimum length. The listed polymers of an amino acid building block may contain the natural and abnormal amino acid building blocks and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of an amino acid building block. The definition includes both full-length proteins and fragments thereof. The terminology also includes post-expression modifications of a polypeptide, for example, glycosylation, treatment with cellel, acetyl-treated, phosphoryl-treated, and the like. In addition, for the purposes of the present invention, a polypeptide refers to a protein that includes modifications, such as deletion sites, addition sites, and substitutions (generally conservative in nature), in the main sequence, as long as the protein maintains the desired activity. These modifications may be intentional, for example through site-directed mutagenesis, or accidental, such as through host mutations that produce proteins or errors due to PCR amplification.

[0111] A recombinant viral vector refers to a polynucleotide vector that produces a gene recombination that includes one or more heterozygous sequences (i.e., a DNA sequence of no viral origin). In the case of AAV vectors of gene recombination, the recombinant DNA is surrounded

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With at least one inverted terminal repeat (ITR) sequence negating some of the models, the DNA of the gene recombination is surrounded by two ITRs.

12 101 The product gene recombination vector (rAAV vector) refers to a polynucleotide vector comprising one or more of the heterozygous (that is, a nucleic acid sequence of non-AAV origin) surrounded by at least one ITR of 887. rAAV mentioned and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that express appropriate auxiliary functions) expressed in rAAV as a vector incorporation. Cap and Rep (i.e. proteins) cap and AAV rep gene products

The larger polynucleotide (for example, in a chromosome or in another vector such as a plasmolem used for cloning or infecting), then the rAAV vector can be referred to as a 0 * 1-vector that can be saved by transcription and encapsulation in the presence of AAV packing functions and appropriate helper functions . Say the rAAV vector is in any number of images, including, but not limited to, plasmids, linear industrial chromosomes, complex with lipids, coated within lipid particles, and encapsulated in a viral particle, for example the 8867 particle. In AAV capsid to generate a glandular recombinant viral particle (rAAV particle).

[0113] rAAV virus or rAAV viral particle refers to a viral particle consisting of at least one AAV capsid protein and an enveloped rAAV vector genome.

[0114] means a heterozygote derived from a stand-alone entity that is genotyped from that of the rest of the entity to which it is being compared or to which it is inserted or merged. For example, a polynucleotide introduced by genetic engineering techniques in a different cell type is a non-polynucleotide. Homogeneous (and,

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When expressed, it can encode a heteropeptide (polypeptide). Similarly, a cellular sequence (eg, a gene or part of it) that is incorporated into a viral vector is the heterozygous nucleotide sequence with respect to the vector.

[10115 The term a modified gene refers to a polynucleotide that has been inserted into a cell and can be transcribed into RNA and optionally, genetically translated and / or expressed under appropriate conditions. In aspects, it imparts the appropriate characteristic to a cell into which it is introduced, or otherwise leads to the desired therapeutic or diagnostic result. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA or shRNA.

[0116] The terms genome particles (gp), genome equivalents, or genome transcripts according to their use in referring to a viral titre, refer to the number of pherons containing the AAV DNA genome of gene union, regardless of transmission or functions. The number of genome particles can be measured. In preparing a vector defined by procedures such as described in the examples in the present application, or for example, in .lO: lO31-lO39, Clark et al. (1999) Hum.gene Ther; . 6: 272-278, Veldwijketal. (2002) Mol.Ther.

[0117] The term carrier genome (vg) according to its use in the present application may refer to one or more polynucleotides comprising a set of vector polynucleotide sequences, for example, a viral vector. The carrier genome may be encapsulated in a viral particle. Depending on the specific viral vector, the vector genome may include single-stranded DNA, double-stranded DNA, single-stranded RNA, or double-stranded RNA. The vector genome may include autologous sequences associated with the specific viral vector and / or any heterozygous sequences introduced into the specified viral vector through genomic recombination techniques. For example, it might include a genome

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The AAV transporter of the recombinant gene on at least one ITR sequence surrounds the promoter, stuffer, the respective sequence (for example, RNAi), and the polyadenylated pretreatment sequence. The complete vector genome may include a complete set of vector polynucleotide sequences. In some embodiments, the DNA titration of the viral vector can be measured in a range of 7% / ml. Suitable methods for measuring this titre are known in the art (for example, quantitative PCR).

8 1011 The terms infectious unit (1y), infecting particle, or transcription unit, according to their use in referring to a viral titre, refer to the number of AAV gene recombinant particles infectious and specialized for transcription as measured by the infectious center experiment, also known as the transcription center experiment, according to its description For example, in McLaughlin et al. (1988) J. Virol, 62: 1963-1973,

[0119] The term unit of transverse transformation (tu) according to its use in referring to a viral titre, refers to the number of AAV vector particles of an infectious gene union produced in the production of a GM product according to its measurement in functional experiments such as described in the examples in the present application, or For example, in I44: 113-I24, xiao et al. (1997) Exp.Neurobiol; Or in Fisher et al. (70: 520-532) (LFU assaycJ.Virol.

00120 The term ITR is well known in the art and refers to the relatively short sequences found at the ends of the counter-directional viral genomes.

[0121] The ITR (AAV), the term well known in the art, is a sequence of approximately 45 1-nucleotide length present at each end of the negative single-stranded AAV genome. The outermost 125 nucleotides of an ITR can be present in either of two alternate directions ,

<img file="MA39439B2_D0006.tif" />

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Leading to heterogeneity between different AAV genomes and between two ends of a single AAV genome. The most distant 125 nucleotides also contain many short autofineralization regions (labeled regions A, A, c, C, 'B, B' and e), allowing for an essential conjugation to occur Between the braid inside this part of the ITR.

[00122 The terminal resolution sequence or trs is a sequence in the D region of AAV ITR cleavaged by AAV rep proteins during viral DNA transcription. The mutant terminal resolution sequence is resistant to fission by AAV rep proteins.

[0123] AAV adjuvant virus refers to a virus that allows AAV (which is a small, defective virus) to be cloned and packaged by a host cell. The number of helper viruses mentioned were defined, including adenoviruses, herpesviruses and smallpox viruses such as vaccinia. Adenoviruses comprise a number of different subgroups, although adenovirus type 5 from subgroup (Ad5) 5 is most commonly used. Many adenoviruses of human origin, mammalian and avian are known and available from repositories such as 00 (8) Viruses from the herpes group, which are also available from repositories such as ATCC, include, for example, herpes simplex viruses (HSV), EBV (Epstein-Barr) viruses, cytomegalovirus (CMV) and pseudovirus (PRV).

The percentage (%) of conformity of the sequence with respect to the reference to a polypeptide or nucleic acid sequence is defined as the percentage of building blocks of an amino acid or nucleotides in the candidate sequence that matches the building blocks of an amino acid or nucleotides in a reference polypeptide or reference DNA sequence, after Sequence alignment and blanks advance, if necessary, to achieve the maximum percentage match of the sequence, without regard to any conservative substitutions as part of the sequence congruence. Alignment can be achieved for percentage determination purposes For an amino acid or an identical nucleic acid sequence

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In many ways they are within the domain of skill in the field, for example, by using computer programs available on common examination, for example those described in the current protocols in Molecular Table 7.7.1, section Supp.30, 1987), eds.iAusubel et al. An example of an alignment program is PennsylvaniA, Educational Soflw ^ Scientific (ALIGN Plus), which includes the ALIGN program, BLAST-2, BLAST, or DNASTAR (Megalign). Skilled in the field can identify appropriate variables to measure alignment, including any. Algorithms required to achieve maximum solution Of alignment with the entire length of the compared sequence. For the purposes of the present request, the% is calculated to match the amino acid sequence of the amino acid sequence specified to, with, or against the specified amino acid B sequence (which can alternatively be formulated as given as the amino acid sequence that includes or includes passes to match the amino acid sequence to, with, or , Or against the amino acid sequence defined by b) as follows: 100 Once the part /, where X is the number of building units of an amino acid recorded as an identical result by the sequence alignment program in the alignment program A and B, and where Y is the total number of building units of an amino acid in B. It will be perceived that where the length of the amino acid sequence is not equal to the length of the amino acid sequence B, it will not pass to match the B amino acid sequence equal to% to match the amino acid sequence B to A. For the purposes of the present application, the% match of the nucleic acid sequence of a specific nucleic sequence c to, with, or against a specified nucleic acid sequence d (which can alternatively be formulated as given as a DNA sequence C that includes or includes passes corresponding to a specific DNA sequence) To, with, or against a specific D-DNA sequence) as follows: 100 times the streak w /, where W is the number of nucleotides recorded as an identical result by the sequence alignment program in the C and D alignment program, and where Z is the total number of nucleotides in D. It will be perceived

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Where the length of the DNA sequence C does not equal the length of the D DNA sequence, the% to match the L to D DNA sequence will not be equal to L% to match the D to C DNA sequence.

[20125 An isolated molecule (for example, DNA or protein) or cell intended to have been identified, separated, and / or recovered from a component of its natural environment.

[0126] Effective quantity refers to an amount sufficient to produce beneficial or desired results, including clinical results ( eg symptom improvement, clinical endpoint achievement, etc.). An effective amount may be administered in one or more administrations. . In view of the disease, an effective amount is sufficient to relieve, stabilize, or delay disease progression.

[0127] The individual or subject is a mammal. Mammals include, but are not limited to, domestic animals (for example, cows, sheep, cats, dogs, and horses), primates (for example, human and non-human primates such as monkeys), rabbits, and rodents (for example, mice and rats). . In certain instances, the individual or subject is a human

Depending on its use in the current application, treatment is the approach to obtaining beneficial or desired clinical results. For the purposes of this invention, beneficial or required clinical results include, but are not limited to, symptom relief, reduction in disease extent, stabilization (for example, non-exacerbation) of disease status, and prevention of spread (for example, metastatic spread). Disease, delay or slowdown in disease progression, improvement or mitigation or disease condition, and remission (whether partial or lesser), whether detectable or undetectable. Treatment also means prolonging survival compared to the expected survival without treatment. [0129] According to its use in the present application, the term preventive treatment refers to a treatment, whereby an individual is known or suspected to be or is at risk of developing a disorder but has not been demonstrated

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Any symptoms or symptoms of the disorder are minimal. An individual undergoing preventive treatment may be treated before the onset of symptoms.

Depending on its use in the present application, a therapeutic agent (eg, a therapeutic polypeptide, DNA, or transgenic) refers to that which provides a useful or desired clinical outcome, for example the representative clinical results described previously. As such, therapeutic factor 5 may be used in the treatment as previously described

0131] The term central reticulum as used in the present application refers to the outer macula, the inner macula, and / or the fovea. The term central retinal cell types according to its use in the present application refers to the central retinal cell types, for example, RPE and photoreceptor cells. 10

[0132] The term macula refers to a region of the central retina in primates that has a relatively higher concentration of photoreceptors, specifically rod cells and cone cells, compared to the peripheral retina. The term macula may be referred to externality according to its use in the present application also by the name peripheral macular. Depending on its usage in the current application, the term interior spot may also be referred to as the central spot. 15th

The term fovea refers to a small region in the central retina of primates roughly equal to or less than 0.5 mm in diameter that contains a relatively high concentration of photoreceptors, specifically cones, compared to the peripheral retina and macula.

[0134] The term subretinal space according to its use in the present application refers to the location in the retina between photoreceptor cells and retinal pigment epithelium cells. The subretinal space may be 20 potential spaces, for example before any injection of subretinal fluid. Say the subretinal space also contains

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Fluid to be injected into the potential space. In this case, the fluid is in contact with the subretinal space. Cells that come into contact with the subretinal space include cells that identify the subretinal biases, for example RPE and photoreceptors.

[0135] The term bubble as used in the present application refers to the fluid space within the subretinal space of the eye. The bubble of the invention may be formed by the single injection of a fluid into a single compartment, by multiple injections of one or more fluids in the same compartment, or by multiple injections in several compartments, which, when repositioned, form a total fluid volume useful to achieve the therapeutic effect Above the required portion of the retinal space.

6 00 Reinforced rhodopsin kinase (RK) Refers to a polynucleotide metaphor derived from the rhodopsin kinase gene (for example, human RK, represented by 6011 GenBank Entrez Gene ID) that specifically directs expression in rod and cone photoreceptor cells, as well as, for example, retinal cell lineages. 1-5 • 1 m 271. Depending on the use of E in the present application, the rhodopsin kinase promoter may refer to the complete promoter sequence or fragment of the enhancer sequence sufficient to direct expression of the photoreceptor cell, such as the annulus described in et al. (2007), S. Ckhani et al. (2OO3), JE, Invest.Ophthalmol.Vis.Sci.48 (9): 3954-61 and Young

4076-85: (9) 44.Invest.Ophthalmol.Vis.Sci. In some embodiments, the RK booster extends from -12 1 to +180 relative to the transcription initiation site.

[0137] The chicken actin promoter 0-a (CBA) refers to a polynucleotide metaphor derived from the chicken actin 0-gene (eg, gallus gallus beta-actin, represented by GenBank Entrez 396526 ID M0). Depending on its use in the current application, a promoter of 0-chicken actin may refer to a promoter containing an early enhancer of CMV, a promoter and exon I.

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And the intron of the chicken 8-actin gene, and the controversial receptor for the beta-globin gene from rabbits, for example the sequences described in et 81. (1989) Gene 79 (2): 269-77, J.cMiyazaki, depending on its use in the present application, the term may be used Padded CAG Booster. According to its use in the present application, the term early CMV enhancer / chicken actin beta enhancer (CAG) may be used interchangeably.

10138 The reference to a value or variable about in the current order includes (and describes) the models directed at that value or variable in itself. For example, a description referring to about X includes the description of X.

[0139] According to the use of e in the current application, the singular form of the denial, an_j, and the definite article the includes the reference to the plural unless indicated otherwise.

(10140) It shall be understood that aspects and embodiments of the invention described in the present application comprise comprise, consist of, and / or mainly consisting of aspects and models.

0141) is a well-known HepaRan sulfate proteoglycan (HSPG) in the domain in turn as a cellular receptor for RJ particles (1998) J.Virol.72 (2): 1438-, c.and SamulskicSummerford (2 45). The link between the AAV2 particle and 0 ^ works at The cell membrane connects the particle to the cell. Other cell surface proteins, for example, the fibroblast growth factor receptor and integrin ανβ5 may also facilitate cell antagonism. When binding, say the AAV2 particle enters the cell via mechanisms in that cytogenic endocytosis mediated by the receptor by clathrin-coated pits. The AAV2 particle can be released from the endosome vesicle once the endosome has acidified. This allows the AAV2 particle to travel to the region surrounding the nucleus and subsequently the cell nucleus. AAV3 particles are also known to bind to Heparan (et al. (2OO2) J. Virol. 76 (2): 791-801, JE, Rabinowitz).

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[0142] Genetic treatment protocols for ophthalmic disorders require local transport of a vector to cells in the eye (eg, retinal cells). The cells that will be the target of treatment in these diseases may include, among others, one or more cells of the eye (for example, photoreceptors, neurons of the eye, etc.). The methods and the sets of the invention are based, at least in part, on 1 finding that the rAAV capsid is special (for example, those comprising the rAAV capsid protein comprise one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycan). It allows for a wide distribution of the vector between cells of the eye. As such, these capsids may be particularly useful in transferring heterologous DNA into an individual eye, improving the rAAV conversion of cells following delivery of the rAAV particle to an individual eye, improving expression of the heterochromic DNA following delivery of rAAV particles to an individual eye, and / or Treating an individual's eye disorder with rAAV particles.

[0143] Likewise, genetic treatment protocols for CNS disorders require local transmission of a vector to cells at 5% p. Cells that will be the target of treatment in these diseases include, among others, one or more brain cells (for example, neurons). The methods and sets from the invention are based, at least in part, on the finding that the rAAV capsid of specific rAAVs (for example, those comprising the rAAV capsid protein comprise one or more amino acid substitutions at one or more sites that interact with the proteoglycans). Heparan sulfate) Allow large-scale distribution of the vector among 15E0 cells. As such, these capsids may be particularly useful in transporting heterozygous DNA to the central nervous system (CNS) of an individual, improving the rAAV conversion of cells following delivery of the rAAV particle to the individual’s CNS, improving expression.

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For heterozygous nucleic acid following delivery of rAAV particles to the individual's CNS, and / or perturbation treatment of the individual's CNS with rAAV particles.

[0144] AAV capsid (eg, AAVrhSR, AAV2, etc.) are known to contain three capsid proteins: VP2, VP1, and 713. These proteins contain large amounts of the compound amino acid sequence and unique terminal sequences 2E. The AAV2 capsid comprises 60 subunits arranged by acanthedral symmetry (et al. (2002), Q., Xie.

VP2, Proc.Natl.Acad.Sci.99 (16): 10405-10) .VPl, and the presence of VP3 was amplified in a ratio of 1: 1: 10.

[0145] The association between AAV2 capsid proteins and HSPG occurs via electrostatic interactions between AAV2 capsid protein principal building blocks and negatively charged glycosamine glycans (2003) J. Viro! 77: 6995-7006, SRetabOpie; Kern, 2003) J Virol.77: 11072-I10 81, et al.). The capsid residues included in these reactions include R585, 532, R487, R484, and 588%. Mutants in these building blocks have shown to reduce the binding of AAV2 to HeLa cells and Heparan itself (2003). SR et al., 0pie (77: 6995-7006.J. Virol; 2003) J.Virol.77: l 1072-110 81, et al., Kem); 2004/027019 WO 322,629, US Patent Νο.7, 2) .Moreover, without wanting to be bound by any theory, it is believed that substitution (precipitations) of an amino acid in one or more of the corresponding building blocks of amino acids 484 , 487, 532, 585 or 588, the numbering based on the AAV2 VP1 numbering can modify the transformation properties of AAV capsid species that are not associated with HSPG, or it can modify the transformation properties of the AAV capsid species regardless of their ability to bind to the HSPG.

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Specific aspects of the invention relate to the transfer of heterozygous DNA into the eye or central nervous system (CNS) of an individual comprising the administration of a glandular conjugated virus particle of a recombinant product (rAAV) to the eye or CNS of the individual. In some embodiments, the rAAV particle includes the rAAV capsid comprising the rAAV capsid protein comprising one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycan. In some embodiments, the rAAV particle of the invention comprises an AAV capsid of serotype 2 (8872). In some embodiments, the rAAV particle of the invention comprises a serotype AAV capsid (AAVrhSR) rh8R.

[0147] According to the description in the present application, rAAV particles are indicated by mutants in capsid proteins in building blocks interacting with HSPG or at one or more of the corresponding building blocks of amino acids 484, 487, 532, 585 or 588, and numbering based on the VP1 numbering AAV2 has beneficial properties, eg enhanced expression and / or reduced inflammation. Consequently, in some embodiments, when transporting an heterologous DNA encoded by the rAAV vector is expressed at a coherent level of expression, compared to the expression level of the heterozygous nucleic acid containing the rAAV particle comprising the rAAV capsid comprising the rAAV reference capsid protein (for example, a protein A capsid of the untreated type (rAAV). In some embodiments, the expression of DNA is increased by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% In some embodiments, the rAAV particle is transported induced Low inflammation, compared to a rAAV particle comprising rAAV reference capsid protein (eg, untreated rAAV capsid protein). In some embodiments, neuritis is reduced by at least about 10%, at least about 25%, at least around

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50%, at least about 75%, or at least about 100%. A suitable rAAV reference capsid protein may include any capsid protein lacking one or more amino acid substitutions in one or more sites that interact with the heparan sulfate proteoglycan ( A reference capsid may thus contain one or more of the background substitutions that do not modify association (HSPGj

10148 In some embodiments, the invention provides methods for transferring heterologous DNA to the eye of an individual that include administration of a gland-associated virus particle of the recombinant product (rAAV) into an individual's subretinal space where the rAAV particle comprises a) rAAV capsid comprising the rAAV capsid protein comprising One or more amino acid substitutions in one or more sites interacting with heparan sulfate proteoglycans, and b) the rAAV vector comprising heteronuclear acid and at least one inverted AAV terminal replication.

10149 In some embodiments, the rAAV particle of the invention comprises a serotype 2 (8872) AAV capsid. In some embodiments, one or more amino acid substitutions are substitutions of the amino acid building blocks of either VP2, VP1 and / or VP3 with AAV2, wherein substitutions of amino alter the interaction of the rAAV particle with HSPG (for example, reduce or eliminate the binding to HSPG) In some embodiments, one or more amino acid substitutions are substitutions of the amino acid building blocks for AAV2 711. In some embodiments, one or more amino acid substitutions are building block substitutions for the amino acid for AAV2 712. In some embodiments, one or more Of acid substitutions In some embodiments, one or more of the amino acid substitutions are the substitutions of the amino acid residues of a combination of VP2, VP1 and VP3 to 8872.

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In some embodiments, one or more amino acid substitutions are the amino acid building block substitutions of either VP2, VP1 and / or VP3 for AAV2 ^ Some embodiments, one or more amino acid substitutions are building block substitutions for an amino acid for any of the capsid proteins of the sequence ID No .: 3,1 and / or 5. In some embodiments, the rAAV particles of the invention comprise capsid proteins of a sequence with ID No. 4,2 and / or 6.

In some embodiments, the rAAV particle of the invention includes the AAV capsid serotype 3 (88273). J In some embodiments, one or more amino acid substitutions are the building block substitutions of an amino acid for any one of VP2, VP1 and / or VP3 for AAV3, where Amino replacements alters the rAAVwithHSPG particle interaction (eg, lowers or de-binding to 1506). In some embodiments, one or more of the amino acid substitutions are the amino acid building block substitutions of 711 AAV3. In some embodiments, one or more of the amino acid substitutions are the amino acid residues of 712AAV3. In some embodiments, one or more of the amino acid substitutions are the substitutions of the amino acid building blocks for AAV3 713.In some embodiments, one or more of the amino acid substitutions are the substitutions of the amino acid residues of the combination of VP2, VP1 and VP3 to 8873. , One or more amino acid substitutions are the substitutions of the amino acid building blocks of any one of VP2, VP1 and / or VP3 for AAV3 (some embodiments, one or more amino acid substitutions are the building block substitutions of an amino acid corresponding to the protein capsid For sequence ID number: 7.

10151 In some embodiments, the rAAV particle of the invention includes the AAV capsid serotype AAVrhSR (rh8R), for example, according to what is described in USPG prismatic No.

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17417 200903 In some embodiments, one or more amino acid substitutions are the substitutions of the amino acid building blocks of any one of VP2, VP1 and / or VP3 for AAVrhSR, wherein substitutions of amino substitutes the rAAV particle interaction with HSPG (for example, reducing or ablating the binding) In some embodiments, one or more amino acid substitutions are the amino acid residues of 711 AAVrhSR. In some embodiments, one or more amino acid substitutions are the amino acid substitutions of VP2 AAVrhSR. In some embodiments, one or more amino acid substitutions are the amino acid substitutions of VP2 AAVrhSR. , One or Most of the amino acid substitutions are the substitutions of the amino acid AAVrhSR 713 G. In some embodiments, one or more amino acid substitutions are the substitutions of the amino acid building blocks of a combination of VP1, VP2 and VP3 for AAVrhSR. In some embodiments, one or more substitutions Amino acid substitutions of the amino acid building blocks of any one from VP2, VP1 and / or VP3 for AAVrhSR. In some embodiments, one or more amino acid substitutions are the substitutions of the amino acid building blocks of a capsid protein represented by a sequence ID No .: 9. In some embodiments, the rAAV particles from the invention comprise capsid proteins of a sequence with ID numbers: 10 and / or 11.

[0152] In some embodiments, the invention introduces rAAV particles for subretinal transport of therapeutic DNA, wherein the rAAV particles comprise one or more of the amino acid substitutions of capsid proteins that reduce or eliminate the binding of the rAAV particle to the heparan sulfate proteoglycan. In some embodiments, the rAAV particle includes an AAV2 capsid or AAV3 capsid; in some embodiments, one or more amino acid precipitates reduce the binding of the rAAV particle to heparan sulfate proteoglycans by at least about 10%, at least about 20%,

Name M.

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At least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least About 90%, or at least about 100%, at least, about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. Amino acid precursors The rAAV particle binding to the proteoglycan heparan sulfate is at least about 10%, at least about 15%, at least about 20%, and so on. Least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% (compared to the correlation to include a particle rAAV on a capsid of the untreated type). In some embodiments, one or more amino acid substitutions reduce the binding of the rAAV particle to the heparan sulfate proteoglycan in any of about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%. %, About 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 10% to about 90%, About 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, About 70% to about 90%, about 80% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to

<img file="MA39439B2_D0007.tif" />

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About 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%. %, About 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, About 40% to about 60%, about 5% 50 to about 60%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 10% to about 40%, about 20% To about 40%, about 30% to about 40%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 20%, (compared to the correlation for the 887 particle) to include a capsid of the untreated type). In some embodiments, 10 one or more amino acid substitutions resulted in an undetectable binding of the rAAV particle to the heparan sulfate proteoglycan compared to the binding of the untreated rAAV particle. The measurement of the binding of AAV particles to the HSPG is in the field; For example, binding to heparan sulfate chromatography media or binding to a cell known to express HSPG on its surface. For example,

See 15 (2003), et al., Kj J. Vîrol. 7: 6995-7006, SR et alOpie.

0J.Vi 01.77: 11072-110 81

[00153 In some embodiments, the invention introduces rAAV particles for subretinal transport of therapeutic DNA, wherein rAAV particles comprise one or more of the amino acid substitutions of capsid proteins that reduce or eliminate the binding of the rAAV particle to the heparan sulfate proteoglycan, wherein one or more amino acid substitutions It is in position 484, 487, 20

532, 585 or 588, numbering based on AAV2 numbering) some models, one or more

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The amino acid replacements are at position 484, 487, 532, 585 or 588D VP1D 8872. In some embodiments, one or more of the amino acid substitutions is at position 484, 487, 532, 585 or 588g VP2g AAV2, with a construct number On VP1 d AV2 (in some embodiments, one or more amino acid substitutions are at position 484, 487; 532; 585 or 588 for VP3 and AAV2, numbered based on VP1 D-8872; in some embodiments, one or more amino acid substitutions Be in position 487, 532, 585 or 588 for VP1 for VP2, AAV2 for AAV2, and UA or VP3 for AAV2, with a build number On the VP1 to 8872. In some embodiments, VP1 for rAAV2 comprises the amino acid sequence of the sequence ID: 1.

In some embodiments, one or more of the amino acid substitutions is at position 484, 487, 532, 585 or 588n VP1 for AAV3, numbered based on VP1 and AAV2 ^ in some models; One or 1 more amino acid replacements are at position 484 487, 532, 585 or 588 and VP2 for AAV3, numbering based on AAV2 3 VP1 ^ In some embodiments, one or more amino acid replacements are at position 484, 487, 532, 585 or 588 for VP3 for AAV3, numbering based on VP1 to 8872 In some embodiments, one or more of the amino acid substitutions is at position 484, 487, 532, 585 Or 588 for VP1 for VP2, AAV3 for AAV3, and / or VP3 and AAV3, numbering based on VP1 for 00rAAV2. Some embodiments, VPlnrAAV2 includes the amino acid sequence of the sequence ID: 1.

In some embodiments, the AAV particles of the invention comprise a capsid with one or more amino acid substitutions being at position 485, 488, 533; 586 or 589; Numbered based on the AAVrhSR numbering. In some embodiments, VP1 of AAVrhSR comprises the acid sequence.

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In some embodiments, one or more amino acid substitutions include the substitution of the positively charged amino acid building block with the positively charged amino acid building unit. In some embodiments, the building unit of a positively charged amino acid is replaced by the building unit of an amino acid that is non-hydrophilic. In additional embodiments, one or more amino acid replacements include an arginine or lysine subunit substitution. In additional embodiments as well, one or more amino acid substitutions includes the substitution of an arginine or lysine building block with an alanine building block. In the other embodiments, one or more amino acid substitutions includes the substitution of the building block of a positively charged uncharged amino acid with the building block of a positively charged amino acid. In some embodiments, the building unit of an amino acid other than hydrophilic is replaced by the building unit of a positively charged amino acid. In additional embodiments, one or more amino acid substitutions comprise the substitution of an alanine residue; in additional embodiments as well, one or more amino acid substitutions comprise the substitution of an arginine or lysine building unit with an alanine residue. In some embodiments, one or more amino acid replacements include substitution at the R533 and / or 586 locus, with a numbering based on VP1 for AAVrhSR. In additional embodiments, the AAV capsid includes the amino acid replacements A586R and / or R533A, numbering based on

VP1 n AAVrhSR. In some embodiments, the rAAV particle includes the rAAV capsid protein of a sequence with number IDs: 10 and / or 11.

10156 In some embodiments of the invention, one or more amino acid substitutions comprise the substitution of the building block of a positively charged amino acid (for example, an amino acid of a positively charged side chain) with the building unit of an amino acid that is not a positively charged (for example, an amino acid that is not It contains positively charged side chain) amino acids

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Positively charged include arginine, histidine and lysine. Examples of positively charged amino acid building blocks include negatively charged amino acids (aspartic acid and glutamic acid), amino acids with uncharged polar side chains (serine, threonine, asparagine, and glutamine), and hydrophilic side-chained amino acids (alanine, valine). , Iso-leucine, leucine, methionine, phenylalanine, tyrosine and tryptophan), glycine, cysteine, and proline; in some embodiments, one or more subunits of the positively charged amino acids for the AAV capsid are substituted by the unit. The structural properties of an amino acid are non-hydrophobic. In some embodiments, one or more of the amino acid substitutions includes the substitution of the building block of arginine or lysine. In additional embodiments, one or more amino acid substitutions include the substitution of an arginine or lysine building block with an alanine building block. In the other embodiments, one or more amino acid substitutions comprise the substitution of the positively charged amino acid residue with the positively charged amino acid building unit. In some embodiments, the hydrophobic non-hydrophobic amino acid residue is replaced by the positively charged amino acid residue. In additional embodiments, one or more substitutions of an amino acid include an alanine building block substitution. In additional embodiments as well, one or more amino acid replacements include the substitution of an arginine or lysine building block with an alanine building block.

[0157] In some embodiments, one or more of the amino acid substitutions includes substitutions at the position R585, 532, 527, R487, R484 and / or R588 for VP2, VP1 and / or VP3, numbering based on VP1 for 8872. Embodiments, one or more amino acid substitutions comprise substitutions in the position R585, 532, 527, R487, R484 and / or R588 for VP2, VP1 and / or VP3n AAV2, with a numbering based on VP1 for 8872. In some embodiments, it includes

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One or more amino acid substitutions for the substitution of 527, R487, R484, 532 and / or R588 for VP2, VP1 and / or VP3 for AAV2, by numbering based on sequence ID: 1. In some embodiments, one or more of the amino acid substitutions includes one or more substitutions of R585A, R487A, R484A and / or R588A for VP2, VP1 and / or VP3 for AAV2, numbered based on VP1 for 8872. In some embodiments, one or more of the amino acid substitutions includes substitutions at the position R585, 532, 527, R487, R484 and / or R588 for VP1, VP2 and / or VP3 for AAV3, numbered based on VP1 for 8872 in some embodiments. One or more of the amino acid substitutions includes one or more substitutions of R487A, R484A, R585A and / or R588A for VP2, VP1 and / or VP3 for AAV3, numbering based on VP1 for 8872. In some embodiments, the rAAV particle includes the rAAV capsid protein of a sequence with number IDs: 2, 4 and / or 6.

In some embodiments, one or more of the amino acid substitutions includes a substitution in the 586, R533, R488, R485 and / or 589 for VP2, VP1 and / or VP3 loci, with a numbering based on VP1 for AAVrhSR. In some embodiments, it includes a substitution or Most of the amino acid substitutions are on substitutions in the position 586, R533, R488, R485 and / or 589 for VP2, VP1 and / or VP3, numbering based on VP1 for AAVrhSR, numbering based on sequence ID number: 9. In some embodiments, one or more amino acid substitutions comprise one or more R533A and / or A586R substitutions for VP2, VP1 and / or VP3 for AAV2, numbered based on VP1 for AAVrhSR. In some embodiments, a particle is included rAAV on the rAAV capsid protein for the sequence ID: 10 and / or 11

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0159 In some embodiments, the AAV capsid includes one or more amino acid substitutions in one or more sites of interaction with HSPG (some embodiments, the AAV capsid includes one or more amino acid replacements in one or more of the mitotic sites or De-binding to HSPG. In some embodiments, the AAV capsid includes one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions that either reduce or de-link to HSPG (some embodiments, include the AAV capsid) On one, two, three, four, five, six, seven, Eight, nine, or ten amino acid replacements that reduce or de-link with] 135. In some embodiments, the AAV capsid includes replacements at position R484 and R487, numbering based on VP1 g 0rAAV2 ^ t. In some embodiments, the AAV capsid includes substitutions at position R484 and R487, numbering based on VP1 to 872 m8]. In some embodiments, the AAV capsid includes substitutions at position R484 and R487. Contains replacements at position R585 and R588, numbering based on VP1 to 2 (some embodiments, the AAV capsid includes replacements at position R585 and R588, numbering based on VP1 to rAAV2). In some embodiments, the AAV capsid includes replacements R484A and R487A, numbering based on Vp1 to AAV2 (some embodiments, including the AAV capsid On the replacements R484A and R487A, numbering based on VP1 for AAV2 (some embodiments, the AAV capsid includes the replacements R585A and R588A, numbering based on VP1 to 388722. In some embodiments, the AAV capsid includes the R585A and R588A replacements, with rA numbering based on V2P1.

[00160 Heparan sulfate proteoglycans (HSPGs) are known to be expressed in many tissues in the body and they play an important role in the extracellular matrix, cell adhesion, and cell signaling. In some embodiments, heparan sulfate proteoglycans are expressed in one or more of the 00CNS cells of specific models, one or more of the CNS cells being the neuron.

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10161 In some embodiments, the invention introduces rAAV particles for CNS transport to therapeutic DNA, wherein rAAV particles comprise one or more amino acid substitutions of capsid proteins that reduce or eliminate the rAAV particle attachment to the proteoglycan heparan sulfate. In some embodiments, one or more of the amino acid substitutions is at position 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585 and / or 588, with numbering based on VP1 for 8872, according to its use. In the present application, the numbering based on VP1 for AAV2 refers to the amino acid of the denoted capsid protein corresponding to the denoted amino acid from VP1 to 88722. For example, if one or more amino acid substitutions are at position 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585 and / or 588, numbering based on VP1 for AAV2, then it is One or more amino acid substitutions of the amino acid (amino acids) of the mentioned capsid protein the corresponding amino acids 347, 350, 390, 395, 448, 451, 484, 487, 527, 532; 585 and / or 588 of VP1 for AAV2 (in some embodiments, one or more of the amino acid replacements are at position 32532, 27527, R487, R484, R451, R448, 395, 390, R35O, R347, R585 and / or R588, with the numbering Based on VP1 for 8872, in some embodiments, one or more amino acid replacements are at position 484, 487, 532, 585 or 588 to 8872. In some embodiments, one or more of the amino acid substitutions is at position 484, 487, 532, 585 or 588 for VP1 for VP2, AAV2 for AAV2, and / or VP3 for AAV2, numbered based on VP1 for 8872. In some embodiments, VP1 for AAV2 (eg, 1AAV2) comprises the amino acid sequence of the sequence ID: 1.

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10162 In some embodiments, the AAV capsid includes replacements at the positions R487, R484, R585, 532 and / or R588, numbering based on VP1n rAAV2.0. In some embodiments, the rAAV particles of the invention include sequential capsid proteins with ID numbers: 2, 4 and / Or 6. In some embodiments, the AAV capsid includes substitutions of positions R484 and R487 or R585 and R588 with numbering based on VP1 to 388272. In some embodiments, the AAV capsid includes the R484A and R487A replacements, or the R585A and R588A replacements, numbering based on VP1 to 8872.E In some embodiments, the AAV capsid includes the R585A and R588A amino acid replacements, with the numbering based on VP1 to 8872. In some embodiments, the AAV capsid includes the amino acid 532Α, numbering based on VP1 to 88722.. In some embodiments, one or more amino acid substitutions includes R451A, R448A, 395Α, 390Α, R350A, R347A 532Α, 527Α, R487. , R484A, replaces R585A and / or R588AS, with numbering based on VP1's AAV2.

10163 In some embodiments, the invention introduces rAAV particles for CNS transport to therapeutic DNA, wherein rAAV particles comprise one or more amino acid substitutions of capsid proteins that reduce or eliminate the rAAV particle attachment to the proteoglycan heparan sulfate. In some embodiments, the rAAV particle of the invention comprises a capsid ^ rh8R serotype (AAVrh8R), for example, as described in USPG Publication 20090317417. In some embodiments, one or more amino acid substitutions are substitutions of the amino acid building blocks of any one of VP2, VP1 and / or VP3 for AAVrh8R, wherein the amino substitutions alter the interaction of the rAAV particle with HSPG (for example, reduce or eliminate the binding to HSPG). 5. In some embodiments, one or more amino acid replacements are

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Amino acid substitutions of 711 AAVrhSR. In some embodiments, one or more amino acid substitutions are the amino acid residues of VP2 AAVrhSR. In some embodiments, one or more amino acid substitutions are the amino acid substitutions of VP3 AAVrhSR. 5. In some embodiments, one or more of the amino acid substitutions are the substitutions of the amino acid residues of a combination of VP1, VP2 and VP3 for AAVrhSR. In some embodiments, one or more of the amino acid substitutions are the substitutions of the amino acid residues. Amino for any one of VP2, VP1 and / or VP3 for AAVhR ^ In some embodiments, one or more amino acid substitutions are the substitutions of the amino acid building blocks of a capsid protein represented by a sequence ID number: 9. In some embodiments, the rAAV particles of the invention include On capsid proteins that have an ID number: 10 and / or 11.

64 101 In some embodiments, particle 3887 includes the capsid of 887 AAVrhSR (rh8R). In some embodiments, one or more amino acid substitutions are at positions 586 and / or 589, numbering based on AAVrhSR 711. In some embodiments, the numbering is based on In some embodiments, one or more amino acid replacements include a substitution at the position 586 and / or 589, and the numbering is based on the AAVrhSR numbering 711. In some embodiments, one includes one Or more than one of the amino acid replacements Replacement of A586R, numbering based on VP1 for AAVrhSR. In some embodiments, one or more amino acid substitutions include the replacement of T589R or 589Κ, numbering based on the VPl of the AAVrhSR.

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[0165] According to what has been discussed above, without wanting to be bound by any theory, it is believed that the substitution (substitution) of an amino acid at one or more of the corresponding building blocks of amino acids 484, 487, 532, 585 or 588, and the numbering is based on the numbering 711 AAV2 can modify the conversion properties of AAV capsid species that are not associated with HSPG, or it can modify the transformation properties of AAV capsid species regardless of their ability to bind to HSPG. Some embodiments include one or more amino acid substitutions of one or more corresponding amino acids. For the amino acid to laminate in Figure 20: For example, in some embodiments, one or more amino acids are substituted at the site (s) corresponding to the amino acids 585 and / or 588 (numbering based on VPl for 2) with the arginine building blocks (eg, S586 and / Or 589 for AAVl or AAV6; S586 and / or 589 for-AAV9; 586 and / or 589 for AAVrhSR; Q588 and / or 591 for 8878; and Q588 and / or 591 for AAVrhlO). These modified capsids can be used, from Among other things, it improves the intravitreal conversion targeting the retina. In other embodiments, one or more amino acids (for example, arginine or lysine) are replaced at the corresponding position (s) of the amino acids 484, 487, 527 and / or 532 (numbering based on VPl for AAV2) with an acid (s). A positively charged amino such as alanine (for example, 528, R488, R485, and / or 533 for AAVl or AAV6; 528, R488, R485, and / or R533 for AAV9 or AAVrhSR; and K53O, R49O, R487, and / (R535 for AAV8 or 8871010). These modified capsids can be used, among other things, in improving the subretinal or CNS conversion.

[0166] In some embodiments, the rAAV particle includes the AAV capsid of serotype 1 (8871). In some embodiments, one or more amino acid substitutions are at the sites.

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586 And / or 589, numbering based on VP1 AAVl. &lt;_) In some embodiments, VPl of 1 AAV comprises the amino acid sequence of a sequence with ID number: 2 1. In some embodiments, one or more amino acid substitutions comprise a substitution at the position S586 and / or Τ589, numbering based on the AAVl numbering 711. In some embodiments, one or more amino acid substitutions include a substitution of S586R or S586K, numbering based on VPl for 88671. In some embodiments, one or more amino acid substitutions comprise a T589R substitution, numbering based on VPl for AAVl (some embodiments, the rAAV particle includes an AAV capsid serotype 6 (8876). In some embodiments, it is one or Most of the amino acid replacements at sites 586 And / or 589, the numbering is based on AAV6 711. In some embodiments, the numbering is based on the VPl of AAV6 that includes the amino acid sequence of the sequence ID: 13. In some embodiments, one or more amino acid substitutions include a substitution at the position S586 And / or 589, numbering based on AAV6 711. In some embodiments, one or more amino acid replacements include a S586R or S586K substitution, numbering based on a VPl of 88776. In some embodiments, one or more amino acid replacements include Replaced T589R, numbering based on VPl For 88776. In some embodiments, the rAAV particle includes an AAV capsid of serotype 8 (8878). In some embodiments, one or more amino acid substitutions are at positions 588 and / or 591, numbering based on the numbering AÂV8 711. Embodiments, the numbering is based on VPl for AAV8 includes the amino acid sequence of the sequence ID: 14. In some embodiments, one or more amino acid substitutions include substitution at position Q588 and / or 591, numbering based on the numbering AAV8 711. In some embodiments, it includes one or more amino acid substitutions Upon replacing the Q588R or Q588K, the numbering is based

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In some embodiments, one or more amino acid replacements comprise a T591R substitution, numbered with VP1 for 88788. In some embodiments, the rAAV particle includes an AAV capsid serotype 9 (88679). In some embodiments, one or more amino acid substitutions are at positions 586 and / or 589, numbering based on VP1 numbering 88799. In some embodiments, the numbering is based on VP1n AAV9 comprising the amino acid sequence of the sequence ID: 15. In some embodiments, one or more amino acid replacements include substitution at the position S586 and or 589, numbering based on AAV9 numbering 711. In some embodiments, one or more amino acid substitutions include substitution of S586R or S586K, numbering based on VP1 g. 8879 In some embodiments, one or more amino acid substitutions comprise the substitution of A589R or 589Κ, numbering based on VP1 for 88799. In some embodiments, the rAAV particle includes the AAV capsid of the serotype rhio AAVrhlO. In some embodiments, one or more amino acid substitutions are at positions 588 and / or 591, numbering based on AAVrhlO 711. In some embodiments, the numbering is based on VPln AAVrhlO comprising the amino acid sequence of the sequence ID: 16. In some embodiments, one or more amino acid substitutions include substitutions for locus Q588 and / or 591, numbering based on AAVrhlO numbering. Some embodiments, one or more amino acid substitutions include substitution of Q588R or Q588K, numbering based on VP1 for AAVrhlO. In some embodiments, one or more amino acid substitutions include an A591R substitution, numbering based on the VPl of AAVrhlO.

4. Methods of treatment

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10167 Genetic treatment protocols for retinal diseases, such as LCA, retinitis pigmentosa, and age-related macular degeneration require local transmission of the vector to cells in the retina. The cells that will be the target of treatment in these diseases are either the retinoblastoma receptor cells or the RPE cells in the sensory neuron retina. Transfer of gene therapy vectors into these cells requires injection into the subretinal space between the retina (RPEj). In some embodiments, the invention provides methods for transferring rAAV vectors into retinal cells where rAAV vectors encapsulated in the AAV capsid comprise one or more substitutions of building blocks. For an amino acid that reacts with .HSPG

[0168] In some embodiments, the invention provides methods for treating a perturbation in a person's CNS involving the transfer of a combination comprising rAAV particles to the individual's CNS system, wherein rAAV particles comprising (A) rAAV capsid comprising the rAAV capsid protein comprising one or more substitutions The amino acid in one or more sites that interact with heparan sulfate proteoglycans, and (b) the rAAV vector comprising heteronuclear acid and replicating at least one AAV terminant. Moreover, methods can be used to transport heterozygous DNA into the central nervous system (CNS). For the individual, ways to improve Transformation of rAAV of cells following delivery of the rAAV particle to the CNS apparatus of an individual, and methods for improving the expression of heterologous nucleic acid following delivery of rAAV particles to the individual's CNS apparatus described in the present request to transfer, for example, the heterocyclic DNA, for example that encoding a therapeutic polypeptide or acid Therapeutic nuclear: These methods can be used, among others, in the treatment of a disorder in CNS (in some models, the individual is a human being.)

Therapeutic vectors

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69 101 The invention introduces methods for the genetic treatment of ophthalmic disorders whereby rAAV particles comprising the therapeutic vectors are transported to the retina of an individual. The advanced transformation of retinal cells can be achieved by encapsulating rAAV vectors in the capsid of rAAVs (for example, rAAVrl particles, rAAV22, etc.) where they are substituted One or more capsid amino acids that interact with HSPG such that rAAV particles are either less bound to HSPG or ablated. The carrier may include hetero-DNA that encodes a polypeptide (for example, a therapeutic or diagnostic polypeptide) and / or therapeutic DNA The DNA that encodes a therapeutic or diagnostic polypeptide and / or the therapeutic DNA can be generated using methods known in the art , using Standard synthesis and recombinant recombinant methods. In some embodiments, heterochromic DNA encodes a therapeutic polypeptide. In some embodiments, the heteronuclear DNA encodes a diagnostic polypeptide. Unconstrained examples of DNA encoding therapeutic polypeptides include: Nucleic acids to replace a missing or mutated gene known to cause retinal disease, for example CNGB3, CNGA3, RPGRIP, RP2, RPGR, MERTK, RPE65, Prph2, and 812a. Other non-exclusive examples of nucleic acids that encode therapeutic polypeptides include those that encode Neurotrophic factors (e.g. EPO, PEDF, FGF2, CNTF, GDNF), anti-cell turnover genes (e.g. NFkB, BCL-Χ, BCL2), antiangiogenic agents (e.g. indostatin, angiostatin, sFlt), and anti-inflammatory agents (e.g. ILl). -ra, IL10, 114 Other therapeutic polypeptides for ophthalmic disorders include, but are not limited to, Lpcatl, RPGRIP, RSl, PDE6C, GUCY2D, REPI, ABCA4, Myo7a tCHM, RDH12, AIPLl. Some embodiments, the encoded polypeptide is the human variant of the polypeptide. Polypeptide.

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10170 The nucleic acids of the invention may encode polypeptides which are intercellular proteins, fixed in the cell membrane, that remain inside the cell, or are selected by the transformed cell using the vectors of the invention. For the polypeptides secreted by the vector-receiving cell; The polypeptide is preferably soluble (i.e., not attached to the cell). For example, soluble polypeptides are free of the trans-membrane region and are selected from the cell. Techniques for identifying and removing DNA sequences encoding trans-membrane transponders are known in the field. [0171] The vectors that may be given according to the present invention also include vectors comprising DNA encoding RNA (for example, RNAi, ribozymes, siRNA, miRNA, RNA). Antipyretic) which, when transcribed from vector nucleic acids, may treat ophthalmic disorder by interfering with genetic translation or abnormal or excess protein transcription associated with the pathological condition of the invention. For example, nucleic acids from the invention may encode RNA that treats disease by eliminating or highly specific reduction of mRNA that encodes abnormal and / or redundant proteins. RNA therapeutic sequences include RNA, small inhibitor RNA (RNA, small siRNA (miRNA), And / or ribonems (eg hammerhead and heparan ribozymes) that can treat diseases by eliminating or highly specific reduction of mRNA encoding abnormal and / or excess proteins, for example those that occur in various forms of inherited retinal degradation. That can be treated with RNA therapeutic sequences include, for example, autosomal dominant retinitis pigmentosa (ADRP) and diabetic retinopathy. Examples of RNA therapeutic sequences and nucleic acids encoding these sequences that may be used in the invention include those described in, for example, US Patent Application No. 291,6,225, the disclosure of which is incorporated in the present application as a reference in its entirety. In some

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Models, the therapeutic RNA sequence is 708-miR. In some embodiments, the 708-miR is used in combination with DNA encoding untreated rhodopsin, either as a scab of the same rAAV vector or as part of the rAAV second vector. In some embodiments, the DNA encoding untreated rhodopsin lacks the target sequence 708-miR that resides in the 3 non-transgenic regions of the rhodopsin gene. RAAV vectors encoding 708-miR and / or rhodopsin are provided by provisional US patent application serial number 969 / 61, 027, built-in current application for reference in its entirety.

72 101 Specific aspects of the invention relate to the use of rAAV particles (eg, a therapeutic vector) comprising (a) a rAAV capsid comprising an rAAV capsid protein comprising and one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycan , And (b) the rAAV vector comprising heteronuclear acid and repeating two AAV terminals and at least a solution. In some embodiments, heterocyclic DNA encodes a therapeutic polypeptide or nucleic acid. Depending on its use in the present application, the therapeutic nucleic acid may express any therapeutic nucleic acid in the present disclosure or any nucleic acid encoding a therapeutic polypeptide from the present disclosure. Therapeutic DNA may be used, for example, to alleviate symptoms, prevent or delay progression, and / or provide treatment for the disorder (for example, the disorder described in the present application).

10173 Evolved transformation of CNS cells can be achieved by encapsulating rAAV vectors into the capsid of rAAVs (for example, AVrh8R, rAAV2 etc.) in which one or more capsid amino acids that interact with HSPG substitute such that rAAV particles are either less bound to HSPG or ablated. A carrier may include a heterochromic nucleic acid encoding a polypeptide (for example, a therapeutic or diagnostic polypeptide) and / or therapeutic DNA.

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Nuclear that encodes a therapeutic or diagnostic polypeptide and / or therapeutic DNA using methods known in the art, using standard synthesis methods and recombinant product. In some embodiments, heteronuclear induction encodes a therapeutic polypeptide. In some embodiments, the heteronuclear DNA encodes a diagnostic polypeptide. In some embodiments, a CNS-coupled heterochromatic DNA encodes a gene.

10174 In some embodiments, heterozygous nucleic acid encodes therapeutic DNA. In some embodiments, the therapeutic nucleic acid may include but is not limited to RNAi, shRNA, siRNA, RNA, anti-phylactic miRNA, ribosome or D. Azyme. As such, therapeutic DNA may encode RNA which, when transcribed from vector nucleic acids, can treat the disorder. Of the invention (eg, perturbation of the CNS) by interfering with genetic translation or abnormal or excess protein transcription associated with perturbation of the invention. For example, nucleic acids of the invention may encode an RNA that treats the disorder by eliminating or hyperspecific reduction of mRNA encoding abnormal and / or redundant proteins. RNA therapeutic sequences include RNA, small inhibitor RNA (RNA, (miRNA)), and / Or ribonems (eg hammerhead and heparan ribonems) that can treat perturbations by eliminating or highly specific reduction of mRNA encoding abnormal and / or extra proteins.

[0175] In some embodiments, heterochromic DNA encodes a therapeutic polypeptide. A therapeutic polypeptide may provide, for example, a polypeptide and / or enzyme activity that is not present or present at a low level in the cell or organism. Alternatively, a therapeutic polypeptide may provide a polypeptide and / or enzyme activity that indirectly counteracts an imbalance in the cell or organism. For example, a therapeutic polypeptide may provide a disorder related to the accumulation of metabolites caused by insufficient metabolism or The activity is the missing metabolism or activity, or it can provide an enzyme for metabolism or activity

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An alternative that reduces the metabolite. A therapeutic polypeptide may also be used to reduce the activity of the polypeptide (for example, that which is overexpressed, that is activated by a mutagenic gain, or whose activity is otherwise unregulated) by an effect, For example, as a dominant negative polypeptide.

10176 In some embodiments, heterochromic DNA encodes a polypeptide selected from an enzyme, neurotrophic factor 5, a polypeptide lacking or mutagenic in an individual with a CNS-related disorder, an antioxidant, anti-cell lysis agent, an anti-angiogenic agent, and anti-inflammatory agent. These polypeptides can be used to treat CNS disorders by, for example, providing a reduced polypeptide or enzyme activity that is not present, or disorganized in, a perturbation in the CNS, alleviating the cause and / or presentation of a disorder in the CNS, and / or Mitigating CNS damage resulting from perturbation in CNS 10 (for example, cell vanishing, inflammation, or other type of cell death). Non-exhaustive examples of DNA encoding therapeutic polypeptides include: Nucleic acids to replace a missing or mutated gene known to cause disruption in CNS, for example

CNGB3, CNGA3, RPGRIP, RP2, RPGR, MERTK, RPE65, Prph2, and 2812]

Other non-exhaustive examples of nucleic acids encoding therapeutic polypeptides over those of T15 encode neurotrophic factors (e.g. EPO, PEDF, FGF2, CNTF, GDNF), cell-fading genes (e.g. NFkB, BCL-, BCL2), antiangiogenic agents. (E.g. indostatin, angiostatin, sFlt), and anti-inflammatory agents (e.g. TGFp, ILl-ra, IL10,

14) Other therapeutic polypeptides for CNS disorders include but are not limited to 20, AIPLl, Lpcatl, RPGRIP, RSl, PDE6C, GUCY2D, REPI, ABCA4, Myo7a.

CHM, RDH12. In some embodiments, the polypeptide encoder is the anthropogenic heteropeptide

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Polypeptide. In some embodiments, heterotrophic DNA encodes for inhibiting neuronal degeneration (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), Tyrosine hydroxylase, TH 811-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidant, angiogenic polypeptide, antiinflammatory polypeptide, and / or aspartocellase (8528). Examples of antioxidants include but are not limited to. On SODl; S0D2; Catalase; 1 4, 3, Sirtuins, or 5; NRF2; PGCla; GCL (Catalytic subunit); GCL (modified subunit); Adiponectin ,; Glutathione peroxidase 1; And globin is nervous. Examples of anti-angiogenic polypeptides include but are not limited to angiostatin, indostatin, PEDF, VEGF receptor soluble, and PDGF receptor soluble. Examples of antiinflammatory polypeptides include but are not limited to 10-IL17R, soluble IL, TNF-R soluble, TNF-R-! G, 1-IL inhibitor, and IL18 inhibitor. Other representative polypeptides are provided for these classes that may be used in Treatments for CNS disruption are as follows.

[0177] Nucleic acids of the invention may encode polypeptides that are proteins of an intercellular, fixed in the cell membrane, that remain inside the cell, or are selected by the transformed cell using vectors of the invention. For polypeptides secreted by the vector-receiving cell; The polypeptide is preferably soluble (that is, not attached to the cell). For example, soluble polypeptides are devoid of the trans-membrane region and are cell-picked. Techniques for identifying and removing DNA sequences encoding trans-membrane domains are well known in the domain.

1 In some embodiments, it is an heterodynamic DNA that is functionally attached to a promoter. Representative enhancers include, but are not limited to, the cytomegalovirus (CMV) promoter

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Prompt Early, MLV LTR, RSV LTR, Phosphoglycerinase-1 Booster (PGK), Monkey Virus Booster 40 (SV40) and CK6 Booster, Transcretin (TTR) Booster, TK Booster, Tetracycline Response Booster (TRE), HBV Booster, hAAT Booster, LSP booster, chimeric liver-specific enhancers (LSPs), E2F booster, telomerase booster (hTERT); Enhanced CMV / chicken beta-actin / rabbit 3-globin (CAG booster; 10 8 (2): 193-9, 1991, Gene, NÎwaetal) and 1-alpha-prolonging factor booster (151 T1-alpha) (.91 (2): 217-23, 1990, GeneiKim et al and. Gene, Guo et al. 3 (9): 802-10, 1996, Ther). In some embodiments, the enhancer includes a human 1-glucorinidase promoter or a CMV enhancer associated with the - chicken actin promoter (8 and 0). The enhancer may be a foundational promoter, an orbite inducer. In some embodiments, the invention introduces a vector of the recombinant product that includes the DNA encoding the heterogeneous transgenic gene from the present disclosure that is operationally linked to the promoter 0138. Representative enhancers and description can be found, for example. Example, in the order USPG Pub.2O14O335O54.

10179 Examples of foundational enhancers include, but are not limited to, the Russian sarcoma retrovirus booster (LTR) (RSV (optionally with RSV enhancer), cytomegalovirus (CMV) booster (optionally with CMV enhancer) (see, for example, 41: 521). -, CelEBoshart et al (1985 (530)) ;, SV40 booster, dihydrofolate reductase booster, 13-actin booster, phosphoglycerol kina g (PGK) booster, and invitrogen [EFla] booster.

[0180] Inducible enhancers allow regulation of genetic expression and can be regulated by compounds that are provided externally, environmental factors such as temperature, or the presence of a specific physiological condition, for example, acute phase, specific cellular differentiation state, or in the transcription of cells. Just. Motivable reinforcers and triggers are available from a variety of sources

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Commercial, including, but not limited to, Clontech, Invitrogen, and Ariad. Many other systems have been described and can actually be selected by the skilled in the art. Examples of promoters regulated with enhancers available externally include the zinc-stimulated goat metallothionine (MT) booster. Dexamethasone (Dex) mouse induced breast tumor virus booster (MMTV), polymerase booster system WO 98/10088 (7); Ecdison Insect Booster (5 No et

93: 3346-3351 (1996, Proc.Natl.Acad.Sci.USA, al)), a suppressible system for tetracycline.

1992, ρroc.Natl.Acad.Sci.USA, Gossen et al.)))), An induction system

Tetracycline (1995, Science, G0ssen et al.); See also Harvey et

Paul., Curr. Opin. Chem. Biol., Al. ()), -System induction by Wang et (RU486).

1997, Nat. Biotech., Al. And. 1997, Gene TherWang et al. System 10.

Inducible propamycin (. 100: 2865-2872 (1997, J. Clin.Invest., Magari et al)) there are still other types of stimulants that may be useful in this context and that are regulated by a specific physiological condition. For example, temperature, acute phase, cell-specific discrimination state, or only cell transcription.

[0181] In another embodiment, the original enhancer, or a fragment thereof, of the transgene may be used. The original promoter 15 may be preferred when expression of the transcriptional gene is desired to mimic the original expression. The original promoter may be used when expression of the modified gene must be regulated temporarily or in an evolutionary manner, or in a tissue-specific manner, or in response to a transcriptional stimulus. In a supplementary embodiment, the other parent expression controls elements, such as enhancer elements, polyadenylated sites, or Kozak consensus sequences that can also be used to simulate the original expression. 20

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[0182] In some embodiments, the sequencing sequences impart the capabilities of a tissue-specific genetic expression. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in the tissue in a specific way. The mentioned tissue-specific regulatory sequences (for example, enhancers, enhancers, etc.) are well known in the art. Representative tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific enhancers: neurons e.g. a neuron-specific enolase booster (13: 503-15, Cell.Mol.Neurobiol., Andersen et al.) (NSE 1993)) A neuromembranous light chain enhancer (Piccioli et al.).

88: 5611-5 (1991, Proc.Natl.Acad.Sci.USA)), and the vgf neuron specific genetic enhancer (. 88: 5611-5 (1991, Proc.Natl.Acad.Sci.USA, Picciolietal)). In some embodiments, a tissue-specific goat is a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrous acidophilic protein (GFAP), adenomatous polyposis (APC), and ionized calcium-binding adaptogen 1 (1-E10). Other suitable tissue-specific enhancers for the veteran will be evident; in some embodiments, the enhancer is a chicken beta-actin enhancer.

In some embodiments, heterozygous DNA undergoes an enhanced sequence control that is expressed in one or more of the? 3 cells. Many of the promoter sequences listed previously (for example, the CBA promoter) are known in the domain and are expressed in one or more of the 75a 0 cells. In some embodiments, the promoter sequence may be expressed as both the co-occurrence in a living organism, and thus may be expressed in one or more CNS cells thanks to its transfer to the CNS system. In other embodiments, the promoter sequence may be used that specifically expresses in the CNS. In some embodiments, it is a subset of one or more of the 15ME cells, which is an heterologous nucleic acid operatively attached to a suitable promoter for expression of either the therapeutic polypeptide or the therapeutic DNA in one

Mard

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Or more cells? As such, in some embodiments, a therapeutic polypeptide or the therapeutic DNA of the invention may be used in the treatment of CNS disturbance.

In some embodiments, a promoter expresses heteronuclear DNA in the brain cell. A brain cell may refer to any brain cell known in the field, including but not limited to a nerve cell (for example, a sensory neuron, a motor neuron, an intermediate neuron, a dopamine-secreting neuron, an intermediate spinal neuron, a choline neuron, a neuron). GABAergic cell, pyramidal neuron, etc.), glial cell (e.g., microglia, large glial cells, astrocytes, oligodendrocyte, endothelial cells, radial glial cells, etc.), The brain parenchyma, microglial cell, neuroendothelial cell, and / or Purkinjcell cell (in some embodiments, the promoter expresses heterozygous DNA in the nerve cell. In some embodiments, the heteronuclear DNA is expressed exclusively in neurons (for example, it is expressed in the neuron rather than in other CNS cells, for example glial cells) 10185 In some embodiments, the invention introduces the rAAV vectors for use in methods to prevent Or treat and solve one or more genetic defects (for example, inherited genetic defects, somatic gene alterations, etc.) in my breasts, for example, a genetic defect that results in a polypeptide deficiency or an excess of polypeptide in a subject, or for treatment Or reduce the intensity Or the extent of deficiency in a subject who exhibits CNS-related disorder associated with a deficiency of the aforementioned polypeptides in cells and tissues. In some embodiments, methods include administration of the rAAV vector encoding one or more therapeutic peptides, polypeptides, functional RNAs, inhibitory nucleic acids, shRNAs, microRNAs, nucleotides for antiemulsion, etc. in a pharmaceutically acceptable carrier to the subject.

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In an amount and for a period of time sufficient to treat CNS-related disorder in a subject suffering or suspected of having said disorder.

20186 The rAAV vector may include a transgenic, DNA-encoding protein or functional RNA that modifies or treats the RNA-related disorder. The following is a non-exhaustive list of genes associated with CNS-related disorders: Neuronal Fade Inhibitor Protein (NAIP), Nerve Growth Factor (NGF), Glial-Derived Growth Factor (GDNF), Brain-Derived Growth Factor (BDNF), Ciliary Rectal Nutritional Factor (CNTF), Tyrosine Hydroxylase (011, TM-cyclo Hydrolase (GTPCH), Aspartocellase (ASPA), dismutase superoxide (S0D1), antioxidant, anti-angiogenic polypeptide, anti-inflammatory polypeptide, and decarboxylase amino acid (8800). For example, the gene encodes a useful axis in the treatment of Parkinson TH, an enzyme that restricts the rate of dopamine synthesis The transgenic gene encoding GTPCII, which generates co-factor TII tetrahydrobiopterin, is also used in the treatment of Parkinson's disease. The transgenic gene encoding GDNF, BDNF, or AADC, while facilitating the transformation of L-Dopa to DA, can also be used in the treatment of Parkinson's disease. To treat ALS, the gene may encode a useful axis: GDNF, BDNF or c) 71E. Also for the treatment of ALS, the useful mutated gene may encode a functional RNA, for example, miRNA, shRNA, which inhibits the expression of 5001. For treatment of localized anemia, the useful mutagen gene may encode NAIP or G.NGF &lt;be The transgenic gene encoding beta-glucorinidase (GUS) is useful in treating storage disease with specific present particle storage (eg, Mucopolysacharidosis MPS VII (VII)). ^ The transgenic gene encoding the primary drug activation gene is, for example, 1157-thymidinkinase Which converts ganciclovir into a toxic nucleotide that works It disrupts DNA synthesis and leads to cell death, useful for treating specific species

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Of cancer, for example, when given in combination with the initial drug. A transgenic gene encoding an endogenous opioid, such as 8-endophrine, may be useful for treating pain. Examples of antioxidants include but are not limited to SODl; S0D2; Catalase; 4, 3, 1 Sirtuins, or 5; NRF2; PGCla; GCL (catalytic subunit); GCL (modified subunit); Adiponectin ,; Glutathione peroxidase 1; And globin is nervous. Examples of anti-angiogenic polypeptides include but are not limited to angiostatin, indostatin, PEDF, soluble VEGF receptor, and soluble PDGF receptor. Examples of anti-inflammatory polypeptides include but are not limited to 10-IL17R, soluble IL, soluble TNF-R, TNF -R-! G, 1-IL inhibitor, and 118) inhibitor. Examples of transgenes that can be used in rAAV vectors will be evident from the invention of the skilled in the art (see, for example, etakCostantiniLC., Genetic Therapy (2000) 7, 93-109)

[0187] In some embodiments, either therapeutic polypeptide or therapeutic DNA is used in the treatment of 5 to 0 disorder. Without being bound by theory, it is believed that therapeutic polypeptide or therapeutic DNA may be used to reduce or eliminate the expression and / or activity of the polypeptide which is Gain of function is associated with the disorder, or to improve the expression and / or polypeptide activity to complement a deficiency associated with the disorder (for example, a mutant in a gene that demonstrates expression of similar or related activity). Non-exhaustive examples of perturbations of the invention that can be remedied by a therapeutic polypeptide or therapeutic DNA of the invention (model genes that can be targeted or provided in parentheses for each disorder are provided) include angina (e.g., 3-caspase, PUMA, HIFla, PARI, Askl, Beclinl, and / or any genes described in AMand, Fukuda 2013 (Genes (Basel) 4: 435-456, Badaut). L), Huntington's disease (mutant HTT), epilepsy (eg, ADK, NMDAR) , SCNIA, and / or any genes described in D. (2O1O), Boison

L g

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51: 1659-1668 Epilepsia), Parkinson's disease (alpha-synuclein), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis; S0D1), Alzheimer's disease (tau, starch protein), basal cortical degeneration or tau (CBD), Basal cortical nodal deterioration or CBGD (tau), frontotemporal dementia or tau (FTD), progressive supranuclear palsy or tau (PSP), multiple system atrophy or MSA (alpha-synuclein), brain cancer (for example, a mutant or gene) An overexpressed tumor is implicit in brain cancer) and present particle storage disease (57). Disorders of the invention include those involving large areas of the cerebral cortex, for example, more than one functional area of ​​the cerebral cortex, more than one lobe of the cerebral cortex, and / or the entire cortex. Other non-exhaustive examples of disorders of the invention that can be treated with a therapeutic polypeptide or therapeutic DNA of the invention include traumatic brain injury disorders and enzymatic impairments, psychiatric disorders (including PTSD), neurodegenerative diseases, and cognitive disturbances (including That dementia,

Autism and depression). Enzymatic defect disorders include without limitation leukodystrophy (including Canavan's disease) and any present particle storage disease described below.

In some embodiments, therapeutic polypeptide or therapeutic DNA is used to treat present particle storage disease. As commonly known in the art, particle storage disease (PMS) is rare inherited metabolic disorders characterized by defects in the function of the particle. These disorders often result from a deficiency of the enzyme required for adequate mucopolysaccharide, glycoprotein, and / or lipid metabolism, leading to pathogen accumulation of cellular material stored in the particle. Non-exhaustive examples of particulate storage pathologies of a present invention that can be treated with a therapeutic polypeptide or therapeutic DNA from r

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The invention (model genes that can be targeted or provided in parentheses for each disorder) are Gaucher's disease type 2 or type 3 (acidic beta-glucosidase, GBA), gangliosidase GM1 (beta-galactosidase-1, GLBl), Hunter's disease (ED 2-Sulfate Ronate, IDS), Krabbe disease (galactosylceramidase, GALC), mannosidase disease (eg alpha-mannosidase, 2Β1), manosidic disease (beta-manosidase, MANBA disease, metachromatic leukodystrophy) (Pseudo-ARSA,), mucinous lipid disease ΙΙ / ΙΙΙ (2-acetyl-glucosamine-1-phosphotransferase, GNPTAB), Niemann-Pick disease (acidic sphingomyelinase, ASM), C disease; Protein C,

NPCl), Pompe disease (alpha-1, 4-glucosidase, GAA), Sandhoff disease (beta-hexosaminidase subunit, HEXB), Sanfilippo disease (72-Sulfo-glucosamine sulfo-hydrolase, MPS3A), Sanfilippo B disease (7-alpha- Acetylglucosaminidase, NAGLU), Sanfilippo C disease (heparan acetyl-008: alpha-gluco saminidase la-acetyltransferase, MPS3C), Sanfilippo D disease (2-acetylglucosamine-6-sulfatase, GNS), Schindler's disease (alpha-2-acetyltransferase, MPS3C) -Acetyl-galactosaminidase, NAGA), Sly's disease (beta-glucorinidase, GUSB), Tay-Sachs disease (subunit Hexosaminidase alpha, HEXA), and Wolman's disease (LIPA, acid-particle lipase).

[0189] Storage diseases are listed with the additional present particles, as well as the defective enzyme associated with each disease, in the following Table 1. In some embodiments, the disease listed in the following table is treated with a therapeutic polypeptide or therapeutic DNA from the invention that complements or otherwise Compensates for the corresponding enzymatic defect.

Table 1. Storage disturbances with lysosomes and paired defective enzymes.

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<td>Defective enzyme.</td><td>Disease storage for particulate matter</td>

<td>Aspartylglucosaminidase</td><td>Aspartylglnsoaminuria</td>

<td> :^؛Mk-6a^؛A1^:Allow^؛E. To &lt;^:٧^NWell: 00 :: k_٠H |: data&gt; i</td><td>0,,: no. 6 0. 1,: 0c. ί</td>

<td>. ; '. Thiophosphatase: ProteinBalmitoyl:</td><td>(CNL!) ^ I in Batten's disease</td>

<td>, '; 0; -i0590.; S0; 0-90 i. '..B'.'</td><td>J '' - m. Lm ;; ^ for his illumination * “Jamal</td>

<td>; . Putin cross the membrane in the case b</td><td>In the latest (13a) Battery ^. '·</td>

<td>Miscellaneous gene. _؛</td><td>Batten, Other Images ((CNL4-CNL8</td>

<td></td><td>Cystinitis</td>

<td>Ceramidase acid</td><td>Farber</td>

<td>G-alpha-1-fucosidase is acidic</td><td>Phocosilic disease '</td>

<td>060 :: 0 '' no,</td><td>Galactosidosialidosis</td>

<td>Glucosidase beta-glucosidase</td><td>Gaucher types 1, 2, and 3</td>

<td>4.40 ptl of lactocyte acid</td><td>:, T6'6jjz'm0; g _؛ T</td>

<td>_J:: w k | 0 ia ha 0; | ds | ua '? L.</td><td>Hunier</td>

<td>...: n.,.? '' E's alfater-odoronidase, 4</td><td>Hurler-Scheie</td>

<td>Galactocerebrosidase</td><td>did not·^,'.' ..., for the public: 6; J Krabbe; Mine: . a</td>

<td>The Alpha-Mannenosidase Fertilizer.</td><td>Al-Fatal disease Almanoside Zacc:</td>

<td>Acid beta-mannosidase.</td><td>: T-6- ^ lm 1; Beta-d manosidosis:</td>

<td></td><td>Maroteaux-Lamy</td>

<td>M. Zak; ' . ';a ?^Mine;R 0006 '· Haszkagi: 6 rs^K Z6--^:</td><td>. White matter is discolored</td>

<td>B11-acetyl-galactosamine — e — sulfides</td><td>44, McCoy's disease</td>

<td>... beta-galactocyl 1 g is acidic</td><td>8 Morquio III disease</td>

<td>XL L-Acetylglucosamine-1-Phosphorous Transferase:</td><td>No ^ -: .. 0.9; Mucocutaneous nebula] 1b / 11</td>

<td> .'^P6 ^ 0 SS .;; ha.a.ma ^ - ';; u: a ...;</td><td>B, Niemann-Pick A</td>

<td>; E :: 'k'; - '^؛K^,:... h: .0.r: a1'.that; he will command you 6 '</td><td>Niemann-PickC</td>

<td>الغ</td><td>?; Pompe</td>

<td>A beta-hexoepaminidase):</td><td>Sandhoff</td>

<td>0, 0. Hepar-n-sulfates. :</td><td>SanfilippoA</td>

<td>GA ~ L-11 ~ AcetylglycoSapidase '</td><td>SanfilippoB</td>

<td>-. Acetylcysteine: 8 and 0-alphaglucosaminidla-aspetyltransferrer</td><td>SanfilippoC</td>

<td>0-Acetylglucosamines-6-capsaicin:</td><td>Sanfilippo D</td>

<td>Alpha-T-acetylgalactosaminases:, B.</td><td>Shindle ^</td>

<td>Alpha-B1-Abetylgalactosaminidase.</td><td></td>

<td> - :^:a . . Alpha-neuroamidase</td><td>Sialidosis: 6 6</td>

<td>Betaskhnokosinidase. :</td><td>Sly</td>

<td>Beta-hexosaminidase 8.</td><td>... Tay-Sachs</td>

<td>To pass .. to fight back. a;',. ...</td><td>Wolman</td>

Huntington's disease

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10190 One example of a disease where the aforementioned vectors may be useful is Huntington disease (HD), caused by a mutant CAG repeat expansion that encodes expanded polyglutamine (polyQ) in the mHTTHD mutant huntingtin is an attractive target for RNA-DNA-based therapies because it is An autosomal dominant disease that results from mutagenesis of a single allele. AAV vectors can provide an ideal transport system for therapeutic DNA and allow for the continuous and continuous expression of these huntingtin-reducing molecules in the brain.

10191 As described in the present application, the intracranial administration of rAAV particles (eg, a therapeutic vector) comprising an rAAV capsid protein containing one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycan results in the conversion of Broad spectrum for neurons. Consequently, aspects of the invention provide methods for transferring heterologous DNA into the central nervous system using the viral particles of the recombinant product described in the present application for treatment of Huntington's disease. In some embodiments, the invention provides methods and combinations for the treatment of Huntington's disease in an individual involving administration to the mammalian combination. Pharmacokinetics from the present disclosure (eg, a pharmaceutical formulation comprising a variant of a viral particle from the present disclosure). In some embodiments, the invention presents methods and combinations for inhibition of expression of htt in mammals with Huntington disease that include administration to an individual of a pharmaceutical formulation from the present disclosure (for example, a pharmaceutical formulation comprising a variant of a viral particle from the present disclosure). The invention presents methods and combinations for inhibiting the accumulation of htt in a cell of a person with Huntington disease that include administration to the individual a pharmaceutical formulation from the present disclosure (eg, a pharmaceutical formulation comprising a variant of a viral particle from the present disclosure).

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10192 In some embodiments, the invention presents methods and combinations to mitigate HD presentation in an individual, including administration of an effective amount of viral particles of a recombinant product that includes one or more amino acid substitutions at one or more sites interacting with the proteoglycan heparan sulfate. To the individual's CNS, where rAAV particles comprise an RNAi-encoding transporter that inhibits the expression and / or accumulation of HTT in the individual. In some embodiments, symptoms of HD include, but are not limited to, dance, stiffness, body movements, etc. the control On them, loss of muscle control, lack of coordination, restlessness, slowed eye movements, abnormal movements, instability, gait gait, abnormal facial expression, problems with speech, difficulties in chewing and / or chewing, sleep disturbance, seizures, dementia, impotence Cognitive (for example, diminished abilities related to planning and abstract thinking, flexibility, acquisition of judgment, sensitivity to interacting with others, self-control, attention, learning, memory), depression, anxiety, changes in personality, aggression, compulsive behavior, obsessive-compulsive disorder, Hypersexual desire, Psychosis, apathy, irritability, suicidal thoughts, weight loss, muscle atrophy, heart failure, decreased glucose tolerance, testicular atrophy and osteoporosis.

[0193] In some embodiments, the invention provides methods for preventing or delaying the development of HD. An autosomal dominant HD is a hereditary disease that can be genetically classified. For example, the number of occurrences can be determined by determining the frequency size based on 108. This type of HTT can be performed in CAG

Diagnosis at any stage of life through direct testing of juveniles or adults (for example, in addition to presentation of clinical symptoms), prenatal examination or prenatal exclusion test (for example, by chorionic villus sampling or amniocentesis), Or preimplantation examination. Additionally, HD can be diagnosed by brain imaging.

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And look for contraction of the caudate nucleus, endocardium and / or enlarged ventricles. These symptoms, together with a family history of HD and / or clinical symptoms, may indicate the presence of HD.

Methods for determining the mitigation of HD symptoms are well known in the field; for example, the Standardized Huntington Rating Scale (UHDRS) can be used to assess motor function, cognitive function, behavioral aberrations, and functional ability (see, for example, Huntington 11: 136- 42 Study Group (1996) Movement Disorders) .32 Development of this classification scale to provide a standardized and comprehensive test for multiple aspects of pathogens, combining elements from tests such as HD Activities and Daily Living Scale, Marsden and Quinn's Dance Intensity Scale, Disability Scales Physicality and Autonomy, HDMotor Rating Scale (HDMRS), HDFCS Scale Functional Capacity (HD), and Quantitative Neurological Test (QNE).) Other useful test for determining the mitigation of HD symptoms includes but is not limited to Montreal Cognitive Assessment, Brain Imaging (on For example, Map Search, Trail Making Test, Category Fluency Test (MRI, Symbol DigitModalities Test, Speeded Tapping Task, Stroop Word Reading Test).

10195 In some embodiments of the invention, the methods and combinations are used in the treatment of a human with T1. According to previously described, HD is inherited in an autosomal dominant fashion and results from the expansion of CAG repeat in the 1111 gene. The onset of HD in juveniles is predominantly inherited from the parents. Isotope species similar to Huntington's disease are also associated with other genetic loci, such as HDL3, HDL2, PRNP, HDLl, and 14 papules. It is believed that other genetic loci may alter the appearance of HD symptoms, including mutations in the GRIK2 genes, MSXl, GRIN2B, GRTN2A ^ APOE and combinations. rAAV

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10196 In some embodiments, the invention provides compositions that include any of the rAAV particles described in the present application. In general, compositions for use in the methods and systems of the invention comprise an effective amount of rAAV particles comprising rAAV vectors encoding a polypeptide and / or RNA, optionally in acceptable excipients. Pharmaceutically. Viral particles comprising AAV capsid (eg, AAV2 capsid or AAVhR) in which one or more amino acids that interact with HSPG have a substitution such that the rAAV particle binding to HSPG is reduced or ablated. According to well known in the field, pharmaceutically acceptable excipients are relatively inert substances that facilitate the administration of the pharmacologically active substance and can be provided in the form of liquid solutions or suspensions, emulsions, or in solid forms suitable for dissolution or suspended in a liquid prior to use. For example, excipients may impart appearance or persistence, or act as a substance to dilute. Suitable excipients include but are not limited to stabilizing agents, wetting agents and emulsifiers, salts of a variety of osmolarity, encapsulating agents, pH-blocking buffers, and buffer solution. For disability. The excipients mentioned include any pharmaceutical agent suitable for direct transport into the eye and which can be administered without unnecessary toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, i.e., from the various TWEEN compounds, and liquids such as water, brine, glycerol and ethanol. Pharmaceutically acceptable salts may be incorporated into the present application, for example, mineral acid salts for example chlorides Aqueous, aqueous bromides, phosphates, sulfate, etc.; And salts of organic acids such as acetate, propionate, malunoate, petzoate, etc. A comprehensive discussion of pharmaceutically acceptable excipients is provided in REMINGTON'S (NJ1991, Mack Pub.Co.) PHARMACEUTICAL SCIENCES.

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00197 Generally, these formulations are formulated for administration by subretinal injection. Accordingly, it is preferable to combine these formulations with pharmaceutically acceptable carriers such as saline solution, Ringer's balanced saline solution (pH 7.4), etc. If not required, formulations can be provided optionally in unit dose form suitable to administer the exact amount subretinal rAAV transport methods.

[0198] Sub-retinal transport methods are well known in the art. For example, see WO 2009/105690, incorporated into the present application for reference. In summary, the general method for transporting rAAV particles (for example, rAAVrhSR particles, rAAV22, etc.) to the subretinal at macula and fovea can be illustrated by the following brief explanation. This example is intended only to illustrate specific features of the method, and is not intended to restrict in any way.

[0199] Generally, the rAAV vector can be transported as a formulation injected into the eye (subretinal) under direct observation using an operating microscope. In some embodiments the vector is encapsulated in the rAAV particle wherein the rAAV particle comprises an rAAV capsid comprising the rAAV capsid protein comprising one or more amino acid substitutions in one or more sites that interact with the heparan sulfate proteoglycan (eg, inhibit Or HSPG ablation), the rAAV vector comprises heteronuclear acid and at least one AAV inverted terminal repeat. This procedure may include vitrectomy followed by injection of the rAAV vector suspension using a micro-cannula through one or more small retinal cells in the subdivision Retina.

10200 Briefly, the infusion cannula can be sutured in place to maintain the normal portion size by infusion (for example, saline solution) throughout the process. Vitrectomy is performed using a cannula with an appropriate hole size (eg 20 to 27 gauge), into which it is replaced

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The volume of vitreous gel removed by infusion of saline or another isotonic solution from the infusion cannula. Vitrectomy is advantageously performed because (1) removal of its cerebral cortex (posterior vitreous membrane) facilitates penetration of the retina with the cannula; (2) Removal and replacement of fluid (eg, saline solution) has room to accommodate injection of the carrier into the eye, and (3) controlled removal reduces the possibility of retinal tears and unplanned retinal detachments.

[0201] In some embodiments, the rAAV formula is injected directly into the subretinal space outside the central retina, by using a cannula with an appropriate puncture size (eg, 2745 gauge), thus forming a bubble in the subretinal space. In other embodiments, the subretinal injection of the combination rAAV is preceded by a subretinal injection of a small volume (for example, about 0.1 to about 0.5 ml L) of a suitable fluid (eg saline or Ringer's solution) into the subretinal space outside the central retina. In the subretinal space it provides a primary fluid bubble within the subretinal space, and causes localized detachment of the retina at the site of the primary bubble This primary fluid bubble can facilitate the targeted transfer of the rAAV composition into the subretinal space (by defining the injection level prior to rAAV transfer), The possible administration of rAAV into the chorionic coating and the possibility of rAAV injection or regurgitation into the vitreous cavity reduced. In some embodiments, this primary fluid bubble may be injected additionally using fluids that contain one or more rAAV formulations and / or one or more additional therapeutic agents by administering these fluids directly into the primary fluid bubble using either the same micro-perforated vials or Other additional. [0202] Intraocular administration of the rAAV formulations and / and the initial small volume of fluid may be performed using a micro-perforated cannula (eg, gauge 27-45) attached to the syringe. In some

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In embodiments, the plunger of this syringe can be operated by an automated device, for example by lowering the foot pedal. A micro-puncture cannula is inserted through a sclerotomy, through the vitreous cavity and into the retina at a pre-defined location in each subject according to the target retinal area (but outside the central retina). Under direct imaging, the carrier suspension is mechanically injected under the sensory nerve retina and causes localized detachment of the retina using autotrophic retinectomy to prevent non-expanding leakage. According to previously mentioned, the rAAV combination may either be injected directly into the subretinal space and create a central extretinal bubble, or the vector may be injected into the primary extrapinal bubble, causing its expansion (and enlarging the retinal detachment area). In some embodiments, injection of the rAAV combination is followed by injection of another fluid into the bubble.

10203 Without being bound by theory, the rate and site of injection (injections) under the retina may produce local shear forces that may damage the macula, fovea and / or the underlying RPE cells. The subretinal injection times can be performed at a rate that lowers or avoids shear forces. In some embodiments, the rAAV combination is injected over a period of about 15-17 minutes. In some embodiments, the vector is injected over a period of about 1720 minutes. In some embodiments, the rAAV combination is injected over a period of approximately 20-22 minutes. In some embodiments, the rAAV formulation is injected at a rate of about 35 to about 65 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 35 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 40 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 45 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 50 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 55 μL / min. In some embodiments, the rAAV combination is injected at a rate of about 60 μL / min. In some embodiments, the rAAV formulation is injected at a rate of approximately 65 μL / min. Will realize that

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The usual skill in the art is that the rate and time of injection of the rAAV combination can be guided by, for example, the size of the rAAV combination or the volume of the bubble necessary to create sufficient retinal detachment to reach the central retinal cells, the size of the cannula used to transfer the rAAV combination, and the ability to safely maintain the cannula position of the invention.

[0204] In some embodiments of the invention, the volume of the injected formulation into the subretinal space of the retina is more than about any one of 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl. l 8, micro - liter 9, micro g 10 micro liter 15 micro liter 20 micro - liter 25 micro liter 50 micro liter 75 micro liter 100 micro liter 200 micro liter 300 micro liter 400 Mick Rowe liter 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, or 1 ml liter, or any quantity in between.

0205]] In some embodiments, the methods involve administration to the eye (eg, subretinal and / or intravitreal administration) an effective amount of viral particles of the recombinant product comprising one or more amino acid substitutions of one or more From the sites that interact with proteoglycans heparan sulfate includes a vector encoding heterozygous DNA. In some embodiments, the combination viral titers are at least about right 105,12.

X 12,11 10 X 12,10 10 «12,9 10 X 12,8 10 X 12,7 10 X 6

15; 12 20 10 X; 12 12 10 X 12.30 10 X 12.25 10 X, or 10x50 12 ML copies of the genome. In some embodiments, the viral titers of the combination are from 1075 12 to 106 12, 12 10x6 to 7 12 10 X 12.7 10 X to 8 X 10 12.8 12 10 X to 109 12.9 * 10 12 to 10 10 10 X 12 10, 10 X 12 to 11 12 10 X 12,11 10 X 10 to 15 12 10 X 12,15 10 X 20 X

<img file="MA39439B2_D0008.tif" />

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10 12,20 12 10 X to 25 12 10 «12,25 10 X to 30 X 30, 12 10 X 10 12 to 50 for 10 10 12, or 50 X 12 10 X to 100 12 X 10 X copies / milliliter of genome. In some embodiments, the viral titre of the combination is approximately i.e. from 5 12 10 X to 10 12 10 X 12,10 10 X to 25 12 10 X, or 25 12 10 X to 50 10 X 12 copies / ml of genome. In some embodiments, the viral caliber of the combination is at least about any of 5 x 109 (10 * 9, 109 * 109.8 * 109.7 x 6, 109 x 11) 109 109 x 109.30 x 109.25 x 109, 20 X 109,15 X, or 50 X 109 units / milliliter of conversion. In some embodiments, the viral caliber of the combination is anywhere from 5 109 X 6 109 X 6 109 X 7 109 7 X 109.7 X 8 109 X 8 109 X 109 X 9 109 X 109 9 X 10 10 X 10 X 109. 10, 109 X to 11 * 11 (109 A 109 to 15 109 X 109) 15 X to 109 X 109 (20 X 20 to 25 X 109,25 X 109 to 30 109 * 109,30 X 50 to 109 X or 50 109 X To 1 00 X 109 units / milliliter of conversion. In some embodiments, the viral titer of the combination is approximately that from 10975 to 10 * 10 (109 109 X to 15 109 X 109) 15 X to 25 * 109, or 25 109 X to 50 X 109 units / milliliter of conversion. , The viral titers of the combination are at least 5 1010.7 x 6, 1010 x 10

1010,20 x 1010 (15 x 1010, 11 x 1010,10 x 1010.9 '' 1010.8 x

1010 X 1010,40 X 1010,30 X 1010,25 X, or 50 X 1010 units / milliliter of infection. In some embodiments, the viral titer of the combination is at least, i.e. from about 5 x 1010 to 6 1010 x 6, 1010 x to 7 1010 x 1010 (7 x to 8 x 1010.8 1010 x to 9 1010 x 101 0.9 to 10 1010 x 1010 (10 x

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To 11 1010 X 1010, 11 X To 15 1010 &lt;1010,15 X To 20 X 1010.20 1010 X To 25 X 1010,25 1010 X 30 To 1010,30 X »1010 To 101040,101040 To 50 1010, Or 1010450 To 100 &gt; 0 101 units / ml of infection. In some embodiments, the viral titers of the combination are at least from about 5 * 1010 to 10 1010 X 1010,10 X to 15 X 1010,15 X 1010 to 25 X 1010, or 25 X 1010 to 50 X 1010 units /

Milliliters of infection.

[0206] In some embodiments, methods include administration into the eye (eg, subretinal and / or intravitreal) of an individual (eg, a human) an effective amount of viral recombinant viral particles comprising one or more Of amino acid substitutions at one or more sites that interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles given to an individual is at least about any from 1 * 810 to about 101 13 copies / kg body weight. In some embodiments, the dose of viral particles given to an individual is about any from 1 07 1 8 to about 1 km 108 13 copies of a kg genome of body weight.

One or more (eg, 2, 3, or more) bubbles may be formed. Generally, the total volume of the bubble or bubbles formed by the methods and systems of the invention does not exceed the fluid volume of the eye, for example about 4 ml in a human subject. Typically, the total volume of each individual bubble can be at least about 0.3 mL, or at least about 0.5 mL let. The retinal detachment can be facilitated with a volume sufficient to detect central retinal cell types and to form a bubble consequently sufficient for optimal treatment. The average skill in the field will realize that he is in

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Formation of a bubble according to the methods and systems of the invention. The appropriate intraocular pressure must be maintained in order to avoid damaging the eye structures. The volume of each individual bubble may range, for example, from about 0.5 to about 1.2 mL to about 0.8 to about 1.2 mL to about 0.9 to about 1.2 mL to about 0.9 to about 1.0 mL to about 1.0 to about 2.0 mL Thus, in one example, for a total injection of 3 mL of rAAV combination suspension, 3 bubbles could consist of about 1 mL of each. The total volume of all bubbles in combination might range, depending on For example, from about 0.5 to about 3.0mlL, about 0.8 to about 3.0mlL, about 0.9 to about 3.0 mL to about 1.0 mL to about 1.0 to about 3.0 mL to about 0.5 to about 1.5 mL to about 0.5 to about 1.2 mL to about 0.9 to about 3.0 mL to about 0.9 to about 2.0 mL to about 0.9 to About 1.0 ml liters.

[0208] In order to be able to safely and efficiently convert to target retinal areas (eg, central retina) outside the edge of the original site of the bubble, the bubble can be manipulated to reposition the bubble according to the target area of ​​the transfer. Bubble treatment can occur by reliever bubble formed by the size of the bubble, repositioning the eye containing the bubble, repositioning the head to the human with one or two eyes containing one or more bubbles, and / or by air-fluid exchange. This is very relevant to the central retina since this region typically resists detachment by subretinal injection. In some embodiments, air-fluid exchange is used to reposition the bubble; Fluid from the infusion cannula is temporarily replaced by air, for example, from the force of air over the surface of the retina. Because a volume of air replaces the fluid from the vitreous cavity from the surface of the retina, the fluid in the vitreous cavity can flow out of the cannula. Temporary lack of stress

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The fluid from the vitreous cavity causes the bubble to move and move by gravity to an independent part of the eye. By correctly positioning the eyeball, the rAAV combination bubble is processed under the retina to include adjacent areas (eg, macula and / or fovea). In some cases, the mass of the bubble is sufficient to cause it to move by gravity, even without the use of fluid-air exchange. The movement of the bubble to the desired location can be further facilitated by changing the position of the subject's head, to allow the bubble to move by gravity to the desired location in the eye. Once the desired bubble preparation has been achieved, the fluid is returned to the vitreous cavity. The fluid is suitable, for example, a fresh saline solution. Generally, the subretinal rAAV combination can be placed in the same position without retinal fixation to retinectomy and without eye trauma, and the retina will reconnect immediately within about 48 hours.

[0200] By safely and effectively converting eye cells (for example, RPE and / or photoreceptor cells for example, to macula and / or fovea) using a vector comprising a therapeutic polypeptide or RNA mutation, the methods of the invention can be used to treat an individual; For example, a human being, a sublimate of an ophthalmic disorder, where the transformed cells produce a therapeutic polypeptide or RNA sequence in an amount sufficient to treat an ophthalmic disorder. In some embodiments, the ocular cell transformation is enhanced by the use of rAAV particles (e.g., rAAVrhSR particles, rAAV22 etc.) comprising AAV capsid proteins comprising one or more amino acid substitutions that interact with HSPG (e.g., inhibit or By capturing HSPG binding () in some embodiments, rAAV particles exhibit low binding to HSPG; for example, they are lower than about 10%, 25%, 50%, 75%, 100% or any number in between. The binding of rAAV to HSPG is reduced from about 5% to about 100%, by

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About 10% to about 50%, by about 10% to about 30%, by about 25% to about 75%, by about 25% to about 50%, or by about 30% to about 50%.

[0201] An effective amount of rAAV (in some embodiments as particles) is administered, depending on the goals of the treatment. For example, where a low conversion percentage can achieve the desired therapeutic effect, then the treatment goal is generally fulfilled or this conversion level is skipped. In some cases, this conversion level can be achieved by converting only from about 1 to 5% of cells. Targeted, in some models at least about 20% of the cells of the desired tissue type, in some models at least about 50%, in some models at least about 80%, in some models at least about 95%, in some models at least about 99% % Of cells of the desired tissue type. As previously discussed, substitution of one or more amino acids of the rAAV capsid that interacts with HSPG improves the conversion of 38,827. As a guide, the number of particles given per injection generally ranges from about 6 10x1 to about 14 x 10 x 14 particles, between about 7 10x1 and 1071 13 particles. Between about Oxl 9 10x1 12 particles, or about 11 10x1 particles, the rAAV formulation can be administered by one or more subretinal injections, either during the same procedure or separately by days, weeks, months, or a year. In some embodiments, several vectors can be used to treat a human. [0202] In some embodiments, the administration of an effective amount of rAAV viral particles comprising rAAV capsid with one or more amino acid substitutions that interact with HSPG converts photoreceptor cells into or near From the site of giving. In some embodiments, more transfers are made around 5%, 10%, 15%, 20%, 25%, 30%, 35%,

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40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 1 00% of the photoreceptor cells. In some embodiments, about 5% is converted to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30%. To about 50% of photoreceptor cells. Methods for identifying photoreceptor cells transformed by AAV viral particles comprising rAAV capsid with one or more amino acid substitutions that interact with HSPG are known in the art; For example, immune tissue chemistry or the use of styluses, for example, an enhanced green fluorescent protein can be used to detect transduction Viral particles comprising rAAV capsid with one or more amino acid substitutions that interact with HSPG.

[10203 In some embodiments of the invention, methods involve the subretinal administration (eg, subretinal space) to the breasts of an effective amount of AAV viral particles comprising rAAV capsid with one or more amino acid substitutions that interact with HSPG to treat an individual with the disorder the eyes; For example, a person with an eye disorder. In some embodiments, the composition is injected into one or more subretinal sites to allow expression of heterozygous DNA in photoreceptor cells. In some embodiments, the composition is injected into any one of one, two, three, four, five, six, seven, eight, nine, ten or more than ten subretinal sites.

10204 In some embodiments the rAAV viral particles comprising the rAAV capsid are administered with one or more amino acid precipitates interacting with the HSPG to more than one site concurrently or sequentially. In an embodiment, rAAV viral particles are not injected as many times

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Exceeding more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours or 24 hours apart.

Injection routes into the vitreous

10205 The general method for vitreous injection can be illustrated by the following brief illustration. This example is intended only to illustrate certain features of the method, and is in no way intended to be restrictive. The procedure for intravitreal injection is known in the field (see, for example,

S. (2013), XJ. and Al-Qureshi, et Retina 29 (7): 875-912 and Fagan, GA, Peyman. (CUn.Experime66t.OphthalmolA \ 251^٠5W-6

[0206] Briefly, the IVF subject can be prepared for the procedure by dilating the pupil, sterilizing the eye, and administering the anesthetic. Any suitable mydriatic agent known in the art can be used for pupil dilation. Adequate pupil dilation can be confirmed before treatment. Sterilization can be achieved by applying a sterile ophthalmic treatment, for example, a solution containing iodine eg Povidone (Betadine®) E3A • E1 A similar solution can also be used to clean the eyelids, eyelashes, and other nearby tissues (for example, the skin). Any suitable anesthetic may be used, for example lidocaine or proparacaine, at any suitable concentration. Anesthetic can be administered by any method known in the art, including without limitation topical drops, gels or gels, and application of the anesthetic under the conjunctiva. 10207 Before injection, a sterilized eyelid endoscope can be used to separate the eyelashes from the area. The injection site can be distinguished with a syringe. The injection site can be chosen based on the patient's lens. For example, the injection site could be 3-3.5 mm of the limbus in patients with a pseudo-lens or oculomatous, and 3.5-4 mm of the limbus in patients with a lens. The patient could look in the opposite direction of the injection site.

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109 X to 15 109 X 109,15 X to 20 109 X 109.20 X 25 X 109,25 * 109 to 30 * 109,30 X 109 to 50 X 109 or 50 X 109 to 100 X 109 units / milliliter of the transfer. In some embodiments, the viral titer of the combination is approximately anywhere from 5 * 109 to 10 109 X 109.10 X to 15 109 X 109 (15 X to 25 109 X, or 25 109 X to 50 X 109 units / milliliter of conversion. In some embodiments, the viral titration of the combination is at least that of about 1010 (7 x 6, 1010x5

1010 (20 x 10015 x 1010,11 x 1010 110 x 1010.9 x 1010.8 x

1010 X 1010,40 X 1010,30 X 1010,25 X, or 50 X 1010 units / milliliter of infection. In some embodiments, the viral caliber of the combination is at least that is from about 5 x 1010 to 6 1010 x 6, 1010 x to 7 1010 x 1010 (7 x to 8 x 1010.8 x 1010 to 9 1010 x 1010 (9 x to 10 1010 x) 1010 X to 11 * 1010,11 1010 X to 15 1010 X 1010 (15 X to 20 X 1010,20 1010 X to 25 1010 X 1010) 25 X to 30 X 1010 (30 X 1010 to 101040,101040 to 101050, Or 50 10 107 to 100 X 1010 units / mL of infection. In some embodiments, the viral titers of the combination are at least from about 5 1010 X to 10 1010 X 1010 (10 X to 15 X). 1010,15 X 1010 to 25 1010 X, or 25 X 1010 to 50 X 1010 units / milliliter of infection.

10209 In some embodiments, methods involve the administration of an individual (eg, subretinal and / or vitreous) into the eye (eg, a human) an effective amount of viral particles of the recombinant product comprising one or more substitutions.

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The amino acid at one or more sites that interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles administered to the individual is at least about any from 1 8 10 X to about 1 13 10 X copies of a kg genome of body weight. In some embodiments, the dose of viral particles administered to an individual is about right 101 8 to about 1 M 108 13 copies / kg genome of body weight.

10210 During injection, the needle can be inserted perpendicular to the sclera and directed to the center of the eye. The needle can be inserted so that its tip ends in the vitreous, looking from the subretinal space. Any suitable size known in the field can be used for injection. After the injection, the eye can be treated with a sterilizing agent, for example an antibiotic. The eye may also be rinsed to remove excess sterilization agent.

Retinal structure and method for determining the efficacy of rAAV transport

1 0021 The retina is known to contain several layers. Cell layers in the retina can include the inner modulator membrane, nerve fibers, ganglion cells, inner plexiform layer, inner nucleus, outer plexiform layer, outer nucleus, outer determining membrane, and receiving cell For light, the retinal pigment epithelium layers. The layer closest to the vitreous is the inner delimiting membrane. This layer can contain Müller cells, which is a class of glial cells. The layer of nerve fibers can contain neurons from the ganglion cells that make up the optic nerve. The ganglion cell layer can include cells Streptococcus and amacrine cells. The inner plexid layer can contain the nerve connections between the dendrites of the ganglionic and amacrine cells and the neurons of the bipolar cells. The inner nuclear layer can contain the nucleus of an elongated, bipolar cell, and the horizontal cells; the outer plexiform layer can contain the neural connections between

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Horizontal cells are dendritic, photoreceptor cell and cell projections. The outermost nucleus layer can contain photoreceptor cell bodies. The inner marker membrane or the outer marker may include cell connections, for example adherent and bonding junctions, between the Müller cell apex processes and between these processes and the internal divisions of the photoreceptor cell. The photoreceptor cell layer, also known as the rod layer, cone cells, and Jacob's membrane, and photoreceptor cells can contain rod cells and cone cells. The retinal layer most distant from the vitreous is the retinal pigment epithelium (RPE), which may include the hexagonal epithelial cell layer containing the pigment granules.

[0212] The retina is also known to contain many different types of cells. Retinal neurons can include photoreceptor cells, bipolar cells, ganglion cells, amacrine cells, and horizontal cells. The photoreceptor cells are sensitive to light. They may sense light and respond by sending signals to the optic nerve through bipolar cells and ganglion cells. Photoreceptors may include rod cells, which generally sense subduction in low-light conditions, and cone cells, which generally sense color and perception of brighter light. Bipolar cells may receive input from photoreceptors and neural connections on elongated cells or ganglion cells . Streptococcus cells can receive information from amenorphs or horizontal cells, and their neurons can form the optic nerve. Horizontal cells can supplement input from multiple photoreceptors and help control light levels. Amorphous cells form cells between neurons that help in regulating cells bilaterally The electrode provides an input to the ganglion cells. may be

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Reticulocyte glial cells include Müller cells, astrogliosis, and microglia.

10213 The efficacy of rAAV transport by subretinal or intravitreal injection may be monitored by several parameters as described in the present application. For example, after treatment of a subject using the methods of the present invention, the subject may be evaluated, for example, to ascertain improvement, fixation, and / or delay in the development of one or more signs or symptoms of a pathological condition by one or more clinical variables including: In it those described in the current application. Examples of such tests are well known in the art and include practical as well as subjective procedures (for example, what the subject provides). For example, to measure the effectiveness of a treatment on a subject's vision function, one or more of the following may be evaluated: The subject's subjective vision state or improved central vision function (for example, improvement in the subject's ability to read fluently and recognize faces), the subject's visual movement (for example, a decrease in the time required to navigate a maze), visual acuity (for example, An improvement in the subject's LogMAR score), fine visual field measurement (for example, improvement in the subject's dB score), dark-adapted field of vision (for example, improvement in the subject's dB score), micro-matrix mapping (for example , Improvement in the subject dB score ), Goldmarm visual field measurement (for example, reduced dark area size (i.e. areas of blindness) and improvement in ability to resolve smaller targets), optical flicker sensitivity states (e.g. improvement in hertz), autofluorescence, and blasphemy phenological measurements ( For example, improvement in the ERG. 5) In some embodiments, vision function is measured by the subject's visual movement. In some embodiments, vision function is measured by the subject's visual acuity. In some embodiments, it is measured

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Vision function by measuring the precise field of view. In some embodiments, the vision function is measured with a dark-adapted field of vision (DEF) measurement. In some embodiments, vision function is measured by the ERG (some embodiments, vision function is measured by the subject's subjective vision quality.

10214 In the case of diseases resulting from a progressive decline in the function of vision, treatment of the subject at an early age not only slowed or stopped the progression of the disease, but could also reduce or prevent loss of vision function as a result of acquired amblyopia. Amblyopia can be of two types. In studies in nonhuman primates and cats raised in complete darkness from birth until a few months of life, animals even after subsequent exposure to light are functionally irreversible despite functional signals being transmitted by the retina. And this blindness occurs because the nerve connections are stopped and the cortex education gradually from birth as a result of the cessation of the stimulus. It is not known whether this function can be restored or not. In the case of diseases related to retinal degeneration, normal visual cortical circuits or in an appropriate evolutionary manner were first initiated to the point at which the degeneration formed a major impairment of function. The loss of the stimulus of vision in terms of sending a signal in the affected eye that constitutes a defective function or learned (acquired amblyopia), which results in the aging of the brain to cut off the signals, or use that eye. In these cases of acquired amblyopia, it is not known whether, with enhanced signal transmission from the retina, as a result of gene therapy to the opaque eye, it could result in the acquisition of more normal function in addition to slowing the progression or stabilization of the disease. In some embodiments, a sick person is less than 30 years of age. In some embodiments, the treated human is less than 20 years of age; in some embodiments, the treated human is less than 18 years of age. In some embodiments, the treated human is less than 15 years of age. In some embodiments, a curative is less

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From 14 years of age. In some embodiments, the treated human is less than 13 years of age. In some embodiments, the treated human is less than 12 years of age. In some embodiments, the treated human is less than 10 years of age. In some embodiments, a machete is less than 8 years of age. In some embodiments, the treated human is less than 6 years of age.

[0215] In some eye disorders, there is a pathogen-cell phenomenon, in which an improvement in the function of one cell type leads to an improvement in another function. For example, the conversion of the RPE to the central retina by rAAV of the invention could then improve the function of the rods, and in turn, it follows. On improving penis function, improving cone function. Therefore, treating one cell type may lead to an improvement in function in another

[0216] The selection of a particular rAAV vector and combination depends on a number of different factors, including, but not limited to, the human medical history and the characteristics of the condition and the individual being treated. Ultimately it is the responsibility of the attending physician to assess such features and design an appropriate treatment regimen.

[0217] In some embodiments, the person being treated has a hereditary eye disorder, but has yet to show any clinical signs or symptoms. In some embodiments, the person being treated has an eye disorder. In some embodiments, the person to be treated has shown one or more signs or symptoms of an ophthalmic disorder.

8 1 102 Non-exhaustive examples of eye disorders that can be treated by the systems and methods of the invention include: early acute autosomal retinal degeneration (Leber's annular latent), congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone malnutrition, inflammation Retinitis pigmentosa, X-ray retinal cleavage, Usher syndrome, age-related macular degeneration, age-related atrophic macular degeneration, neovascular AMD, myopathy

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Diabetic macula, proliferative diabetic retinopathy (PDR), cystic macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, posterior uveitis, choroid degeneration, Leber's hereditary optic neuropathy.

9 1 102 The compositions from the invention (eg, AAV viral particles comprising subretinal transport or vitreous administration of an AAV capsid may be used with one or more amino acid substitutions that interact with HSPG or at one or more of the homologous sites of 484 amino acids). 487, 532, 585 or 588) either alone or in combination with one or more additional therapeutic agents for treating eye disorders. The interval between sequential administration may be at least in light (or, alternatively, less than a minute, hours , Or days.

[0220] In some embodiments, one or more additional therapeutic agents may be administered subretinal or vitreous (for example, by administration into the vitreous). Non-exhaustive examples of an additive therapeutic agent include polypeptide of neurotrophic factors ( For example, EPO, PEDF, FGF2, BDNF, CNTF, GDNF), polypeptide antiangiogenic agents (eg, sFlt, angiostatin, indostatin), anti-angiogenic nucleic acids (for example, miRNA, siRNA, ribozyme) Acids, for example Anti-angiogenic anti-VEGF nuclei, anti-angiogenic morpholines, eg anti-angiogenic morpholines, VEGF, antiangiogenic antibodies and / or antibody fragments (eg, Fab fragments), eg antibodies Anti-angiogenic and / or anti-VEGF antibody fragments.

Ways to transfer the CNS

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9 1 102 In some embodiments, the administration of an effective amount of viral particles to the recombinant product (for example, AAVrhSR particles, AAV2, etc.) includes one or more amino acid substitutions at one or more sites that interact with the heparan sulfate proteoglycan from Current detection of neuronal transduction (for example, striated ganglion neurons, eg spinal neurons) at or near the site of administration. In some embodiments, more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, are converted. 70, 75% or 100% of neurons. In some embodiments, about 5% is converted to about 100%, about 10% to about 50%, about 10% to about 30%, about 25% to about 75%, about 25% to about 50%, or about 30%. To about 50% of the nerve cells. Methods of identification of neurons transformed by viral particles of recombinant gene recombinant DNA are known in the art; For example, immune tissue chemistry, RNA detection (for example, RNA-seq, Northern blotting, qPCR, in situ hybridization, etc.) or the use of a co-expressed marker.For example, the enhanced green fluorescent protein can be used to detect expression. .

[0220] In some embodiments of the invention, the methods involve administering to the mammalian brain an effective amount of viral particles of recombinant product viral particles of recombinant product comprising one or more amino acid replacements in one or more locations where It interacts with heparan sulfate proteoglycans from the current disclosure for the treatment of a mammalian, eg, human. In some embodiments, the combination is injected into one or more sites in the brain to allow the expression of heterozygous DNA from the current detection in at least neurons. In some embodiments, this is done

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Injecting the combination into any one of one, two, three, four, five, six, seven, eight, nine, ten or more than ten locations in the brain. In some embodiments, the composition is injected into the striated node. In some embodiments, the composition is injected into the dorsal striated ganglion. In some embodiments, the composition is injected into the endometrium. In some embodiments, the composition is injected into the caudate nucleus. In some embodiments, the composition is injected into the endocardium and into the caudate nucleus. In some embodiments, recombinant viral particles comprising one or more amino acid replacements at one or more sites that interact with heparan sulfate proteoglycan are administered to the individual's CNS by stereotaxic injection, for example to the striated node. In some embodiments, recombinant viral particles comprising one or more amino acid replacements at one or more sites that interact with heparan sulfate proteoglycans are administered to the individual's CNS by convection-enhanced transport (CED); For example CED to scrambled node.

[0221] The administration of rAAV particles can be performed by several pathways. In some embodiments, administration includes direct injection into the spinal cord and / or intracerebral administration. In some embodiments, administration is in a selected location of the brain, the medulla, the arch, the cerebellum, inside the cranial cavity, the periocerebellar meninges, the dura mater, the arachnoid mother, the pia mater, the cerebral fluid (? ^ Of the subarachnoid space surrounding the brain, the deep nuclei of the cerebellum , The ventricular system of the brain, for the subarachnoid space, striated ganglia, cerebral cortex, cerebral septum, hypothalamus, hypothalamus, and parenchyma.In some embodiments, administration includes injection of a ventricle into the brain into at least one cerebral lateral ventricle. In some embodiments, administration includes intrathecal injection into the cervical, thoracic, and / or lumbar region. In some embodiments, the administration includes

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Injections into the striatum. In some embodiments, administration includes injection into the hypothalamus. The various techniques and devices suitable for these administration pathways are described in the present application, for example, CED and / or stereotaxic injection.

10222 In some embodiments, recombinant viral particles comprising one or more amino acid replacements are administered in one or more loci that interact with the heparan sulfate proteoglycan in one half of the brain. In some embodiments, the viral particles of the recombinant product are administered into the two hemispheres.

10223 In some embodiments, recombinant viral particles that comprise one or more amino acid substitutions are administered at one or more sites that interact with heparan sulfate proteoglycans at more than one site simultaneously or sequentially. In an embodiment, multiple injections of viral particles of the recombinant product are no more than one hour, two hours, three hours, four hours, five hours, six hours, nine hours, twelve hours or 24 hours apart.

In some embodiments, the invention presents a method for treating a human with a CNS disorder by administering an effective amount of a pharmaceutical formulation comprising viral particles of the recombinant product comprising one or more amino acid substitutions in one or more sites that interact with the proteoglycan heparan sulfate. From the current disclosure for treating a disorder in 5 to 0. In some embodiments, the pharmaceutical composition includes one or more pharmaceutically acceptable excipients.

[0225] Generally, it can be transferred from about 1 μl to about 1 ml of the formulation of the invention (for example, from about 100 μl to about 500 μl of

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Combination). In some embodiments of the invention, the viral particle size of the recombinant product comprising one or more amino acid substitutions at one or more sites interacting with heparan sulfate proteoglycans injected into the striated node is about or more than about any one in 1 μm. Liter, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 L, 400 ميL, 500L, 600 ميL, 700L, 800L, 900 ميL, or 1mlL, or any quantity in between.

In some embodiments, the first volume of viral particles of the recombinant product that includes one or more amino acid primers is injected into one or more sites that interact with heparan sulfate proteoglycans in a first region of the brain, and the second volume is injected from a variant of the RAAV particles in the second region of the brain; for example, in some embodiments, the first volume of heterologous rAAV particles is injected into the caudate nucleus, and the third volume of the composition is injected into the endothelium. In some embodiments, 1 X rAAV particle size from the variant image is injected into the caudate, and 1.5 X 3.5, X 3, X 2.5, X 2, X, or 4 X rAAV particle size from the variant image is injected into the lining, where »is A volume of approximately 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, or more than approximately any one of 1 μL, 2 μL, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl. Μl, 700 μl, 800 μl, 900 μl, or 1 ml liter, or any quantity in between

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10227 The compositions of the invention (for example, viral particles of the recombinant product comprising one or more amino acid replacements at one or more sites interacting with heparan sulfate proteoglycans from the present disclosure) may be used either alone or in combination with one. One or more additional therapeutic agents to treat a CNS disorder (eg, 10). The interval between sequential administration may be at least (or, alternatively, less than) minutes, hours, or days.

00228 In some embodiments, the methods include administering to the CNS an effective amount of recombinant viral particles comprising one or more amino acid substitutions at one or more sites interacting with heparan sulfate proteoglycans comprising a vector encoding heteronuclear acid. In some embodiments, the viral titer of the combination is at least approximately that of 105 12,106 12,7 10 X 12,9 10 X 12,8 10 X

X 12,25 10 X 1220 10 X 12,15 10 X 12,11 10 X 12,10

12.30 X 12 10, or 50 12 X 10 copies / mL of the genome. In some embodiments, the viral titer of the combination is about 12,05 121 to 6 107 12,12106 to 7&gt; 12,710 10 12 to 12 10 X 12,8 10x8 to 10 χ12,9 10x9 12 to 10 1210 * 12,10 10 X to 1011 12,11 12 10 X to 15 X 10 12,15 * 10 12 to 20 12 10 X 12,20 10 X to 25 X 12, 25 10 X 10 12 to 30 12 10 X 30, 12 10 X to 50 12 10 X, or 50 12 10 X to 100 M 0 1 12 milliliter copies of the genome. In some embodiments, the viral titre of the combination is approximately any of 105 12 To 10 10 12,10 12 10 X to 25 12 10 X, or 25 12 10 X to 50 12 X 10 copies / ml of genome. In some embodiments, it reaches the caliber

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The viral combination of at least about any of 5 109, 6 A 7) 109 7 8'109 '

X 109 (25 X 109 (20 X 109) 15 X 109 (11 X 109,10 X 109) 9

109,30 X 109, or 50 X 109 units / milliliter of conversion. In some embodiments, the viral titer of the combination is approximately anywhere from 5 109 x 6 109 x 6 (109 x 7) 109 109.7 to 8 109 x 109 (8 x 9 109 x 109 x 9 to 10 109 10 109 x 10 x 10). 11 to 109 X 109 (11 X to 15 109 X 109) 15 X to 20 109 X 109.20 X 25 to 109 X 109.25 X 30 to 109 X 109 (30 X to 50 109 X 109 or 50 X 109 to 100 109 X units / milliliter of conversion. In some embodiments, the viral titer of the combination is approximately any from 5 109 X to 10 X 109 (10 X 109 to 15 109 X 109,15 X to 25 109 X, or 25 109 X to 50 X 109 units / milliliter of conversion. In some embodiments, the viral titer of the combination cancels at least that of about 5 x 1010,6 1010 (10 x 1010) 9 x 1010 (8 x 1010.7 x).

1010 (30 × 1010) 25 × 1010 (20 × 1010) 15 × 1010 (11

1010.40 m 10 108, or 101050 units / mL of infection. In some embodiments, the viral titer of the combination is at least from about 5 1010 X 6 X 6 (1010 X 1010 to 7 1010 X 101 0.7 X Y 8 1010 X 1010 (8 × 1010) 9 1010 × 10 × 1010,10 1010 × 11 1010 × 1010 (11 × 1010 × 1010) 15 × 1010 × 1010 (15 × 1010 × 1010) 20 × 20 × 1010 (25 × 25 1010 to 30 1010) X 1010,30 X 40 X 1010,40 X 1010 X 50 X 1010, OR 50 X 1010 X 100 X 1010 UNIT / MML of infection. In some embodiments, the viral titers of the combination are at least from about 5 1010 X to 10 X

<img file="MA39439B2_D0009.tif" />

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1010.10 X 1010 to 15 1010 X 101 0.15 X 25 1010 X, or 25 X 1010 to 50 B 1010 units / mL of infection

[0229] In some embodiments, the methods involve administering to the CNS of an individual (for example, a human) an effective amount of viral particles of recombinant product comprising one or more amino acid substitutions at one or more locations that Interact with heparan sulfate proteoglycans. In some embodiments, the dose of viral particles administered to an individual is at least about any from 1, 10 to about 1 13 10 x copies / kg genome of body weight. In some embodiments, the dose of viral particles administered to an individual is approximately any from 1 X 10 to about 1 X 10 copies / kg body weight.

10230 In some embodiments, the methods involve administering to the CNS of an individual (for example, a human) an effective amount of viral particles of the recombinant product that includes one or more amino acid substitutions at one or more sites that interact with proteoglycans sulfate. Heparan to an individual. In some embodiments, the total viral particles are given to an individual at least about 1 14 10 X to about 1 14 10 X genome. In some embodiments, the total viral particles are given to an individual about any from 1 9 10 X to about 1 14 10 X Genomic version.

5. Expression constructs

In some embodiments, the invention provides methods for transporting heterologous DNA into the eye by subretinal transport of a rAAV vector comprising heteronuclear acid and wherein the rAAV vector is encapsulated in the rAAV capsid (for example, rAAV2 capsid, rAAVrhSR capsid, etc.) comprising one or more Some embodiments of the amino acid substitutions that interact with HSPG are presented

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The invention methods for treating a perturbation in a person’s CNS that include transfer of a combination comprising rAAV particles to the individual’s CNS, wherein rAAV particles comprise (A) rAAV capsid comprising rAAV capsid protein comprising one or more amino acid substitutions at one or more loci. That interacts with heparan sulfate proteoglycans, and (b) the rAAV vector comprising heteronuclear DNA and replicating at least one AAV terminal. In some embodiments, heterozygous DNA (eg, a transgenic) is operationally linked to a promoter. Representative enhancers include, but are not limited to, the early CMV promoter, MoMLV LTR, RSV LTR, promoter. Phosphoglycerinase-1 (PGK), monkey virus booster 40 (SV40) and CK6 booster, transethyrin (TTR) booster, TK booster, tetracycline-responsive (TRE) booster, HBV booster, hAAT booster, LSP booster, chimeric liver boosters (LSPs) , E2F booster, telomerase booster (hTERT); Enhancer cytomegalovirus / chicken enhancer Beta-actin / rabbit 3-globin (CAG promoter; 10 8 (2): 193-9, 1991, GeneiNiwa et al) and a 1-alpha-prolonging factor booster (1T1b1-alpha) (.91 (2): 217-23 and Guo et al., 1990, Gene, Kim et al. 3 (9): 802-10, 1996, GeneTher). In some embodiments, the enhancer includes a human 9-glucorinidase promoter or a CMV enhancer associated with an 8- promoter. Chicken Actin (018). It can be a structural reinforcer, an inductor or a suppressor. In some embodiments, the enhancer is able to express the variable DNA in an eye cell. In some embodiments, the promoter is able to express heterozygous DNA in photoreceptor cells or RPE (models, the promoter is a rhodopsinkinase promoter (RR); for example, a human RK promoter. In some embodiments, a promoter constitutes an opsin promoter; for example, Human opsin booster or rat opsin booster.

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The present invention foresees the use of the viral genome of the recombinant product to insert one or more DNA sequences encoding a therapeutic polypeptide and / or DNA for a rAAV fusion particle packing comprising one or more amino acid substitutions that interact with 11508. The genome can comprise Viral recombinant recombinant on any component to stabilize expression of therapeutic polypeptide and / or DNA, for example, enhancer, ITR, ribosome binding element, terminator, enhancer, selector stinger, intron, polyA signal, and / or origin Transcription. 10233 In some embodiments, the rAAV vector is a self-completing rAAV vector, for example one that includes a self-completing recombinant genome (the term self-completing recombinant genomes may be used interchangeably in the present application). AAV viral particles are described with autologous genomes. Methods for using self-executing AAV genomes in patent applications Fig

7,790,154; 7,846,729; 8,093,054; And 8,361,457; et al., Wang Z,

102105-2111 each, (2003) Gene Ther which is integrated into the current application as a reference with its loader. RAAV comprises a self-completing genome that rapidly forms a double-stranded DNA molecule thanks to its partially complementary sequences (for example, complementing the coding and non-coding of the transgenic strands). In some embodiments, the first heterochromic DNA sequence and the second heterochromic DNA sequence are coupled by a mutagenic ITR (for example, the right ITR). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heteronuclear DNA and a second nucleic acid sequence encoding a complementary nucleic acid, where the first nucleic acid sequence can form a base pair in the strand with a second nucleic acid sequence of most or all of its length.

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10234 In some embodiments, the first heterochromic DNA sequence and the second heterozygous DNA sequence coupled by a mutated ITR (eg, right ITR) are linked. In some embodiments, ITR includes the polynucleotide sequence 5'CACTCCCTCTCTGCGCGCTCGCTCGCTCACTACTGAGGCCGGGCGACCAAG

3 - CCCACGCCCGGGCTTTGCCCGGGCG '(Sequence ID No .: 8). The mutagen includes the deletion of a D region that includes the peripheral resolution mapping. As a result, upon transcription of the 887 viral genome, the rep proteins of the viral genome will not cleave into mutated ITR and as such, the viral genome of the recombinant gene recombinant product comprising the following will be packed in a 5 'to 3' order in a viral capsid: AAV ITR, urinary metaplasia The first heterozygous nucleotide including the upregulated sequences, mutant ITR 8867, the second heterozygous polynucleotide in an inverted direction on the first heterozygous polynucleotide and AAV ITR third.

6. Viral particles and viral particle production methods. Viral particles for the rAAV vector

[0235] In some embodiments, the invention provides methods for transporting heterologous DNA into the eye by subretinal transport of a rAAV vector comprising heteronuclear DNA and wherein the rAAV vector encapsulated in the rAAV capsid (eg, rAAVrh8R, rAAV2, etc.) comprises one or More than 110 amino acid substitutions interact with 110. In some embodiments, the invention presents methods and dentures related to the transport of rAAV particles into the CNS apparatus of an individual.

In some embodiments, the rAAV particle includes the rAAV vector. In some embodiments, the viral particle is a recombinant AAV particle comprising of nucleic acid comprising a heteromorphic gene surrounded by one or two inverted terminal repeats.

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ITRs (AAVs) - the DNA is encapsulated in the particle 887. The AAV particle also includes capsid proteins. In some embodiments, the DNA includes the coding sequence (s) of interest (for example, a heterogeneous transgenic) components that are operationally related in Transcriptional orientation, control sequences including contraption and termination of transcription sequences, and thus the formation of discrete expression.

10237 Reduced expression at terminals 5 'and 13' is enclosed by one on top of the two functional AAV ITRs sequences. The term functional AAV ITRs is intended to function as the sequence of ITRs according to its determinant to save, transcribe and mobilize Veron 887. See., 2000, PNAS, Davidson et al 323428 (7) 97;.! J. Virol., Passini et a; 2003, 40. -7034: (12) 77; Gene, and Pechan et al 16: 10-16, 2009, Ther. All have been slashed in the current application for download for reference. To implement some aspects of the invention, recombinant product vectors include at least all AAV sequences necessary for encapsulation and physical structures of infection by rAAV. ^ 8 ^ AAV for use in vectors of the invention shall not include an untreated nucleotide sequence (for example, as described in 5: 793-801, 1994, Hum.Gene Ther.iKotin), and can be altered by insertion, deletion or substitution of nucleotides, or AAV ITRs may be derived from any of the many 887 serotypes. There are more than 40 serotypes of AAV currently known And it continues Definition of a new serotype and heterotypes of an existing serotype. See 99 (18): 2002, PNAS, Gao et al 1854-6 l; 100 (10): 6081-6, 2003, PNAS, Gao et al; &amp;. 2003, J. Virol., Bossis et al, 6799-810: (12) 77.

10238 The use of any serotype of AAV is being studied within the scope of the present invention. In some embodiments, the rAAV vector is an AAV serotype derivative vector, including without limitation, AAV.

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ITRs are AAVrh8, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV2, AAVl ,, AAV DJ, AAV2R471A, AAV12, AAVll, AAVrhlO, AAVIO, AAV9, AAVrh8R

AAV for goat, AAV bovine, or AAV ITRs mouse or similar. In some embodiments, the DNA in AAV that includes ITRs for AAV ITRs is AAV4, AAV3, AAV2, AAVl, AAVrhlO, AAVIO, AAV9, AAVrh8R, AAVrh8, AAV8. 8875, AAV7, AAV6

88711, AAV, AAV DJ, AAV2R471A, AAV12 goat, AAV bovine, or AAV mouse or similar. In specific embodiments, the DNA in AAV comprises 88272 ITR. According to previously described, rAAV particles may additionally comprise a capsid comprising the rAAV capsid protein comprising one or more amino acid substitutions at one or more sites that interact with Proteoglycan heparan sulfate.

[0239] In some embodiments, the vector may include DNA stuffer (some embodiments, say that it encodes for DNA stuffer is a fluorescent green protein. In some embodiments, the DNA stuffer may be located between the reinforcer and the DNA that encodes RNAi.

10240 The different AAV serotypes are used to improve the conversion of specific target cells or to target specific cell types within a specific target tissue (eg, CNS tissue) .0G The rAAV particle includes viral proteins and viral nucleic acids of the same serotype or mixed serotype. For example, In some embodiments the rAAV particle may comprise the AAV2 capsid proteins of the invention and at least one AAV2 or may include AAV2 capsid proteins and at least one AAVl ITR. Any combination of AAV serotypes is provided. To produce the rAAV particle in the current order as if each combination had been explicitly specified in the present order. In some embodiments, the invention presents rAAV particles comprising a capsid

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AAV2 of the invention. In some embodiments, the invention introduces rAAV particles comprising the capsid 886711180 of the invention.

AAV particle production

00241 Various methods are known in the art for the production of rAAV vectors, including transmission of infection, stable-cell lineage production, and infectious hybrid virus production systems comprising adenoma-887 hybrids, virus hybrids ^^ - 0 1997 (J. Virology, JE et al.) )

And Bacovirus-887 hybrids.All rAAV cultures to produce rAAV particles require the following; 1) cells of a suitable family, including, for example, human-derived cell strains e.g. 549, HeLa, or 293 cells; Or cell strains derived from insects, for example 9-SF, in the case of Pneumovirus production systems; 2) a suitable adjuvant virus function, provided by an untreated or mutagenic adenoma (e.g. a temperature-sensitive adenoma), herpesvirus, bacovirus, or a plasmid structure providing adjuvant functions; 3) Replication of AAV, cap genes, and metabolites Gene; 4) A modulating gene (e.g., a therapeutic axis gene) surrounded by at least one AAV of sequence ITRs; And 5) suitable media and media components to support rAAV production. Suitable media known in the industry may be used to produce 3887 vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Dulbecco's Modified Eagle Medium (DMEM), (MEM). Modified Eagle Medium that

And regular formulas such as those described in US Patent No. 6,566,118 and Sf-900 II SFM medium according to the description in US Patent No. 6,723,551, both of which have been incorporated in the present application as a reference in their entirety, especially with regard to common media formats For use in the production of recombinant AAV vectors.

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RAAV particles can be produced using methods known in the art; see, for example, US Patent Numbers 6,566,118; 6,989,264; When implementing the invention, the host cells to produce rAAV particles include mammalian cells, insect cells, plant cells, microorganisms and yeast. The host cells can also be the synaptic cells in which the AAV replication and cap gene are preserved in the cell. The family or progenitor cells in which the AAV vector genome is maintained in stable R. Fill cells and progenitor cells are formed Prototypes are derived from 293, Α549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

10243 In some embodiments, rAAV particles can be produced by the TDF method, for example the TDF method presented below. Briefly, it is possible to infect a plasmid containing the rep gene and a capsid gene, in addition to the adenovirus plasmid (for example, using the phosphate method). Calcium) in the cell lineage (for example, 293-cells), and the virus can be pooled and optionally purified.

In some embodiments, rAAV particles can be produced by the generative cell strain method, for example the method of producing cell lineage is presented below (see also (reference reference in Martin et. Al; 269-24: 253 2013) Human Gene Therapy Methods)). , The infection can be transferred to the cell lineage (eg, HeLa cell lineage) stably using a plasmid containing the rep gene, capsid gene, and the reinforcer sequence - the transgenic gene. Cell strains can be examined to select the leading clone to produce rAAV, which can then be expanded To produce a bioreactor and transport Infection to it by using an adenoma (for example, an untreated adenoma) as an aid to initiate production

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The virus can be aggregated in the next step, the adenoma is not inactivated (for example, by heat) and / or removed, and the rAAV particles can be purified.

10245 In some embodiments, a method is presented to produce any rAAV particle according to what has been disclosed in the present application that includes (a) culturing a host cell under conditions in which rAAV particles are produced, wherein the host cell comprises (1) one or more bundle genes. AAV, where each mentioned AAV package gene encodes AAV transcription and / or protein encapsulation; (2) a precursor rAAV vector comprising DNA encoding a therapeutic polypeptide and / or DNA as described in the present application enclosed in at least one AAV ITR, and (3) an AAV adjuvant function; And (b) Extraction of rAAV particles Produced by the host cell. In some embodiments, at least one AAV ITR mentioned is selected from the group consisting of AAV ITRs namely AAVl, AAVrhSR, AAVrhS, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV2.

8879, AAV DJ, AAV2R471A, AAV12, AAVll, AAVrhlO, AAVIO, from AAV goat, AAV bovine, or from AAV mouse or similar. In some embodiments, said protein encapsulation includes one or more amino acid substitutions at one or more sites that interact with heparan sulfate proteoglycans; in some embodiments, the AAV2 protein encapsulation is the protein encapsulation. In some embodiments, the protein encapsulation is the encapsulation of the AAVrhSR protein.

10246 A suitable rAAV production culture media of the present invention may be supplemented with serum or recombinant serum-derived proteins at a level of 0.5% -20% (v / v or w / v). Alternatively, according to what is known in the art, it is possible. Production of rAAV vectors under serum-free conditions may also be referred to as media without animal-derived products. A person of ordinary skill in the art will realize that they can also complete the circles

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Commercial or conventional designed to support the production of rAAV vectors with one or more of the industry-known cell culture components, including without limitation, glucose, vitamins, amino acids, and / or growth factors, to increase rAAV titers in production cultures.

[0247] rAAV production farms can be grown under a variety of conditions (over a variety of temperatures, for various lengths of time, etc.) suitable for the particular host cell to be used. According to what is known in the art, rAAV production farms include certified farms On conduction they can be cultured in suitable conduction-dependent vessels eg, for example, cylindrical flasks, hollow fiber filters, fine carriers, and fluidized bed or packed bed bioreactors. The rAAV production culture conveyor may also include host cells conditioned with suspension. For example, the SF-9j, 293, HeLa cells that can be cultured in a variety of ways including, for example, flasks with a spinning rotor, stirred tank bioreactors, and single-use systems such as a Wave bag system

[0248] The rAAV vector particles of the invention may be harvested from rAAV production cultures by lysis of host cells of the production farm or by harvesting of spent media from the production farm, provided that the cells are cultured under conditions known in the art to release rAAV particles into media of intact cells, as described. (In more detail in US Pat. No. 18 1, 6,566). Suitable cell analysis methods are also known in the art and include, for example, multiple freezing / thawing cycles, exposure to sound waves, micro-liquefaction, and substance therapy. Chemicals, eg detergents and / or protease enzymes.

10249 In a further embodiment, the rAAV particles are purified. The term purified according to its use in the present application includes rAAV particle preparation abstracted from at least some of the components

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Others can also be present where rAAV particles are naturally occurring or are prepared primarily. Thus, for example, isolated rAAV particles can be prepared using a purification technique to enrich them from a source mixture, for example a farm dissolution product or a floating solution of a production farm. Enrichment can be measured. In several ways, for example, by proportioning DNase-resistant particles (DRPs) or genomic copies (gc) present in solution, or by infection, or can be measured with respect to the second, and is likely to interfere with the material in the source mixture. For example Contaminants, including production farm pollutants or pollutants in the process, including helper virus, media components, etc.

10250 In some embodiments, rAAV production culture harvest is purified to remove host cell fragments. In some embodiments, the production farm harvest is filtered through a series of depth filters including, for example, a DOHC Millipore Millistak + HC Pod Filter, an AIHC Millipore Millistakt HC Pod Filter, and 0.2 μm. M of 1311. * can Opticap XLIO Millipore Express SHC Hydrophilic Membrane Filter Membrane

Leaching is also achieved by a variety of other techniques known in the art, for example, centrifugation or filtration through any cellulose acetate filter for 0.2 μm or larger pore size known in the art.

0251 In some embodiments, the rAAV production farm harvest is additionally processed with Benzonase® to digest any high molecular weight DNA present on the production farm. In some embodiments, Benzonase® is digested under standard conditions known in the art including, for example, a final concentration of 1-2.5 units / mL of Benzonase® at a temperature ranging from ambient temperature to 37 ° C for a period of time. 30 minutes to several hours.

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RAAV particles can be isolated or purified using one or more of the following purification steps: Balanced centrifugation; Anion exchange filtration through flow Tangential flow filtration (TFF) for rAAV particle concentration; RAAV capture by apatite chromatography; The thermostatic shutdown of auxiliary virus And rAAV capture by non-hydrophilic reaction chromatography; Exchange of buffer solution with volume exclusion chromatography (SEC); Nanometric filtration; And rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in a variety of combinations, or in a different order. In some embodiments, the method includes all the steps in the order described below. Methods for purifying rAAV particles are found, for example, in Journal of Virology 72: 2224-2232, Xiao et al. US Patent Nos. 264,989,6 and 948,137,8; And International Application No. 2010/148143 wo.

Also in the present application are pharmaceutical compositions comprising the rAAV particle comprising heteronuclear acid encoding a therapeutic polypeptide and / or the therapeutic nucleic acid, wherein the rAAV particle comprising an rAAV capsid comprising one or more substitutions or amino acids that interact with HSPG, A pharmaceutically acceptable carrier. Pharmaceutical formulations may be suitable for any method of administration described in the current application. For example, by giving a subretinal.

[0254] In some embodiments, the pharmaceutical formulations comprise the rAAV described in the present application and the pharmaceutically acceptable carrier is suitable for administration to a human. Such carriers are known in the art (see, for example, 15th, Remington's Pharmaceutical Sciences 10.1035-103 8 and 1570-1580, Edition). In some embodiments, pharmaceutical formulations include the rAAV described in the application for a current and a pharmaceutically acceptable carrier that is Suitable for injection in ί

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Al Ain: The said pharmaceutically acceptable carriers can be sterile liquids, for example water and oil, including those of petroleum, animal, vegetable or synthetic origin, for example peanut oil, soybean oil, mineral oil, etc. Also, the use of saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions as liquid carriers, especially for injection solutions. The pharmaceutical composition may additionally include additional ingredients, for example preservatives, buffer solution, surfactants , Antagonists Oxidant and stabilizers, anionic wetting agents or laminating agents, viscosity increasing agents, etc. The pharmaceutical compositions described in the present order may be packaged in single dose units or in multiple dose forms. The formulations are generally formulated as a solution that is sterile and is highly surfactant.

7. Systems and Kits 10

RAAV formulations may be contained as described in the present application within a system designed for use in one of the methods of the invention as described in the present application.

Subretinal transport

0256 In some embodiments, the invention provides a system for transferring the subretina of the vector to the eye of the individual.

It comprises a) a formulation comprising an effective amount of rAAV particles, where 1) a protein capsid of 15 rAAV particles comprising one or more amino acid precipitants at one or more sites interacting with heparan sulfate proteoglycans, and 2) the carrier comprising an acid A heteronuclear that encodes a therapeutic or RNA polypeptide and replicates at least one AAV terminant; And b) a networked device for rAAV.

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[0257] Generally, the system includes a micro-perforated cannula, where the cannula has a size of 27 to 45, one or more syringes (for example, 1, 2, 3, 4 or more), and one or more fluids (for example. Example, 1, 3, 2, 4 or more) suitable for use in the methods of the invention.

10258 A micro-perforation cannula is suitable for the subretinal injection of the carrier suspension and / or other fluids to be injected into the subretinal space. In some embodiments, the cannula is on a scale of 27 to 45. In some embodiments, it is a cannula with a fine hole of a scale of 35-41. In some embodiments, the cannula is with a fine hole hole at a scale of 40 or 41. In some embodiments, it is a cannula with a fine hole of a gauge of 41. The cannula is any suitable type of cannula, for example, the de-Juan® or Egle® cannula.

[0259] The syringe may be any suitable syringe, provided it can connect to the cannula to transfer the fluid. In some embodiments, the syringe is an Accurus® system (some embodiments, the system includes a single syringe). In some embodiments, the system includes two syringes; in some embodiments, the system includes three syringes. In some embodiments, the system includes four or more syringes. The 10260 additionally includes a robotic injection pump, which may be energized by, for example, a foot pedal.

10261 Fluids suitable for use in the methods of the invention include those described in the present application, for example, one or more fluids each of which comprises an effective quantity of one or more vectors as described in the present application, and one or more fluids to form a primary bubble (For example, a saline or other suitable fluid), and one or more fluids that contain one or more therapeutic agents.

Fluids suitable for method use of the invention include those described in the present application, for example, one or more fluids each of which comprises an effective amount of

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One or more vectors as described in the present application, one or more fluids to form a primary bubble (for example, a saline or other suitable fluid), and one or more fluids comprising one or more therapeutic agents.

10263 In some embodiments, the volume of fluid comprising the effective amount of the carrier is greater than about 0.8 mL l. In some embodiments, the volume of fluid comprising the effective amount of the carrier is at least about 0.9 mL l. In some embodiments, the volume of fluid comprising the effective amount of the carrier is at least about 1.0 ml. In some embodiments, the volume of fluid comprising the effective amount of the carrier is at least about 1.5 ml. In some embodiments, the fluid volume comprising the effective amount of the carrier is at least about 1.5 ml. The carrier is at least about 2.0 mL. In some embodiments, the volume of fluid comprising the effective amount of the carrier is greater than about 0.8 to about 3.0 mL. In some embodiments, the fluid volume comprising the effective amount of the carrier is greater than about 0.8 To about 2.5 mL l. In some embodiments, The fluid volume comprising the effective amount of the carrier is greater from about 0.8 to about 2.0 mL. In some embodiments, the volume of fluid comprising an effective amount of the carrier is greater than about 0.8 to about 1.5 mL. In some embodiments, the volume of fluid comprising an effective amount of the carrier is greater than about 0.8 to about 1.0 mL l. In some embodiments, the volume of fluid comprising an effective amount of the carrier is about 0.9 to about 3.0 mL. In some embodiments, the volume of fluid comprising the effective amount of the carrier is about 0.9 to about 2.5 mL liters; in some embodiments, the volume of fluid comprising the effective amount of the carrier is about 0.9 to about 2.0 mL liters. In some embodiments, the volume of fluid comprising the effective amount of the carrier is about 0.9 to about 1.5 mL liters; in some embodiments, the volume of fluid comprising the effective amount of the carrier is about 0.9 to about 1.0 mL. In some embodiments, the volume of fluid comprising

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The effective amount of the carrier is about 1.0 to about 3.0 mL. In some embodiments, the volume of fluid comprising an effective amount of the carrier is about 1.0 to about 2.0 mL.

[0264] The pre-bubble formation fluid, for example, ranges from about 0.1 to about 0.5 mlL. In some embodiments, the total volume of all fluids in the system ranges from about 0.5 to about 3.0 mlL.

00265 In some embodiments, the system includes a single fluid (for example, a fluid containing an effective amount of a carrier). In some embodiments, the system includes two fluids. In some embodiments, the system comprises 3 fluids; in some embodiments, the system comprises 4 or more fluids.

10266 Systems of the invention may furthermore be packaged in kits, wherein the kits may additionally contain instructions for use. In some embodiments, the dentures further include a subretinal transport device for rAAV particle compositions. In some embodiments, the instructions for use include instructions according to one of the methods described in the present application. In some embodiments, the instructions for use include instructions for subretinal transport of rAAV particles that include a capsid with one or more amino acid substitutions that alter, reduce or eliminate the rAAV particle binding to HSPG.

Transfer to the CNS device

10267 The present invention provides kits for the transfer of heterologous nucleic acid to the CNS of an individual comprising a combination comprising rAAV particles, wherein the rAAV particle comprises (A) rAAV capsid comprising the rAAV capsid protein comprising one or more amino acid replacements in one or more of the Positions interacting with heparan sulfate proteoglycans, and (b) the rAAV vector comprising heteronuclear acid and repeating one upside-down terminal on

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the least. Also, in the present application, kits are provided for treating a CNS disorder in an individual comprising a combination comprising rAAV particles, wherein the rAAV particle comprises (A) rAAV capsid comprising rAAV capsid protein comprising one or more amino acid replacements in one or more. From the sites interacting with heparan sulfate proteoglycans, and (b) the rAAV vector incorporating heterozygous DNA for treating CNS disruption and at least one inverted AAV terminal replication.

Sets may include either the rAAV particle or the rAAV particle of combinations of the invention. For example, the ensembles may include rAAV particles with a rAAV capsid comprising the rAAV capsid protein comprising one or more amino acid substitutions at one or more sites that interact with the HEPRAN sulfate proteoglycans (for example, one or more of the rAAV capsid protein). Amino acid substitutions that reduce the binding of the rAAV particle to the proteoglycans of heparan sulfate, eg replacements at R585, 532, R487, R484 and / or R588, with a numbering based on VP1 for AAV2) and the rAAV vector includes heteronuclear acid and repeat at least one AAV inverted terminal.

[0269] In some embodiments, the kits also include a CNS transport device for the 3887 particle combination. The particles for the CNS transport (eg, for combination transport involving rAAV particles) are known in the field and may use a pump (eg, osmotic and / or An infusion pump, as described below) and an injection device (for example, a catheter, cannula, etc.). Optionally, imaging technology can be used to guide the injection device and / or monitor the transfer of the infusion material (for example, a combination containing 887 particles). CNS transport can include stereotaxic transport, Or by convection-assisted transport (CED), the injection device can be inserted into a tissue

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CNS in the submissive. An experienced person can determine the appropriate coordinates for placement of the injection device into the target CNS tissue. In some embodiments, positioning is done through an anatomical map obtained for example by CT and / or MRI imaging of the subject's brain to direct the injection device to the target CNS tissue.

[0270] In some embodiments, intraoperative magnetic resonance imaging (iMRI) and / or real-time imaging may be performed for transmission. In some embodiments, the device is used to administer rAAV particles to the mammary by the methods of the invention. IMRI is known in the art as an MRI-based imaging technology for patient imaging during surgery, which helps to confirm a successful surgical procedure (for example, to transfer rAAV particles to the device) CNS) and reduce the risk of damaging other parts of the tissue (for further description, see, eg, 2009), Fiandaca et al. Neuroimage 47 Suppl.2: T27-35). In some embodiments, a tracer agent (eg. MRI vs., improvement factor) jointly with Precipitant (eg, formulation containing rAAV particles) to provide real-time monitoring of the tissue distribution of the infusion material. See, for example, Neuroimage 47 Suppl.2: T27-35 (2009), Fiandaca et al. USPG Pub 2007/0259031; US Patent No. 7,922,999. The use of a tracking agent can indicate the interruption of transport. Other tracking and imaging method known in the industry can also be used to track the distribution of infusion.

[0271] In some embodiments, rAAV particles may be administered by standard stereotaxic injection (the term stereotaxic can be used interchangeably in the present application) using devices and methods known in the field to transport 3887 particles. Generally speaking, these methods can use an injection device, a layout system to transfer an area of ​​target tissue for transport in a series of coordinates (for example,

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Variables along lateral, dorsoventral, and lateral terminal axes), and a stereotaxic positioning device according to the planned coordinates (for example, a stereotaxic device, optionally that includes a probe and a structure to hold the head in place in alignment with the coordinate system). An unconstrained example of a system that could be It is useful for MRI-guided surgery and / or stereotaxic injection into the ClearPoint® System (TN, Memphis, MRI Interventions).

10272 The other, representative, unconstrained method for transporting the rAAV particle to the CNS is convection-enhanced transport (TEL). According to its use in the present application, the term convection-enhanced transport (CED) can refer to the transfer of a therapeutic agent to the CNS by an infusion at a rate at which hydrostatic pressure leads to a load distribution. In some embodiments, the infusion is greater than 0.5 μL / min. However, any appropriate flow rate can be used so that intracranial pressure is maintained at levels appropriate to prevent brain tissue being injured. A CED may be performed, for example, by using an appropriate catheter or cannula (for example, a cannula with a graduated design free of reflux. ) By placing the tip of the cannula at least in close proximity to the target 05a0 tissue (for example, the tip is inserted into the CNS tissue). After the cannula is placed, it is connected to a pump that transfers the therapeutic agent through the tip of the cannula to the target CNS tissue. Graded can be maintained Pressure from the tip of the cannula during infusion. In some embodiments, the leak can be monitored by a detectable tracer by means of, for example, intraoperative MRI (iMRI) or other real-time MRI technology.

[0273] CED is based on the infusion of infusion material (eg, formulation containing rAAV particle) into the CNS under the influence of pressure in which the hydrostatic pressure of the fluid between the cellular tissues is overcome. The CNS leakage material comes into contact with the vessel walls, and which

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It is used as a pump to distribute the infusion through convection and improve its transfer range (see, for example. 47 Hadaczek et; 2006) 302-17: 291 £ ¾7 Bankiewicz et alS) Hum.Gene, 2000) Exp.NeurolA64: 2-) A); 194 (2): 476-483ί LM et “7, Sanftner;

5o777ze 14 (4): 571-577 (. JR et. “/. Forsayeth (2006); US Patent Application Publication No. 0141980/2002; US Patent Application Publication No. 0259031/2007; and wo 2010/088560).

In some embodiments, a convection-assisted transmission device includes an osmotic pump and / or an infusion pump. An osmotic pump and / or infusion pump shall be commercially available (eg, from CA, ALZA Inc.în Palo Alto, Hamilton Coip.tALZET® Corp.). Pump systems may be implantable. Representative pump systems can be found at For example, in the US Patent No. 239, 51 3, 7; 7,341,577; 6,042,579; 815, 5 73, 5; And 4,692,147. Representative devices for CED, including progressive and anti-reflux cannulas, can be found in WO and 2006/042090 WO, which are listed in the current application for reference in their entirety.

(00275 In some embodiments, a convection-assisted transfer device includes an anti-reflux cannula (eg, progressive and anti-reflux cannulas). Further descriptions and representative anti-reflux cannulas can be found, for example, in Krauzeetal., Methods 362-465. : 349.Enzymol; 2006/0135945 USPG Pub USPG Pub; For PCT / US08 / 6401. In some embodiments, only one cannula is used. In other embodiments, more than one cannula is used. In some embodiments, a convection-assisted transfer device includes an anti-reflux cannula connected to a pump that produces sufficient pressure to make the infusion flow from

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Through the cannula into the target tissue at controlled rates, i.e. any suitable flow rate can be used so that intracranial pressure is maintained at levels appropriate to prevent brain tissue being injured.

10276 In some embodiments, penetration of the infusion material is further enhanced by the use of a facilitating agent. The facilitating agent can further facilitate the transfer of the infusion material to the target tissue (for example, the target CNS tissue). The unconstrained example of a facilitating agent is heparan with a low molecular weight (see, for example, US Patent No. 7,922,999).

10277 In some embodiments, the kits also include instructions for the CNS transport of the 8867 particle composition.] The kits described in the present order may also include other materials required from a commercial and user point of view, including other buffers, dilution agents, filters, Needles, syringes, and package leaflets with instructions to perform any methods described in the present application. Suitable packing materials may also be included and can be any packaging materials known in the industry, including, for example, vials (eg sealed vials), containers, ampoules. , Suitable bottles, jars, and packaging (for example, Mylar or plastic insulated bags), etc. These manufacturing products may also be sterilized and / or isolated. In some embodiments, the kits include instructions for treating a CNS disorder described in the present application using any of the methods or rAAV particles described in the present application. Kits may include a pharmaceutically acceptable carrier suitable for injection into an individual's CNS, and one or more of: buffer solution, diluent agent, filter, needle, syringe, and package leaflet with instructions for performing injections into the individual's CNS. Excipients

R

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[0278] In some embodiments relating to subretinal transport and / or CNS transport, the kits also contain buffer solutions and / or excipients that are pharmaceutically acceptable. According to what is well known in the field, pharmaceutically acceptable excipients are relatively inert substances that facilitate pharmacologic administration of the active ingredient and can be provided in the form of liquid solutions or suspensions, emulsions, or in solid forms suitable for dissolution or suspended in a liquid prior to use. For example, an excipient can give appearance or stability, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting agents and emulsifiers, salts of osmotic diversity, encapsulating agents, buffer buffers, and buffer buffers. The excipients mentioned include any pharmaceutical agent suitable for direct transport into the eye and which can be administered without unnecessary toxicity. Pharmacologically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline solution, glycerol and ethanol. Pharmaceutically acceptable salts in the present application, for example, mineral acid salts e.g. aqueous chlorides, aqueous bromides, phosphates, sulfates, etc .; And salts of organic acids, for example, acetate, propionate, malunoate, Benzoates, etc. Thorough discussion available

Pharmacologically acceptable excipients at Mack (REMINGTON'S PHARMACEUTICAL SCIENCES. NJ1991, Pub.Co).

10279 In some embodiments related to subretinal transport and / or CNS transport, pharmaceutically acceptable excipients may include pharmaceutically acceptable carriers. Such pharmaceutically acceptable carriers may be aseptic liquids, such as water and oil, including petroleum of animal origin. Or vegetable or synthetic, such as peanut oil, soybean oil, mineral oil, and the like. Salt solutions, aqueous dextrose, polyethylene glycol (PEG) can be used

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Glycerol solutions are also liquid carriers, particularly in solutions for injection. Additional ingredients may also be used, for example preservatives, buffers, buffers, surfactants, antioxidants, stabilizers, non-anionic or purifying agents, viscosity agents and the like. The kits described in the present application may be packaged in unit doses. Single or multi-dose. The contents of the kits can generally be formulated as a sterile or highly isotonic solution.

Examples

[0280] The invention may be fully understood with reference to the following examples. They shall not, however, be seen as restricting the scope of the invention. It shall be understood that the examples and models described in the present application are for illustrative purposes only, and that various modifications or changes in their light will be proposed to the skilled in the field and will not be included within the scope and jurisdiction of this application and the scope of the attached protection elements. Example 1: Mutations in arginine subunits required for AAV2-mediated normalization to reduce HSPG binding in cultured cell strains

[0281] Treatment vectors with AAV2 gene are currently being used in clinical trials of ocular variants. These vectors can be transported via the in vitro administration pathway, which in mice and nonhuman primates, by converting retinal ganglion cells and Müller cells, or via the subretinal administration pathway, It targets retinal pigment epithelial cells and photoreceptor cells. Different AAV serotypes employ different cell surface receptors and co-receptors for infection. It is known that the primary cell surface receptor for AAV2 is the heparan proteoglycansulfate (RJ (1998), c.and Samulski, Summerford) (HSPG 1438-45: (2) 0.72 a107). Understanding the mechanism by which the AAV2 transporter transforms the retina is important for the development of

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Additional AAV gene therapy vectors.

10282 To investigate the role of HSPG binding in the AAV2 transformation of the retina, AAV2 vectors were generated using capsid proteins that carry mutations in the arginine building block known to be required for HSPG 88672 HBKO (HSPG) binding. These vectors resulted in a significantly reduced conversion of 293 and HeLa cells in culture medium, compared to the untreated type 8872. Amazingly, the AAV2HBKO vector conducting subretinal to the mouse eye resulted in a 2-l increase in conversion compared to the untreated type 8872. When transporting the AAV2HBKO conveyor in vitreous, no appreciable conversion was found. These results suggest that the amino acid mutations required for HSPG binding have adverse effects on the conversion adequacy of the subretinal versus the vitreous injection of 887 particles.

Methods Create a mutant plasmid of 2 arginine

10283 The p! M45BD plasmid, AAV2 rep / cap, was mutated using the Quikchange Kit Primer Design. (Agilent Technologies) Lightning Multi Site Directed Mutagenesis Kit

PCR mutagenesis to advance changes in arginine compounds 585 and 588 to alanine compounds. Positive mutants were elated by the sequence procedure.

Generation of rAAV vectors

[0284] AAV vectors of gene recombinant expression of either an evolved green fluorescent protein (EGFP) or a hybrid soluble VEGF receptor (sFLT02) were produced by triple infection transfer to 293 cells using the p! M45BD plasmid or rep / cape.

And pAdHelper. The transgenes underwent a control of either 8-chicken actin (CBA) or enhancers

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Rhodopsin kinase (RK) is human.

In vitro conversion experiments

[0285] 293 cells or HeLa cells were placed in dishes of 24 eyes (2-1 5 10 cells per eye). After 24 hours of placement, the cells were infused with 1 10 x 3 24 / cell (b) 149 Ad5ts. Transfection efficiency was measured 48 hours after infection by either EGFP fluorescence or by ELISA to quantify SFLT02 in media (VEGF RI ELISA). Soluble human by R&amp;D systems. Animals

[0286] 6 / C57BL adult mice purchased from Jackson Laboratories Bar) Free access to food and water provided to the animals. Genzyme and kept in a barn (MEHarbor

During the study period. A. Conduct all procedures according to an Institutional approved protocol. Animal Care and Use Committee. Vitreous injection

[0287] Narcolepsy was induced and maintained with 3.5% isoflurane loaded at 800 ml L / min of oxygen delivered to the animal via the nose cone. One μL of test material was injected into the vitreous humor using a Hamilton syringe installed using a 33-gauge beveled-tip needle (Reno, Hamilton Co, 27). The needle was directed through the ocular sclera approximately 2 mm below the limbus and carefully advanced into the vitreous chamber to avoid contact with the lens. Eye. M Transfer test material over the course of 1-2 s. After the injection, the needle was held in place for approximately five seconds before withdrawal. M) Allow the animal to recover from anesthesia before returning it to its cage.

Subretinal injection

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10288 Mydriasis and ciliary muscle were stimulated with topical use of tropicamide (Alcon, TXiFort Worth). Narcolepsy was stimulated and maintained with 3.5% isoflurane carried in 800 mL / min of oxygen delivered to the animal via a cone. The eye was fixed with inverted ring forceps (FL). , Sarasota, World Précision Instruments) and an experimental incision of approximately 2 mm below the limbus in the sclera was made using a 30-gauge needle. The blunt-tipped 33 gauge needle was guided through the incision and advanced backward until its tip penetrated the neural retina. Sensual background. One μl of test material was transferred over a 1 second course. The needle was held in place for approximately five seconds before withdrawal. The animal was allowed to recover from anesthesia before being returned to its cage.

Histology for EGFP

00289 An untreated EGFP signal was observed with epifluorin fluorescent fluorescence microscopy on eyes fixed with formalin treated for paraffin embedding.

Results

[0290] As shown in Fig. 1, five capsid building blocks were shown to be of interest for AAV2 binding to 0 No. An AAV2 mutant carrying two amino acid substitutions was generated in these building blocks: R585A and R588A (the numbering is based on the 1M amino acid sequence. Test of this mutant, referred to by the name HBKO, for its ability to transform cells in culture medium.

10291 Human cell strains were cultured in culture medium and transformed with the untreated type or AAV2 particles of the HBKO mutant. To measure the conversion efficiency, the viral genomes of each of the 2 particle types were modified to include the transgenes using an ubiquitous CBA booster to direct the expression of a soluble Fit (human VEGF receptor 1). 48 hours after the conversion, this was done.

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Measure the conversion efficiency using an ELISA-based immunoassay to quantify the Fit of the product. Figure 2 shows that the HBKO mutant demonstrated a significantly reduced ability to transform 293 human cells into culture medium. Using stoichiometric numbers of AAV particles and 293 cells, the 99.6% HBKO mutant showed a decrease in conversion efficiency compared to the untreated type.

00292 To measure this effect in multiple human cell strains, 293 cells and HeLa cells were transformed according to their previous description. In these experiments, have been modified vectors to express EGFP, rather than Fit, from enhanced 038.aodh Figure 3 that the type is not the processor AAV2 be able to convert each of the cell strains, according to measured by glittering fluorescent EGFP. ^ Contrast, was a mutant HBKO impairment Z Great at converting both cell strains. These results confirm that the HBKO mutant has a significantly reduced ability to convert fetal kidney cancer and cervical cancer cell strains in culture medium.

Example 2: Mutations in arginine subunits required for HSPG binding have adverse effects on 2-mediated conversion after injection into vitreous versus subretinal

A 20293 was tested for the ability of AAV2 particles of the HBKO mutant to convert eye cells upon vitreous injection and subretinal injection in mouse. Figs 4a and 4 compare the conversion efficiency of untreated type and HBKO particles to the AAV2 mutant when injected into vitreous or subretinal injection. For these experiments, experiments on conversion efficiency were performed by measuring the expression of Soluble Fit (sFLT) for the transduction using AAV2 particles carrying an axis gene that used the ubiquitous CBA promoter to direct the expression of 114. Using in vitreous syringes, the HBKO mutant showed a significantly reduced ability to transform cells (Fig. 4a).

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Surprisingly, the HBKO mutant exhibited improved conversion after subretinal injection (Fig. 4b). This increase was consistently observed when the amount of the injected AAV2 vector genomes varied 10-fold (10 8 and 109 vg, according to its lysosomes). These results conflict with those observed in strains. Cell cultured and vitreous injection; these data indicate the potential importance of HSPG binding ability to mediate eye cell type transformation and retinal layers with different types of 5 ocular injections.

[0295] To visualize the retinal cell transformation, using the non-fluorescent type or AAV2 particles of the HBKO mutant carrying a two-axis expression of EGFP from the ubiquitous CBA promoter. As shown in Figure 5, AAV2 particles of the untreated type were able to transform the retina after injection into the vitreous, according to ^ Thus, the HBKO 10 particles of the AAV2 mutant showed an undetectable GFP fluorescence upon injection into vitreous. These results are in agreement with those shown in Fig. 4a.

10296 Figure 6 quantifies the transformation efficiency of an untreated species (AAV2 CBA) and the amplitude of HBKO (8872 CBAHBKO) AAV2 particles upon subretinal injection. To measure transposition, each of the AAV2 particles comprised transgenomes of each of the two AAV2 particles expressing Fit using the ubiquitous 15 08 promoter. Two quantities of vector genomes were used in the injections, as shown in Fig. 6. These results confirm the observation shown in Fig. 4B and illustrate the surprising result indicating that mutagenesis of the building blocks required for HSPG binding increases the ability of AAV2 to transform cells after subretinal injection.

[0297] to visualize the conversion; The untreated type or AAV2 particles were used for a 20 HBKO mutant carrying a two-axis expression EGFP expressing an ubiquitous CBA promoter. As can be seen in Fig. 7, m

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Improvement of GFP fluorescence in the retina upon transformation with AAV2 particles of the HBKO mutant, compared to the untreated type.

10298 In light of the knowledge that a subretinal injection targeted cell layers using photoreceptor cells, the ability of AAV2 particles of the HBKO mutant to transform photoreceptors was studied. In the eye, the rhodopsin kinase (RK) booster is known to specifically direct expression in photoreceptor cells, such as cone and rod cells (et 81. (2007), SC, Khanî).

3954-61. '(9) 48.4l ^. (Invest.Ophthalmol.Vis.Sci). On it, the untreated type and AAV2 particles of the HBKO mutant were generated using vector genomes carrying an axis gene using the RK promoter to direct the expression of A! As shown in Fig. 8, AAV2 particles of the HBKO mutant showed an advanced cellular transformation of the photoreceptor after subretinal injection, compared to the untreated type. This observation was consistently observed upon injection with 10 times different amounts of vector genomes (10, 8 and 109 g according to their coding). These results illustrate the result. The amazing thing is that the building blocks have gone off

Required for HSPG binding improves the ability of AAV2 particles to convert photoreceptor cells to the retina after subretinal injection. Mutagenesis of these building blocks represents a potential method for improving photoreceptor cell conversion to AAV2-mediated gene therapy.

10299 AAV is a single-stranded, non-enveloping DNA virus that is a member of the parvovirus group. Demonstrates the different serotypes of AAV including AAV2, AAVl, 48874 AAV5; 76 e, insist for different properties of the texture distribution. The multiple tissue transpiration of these AAV capsid aids the use of AAV-based vectors in large-scale gene transfer applications both in laboratory and in the organism of the liver, muscle, skeleton, brain, and retina; Heart and Spinal Cord (14: 316-, (2006)) Molecular Therapy, et al., Z., Wu

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327) .Virus binding to a family cell requires special interactions of the virus capsid with the receptor cellular molecules. Capsil AAV2 previously demonstrated the use of heparan proteoglycansulfate (HSPG), integrin 5 7 and the human fibroblast growth factor receptor 1 as primary and secondary receptors to mediate cell entry into the c.and vector gene therapy. RSamulski (1998), Summerford (AAV2)

J. Virology; 1445-1438: 72; 5: 78-82, Nat. Medicine, c.etal., Summerford; 5, Qing

5: 71-77, Nat. Medicine, et al K). A mutagenesis analysis of AAV2 proteins revealed that a group of essential amino acids, specifically arginine, R487, R484 R588, R585 and lysine الهيب532 contribute to the binding of heparan and the conversion of hepatocytes in the laboratory and in living organism. For the 8872 vectors, mutants in these building blocks of an amino acid perform a significantly reduced conversion of the liver to the 8872 based vectors by administering via the J-vein pathway 10 and increasing the transfer of a gene to the heart, muscle, and skeletal system (77: J. Virology, A.eta!, Kem. 70). : Cardiovascular Research, OJ.etal., Mullerl 1072-110 81

The role of these major amino acids in the conversion of AAV2 transporters in the retina was investigated by both the vitreous administration pathway and the retinal subclass of the vector. Mutagenesis of R585 and R588 largely eliminated the conversion of 293 cells and HeLa cells in vitro (Figures 2 and 3). 15 DalkaraD suggests 17: Molecular Therapy, et al. That the vitreous transported AAV2 vectors failed to penetrate the outer retina due to binding to heparan sulfate proteoglycans available in the inner retinal retinal membrane. As evidenced in the present examples, upon vitreous transport of the R585 / R588 mutagenic vectors the vectors are less able to Transposition of the inner retina compared to the noninvasive 72e8 vectors (Figures 4a and 5), indicating that binding to 3508 is significant in vitreous transformation of the retina 20. The subretinal transport based on the AAV2 vectors leads to cell transformation

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Retinal pigment epithelial (RPE) transduction and some of the transposition of the reperient cell. Surprisingly, it has been found that the R585 / R588 mutated vectors act to transform the outer retina at least 10 times better than the noninvasive AAV2 vectors (Figures 4b, 6 ​​and 7). By using the RhoSyncinase promoter of the Idiom receptor (RK), the expression of an axis gene in the photoreceptor significantly increases with the AAV2 R585 / R588 mutant vector (Fig. 8). These vectors will be dependent on the mutants in the main amphiphenic subunits of the AAV2 capsid. Extremely useful in transforming the outer retina especially the photoreceptor cells in the treatment of a variety of retinal disorders.

Example 3: Expression of a widespread GFP after transduction of the 2ΗΒΚΟ vector in the striatum

[0301] With site-guided floats inserted into the AAV2 capsid, an AAV2 HBKO mutagenesis vector that is unable to bind to Meparan was generated. The transfer properties of this HBKO vector were evaluated in both the untreated mouse and the HD mouse model (YAC128) using single injection processes In planned objects.

Methods Composition of a mutant AAV2 arginine plasmid

[0302] The 45ΒΙ elasmid was mutated, AAV2 rep / cap; Using a spikes configuration kit

Agilent) from the Quikchange Lightning Multi Site Directed Mutagenesis Kit directed site

Technologies) .PcR mutagenesis primer was designed to introduce changes of arginine to alanine at building blocks 585 and 588. Positive mutants were confirmed by the sequence procedure.

AAV vectors

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The recombinant AAV vectors were produced by triple transmission (using phosphatcalcium) to 293 cells of a human fetal nephrogenic carcinoma (-293) according to what was described previously in (2232-72: 2232.! Xiao et al. (1998)). In short, a plasmid containing a representative serotype 2 gene and a capsid gene of either serotype 1, or 2, was used along with adenovirus adjuvant (CA) plasmid w, Palo Alto, stratagene according to its use. Chicken beta-actin promoter (CBA) .Viruses were synthesized with hiccups B 72 hours and was purified by column according to what was described above. (Xiaoetal (1998) Ι.νίο1.72: 2224-2232) Animals

10304 All procedures were performed using an Institutional Animal approved protocol

Sanofi Company (Department of Health, Care and Use Committee at Genzyme

NIH Publication 8623), and Human Services. The mice used included the YAC128 mice (a yeast synthetic chromosome involving the HTT axis gene of a full-length human mutant with 128 CAG repeats on a pure FVBZNJ background) and mice of the same untreated FVBZNJ species al. (2003) Hum.Mol.Genet. 12: 1555-1567 (Slow; e; Van Raamsdonk et

YAC128 was obtained from both mice. 14: 3823-3835) '' /. &lt;2005

And their FVB / NJ counterparts from the same family from the colony that was housed at Charles River Laboratories. The mice were kept in a light-dark cycle for 12 hours so that food and water were available as desired. All behavioral tests were performed during the animals' light cycle (between 8 am and 4 pm). The N values ​​for all experiments are shown in Tables 2 and 3 below.

Table 2. AAV particle-treated wild mouse showing GFP (Experiment 1).

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<td>(25 * () Dose</td><td>Value • (WT mice)</td><td>treatment</td>

<td>10x6</td><td> 6</td><td>AAV2HBKO-CBA-GFP</td>

<td>06 and</td><td> 6</td><td>AAV2-CBA-GFP</td>

Table 3. A YAC128 furnace treated with AAV particles showing GFP, HttmiRNA (Experiment 2).

<td>Dosage (DRPs)</td><td>YAC128 value Mice)</td><td>treatment</td>

<td>10x6</td><td> 8</td><td>AAV2HBKO-CBA-miRNA-Htt- GFP ________</td>

<td>1.5 A 10 10</td><td> 8</td><td>ΑΑνΐ-CBA.miRNA-Htt-GFP</td>

<td>Non-user</td><td> 8</td><td>Untreated</td>

Surgical procedures

The animals were anesthetized with 3% isoflurane and placed in the context of stereotaxic surgery. Intracranial injections were performed according to what was described earlier by Stanek)

2014) .5 (Hum.Gene 77ίβ7. 25: 461-474). In short, 3 μL of viral vectors 5 of the recombinant product were injected into the striated node (AP, ml, TD 2.00; DV, 2- 5 of the important and dural; with a cutting rod, 0.0 (using a 10 μL Hamilton syringe at 0.5 μL / min. The needles were left in place for 1 minute after completion of the infusion. 1 hour before surgery and for 24 hours after surgery, the mice were given ketoprofen) 5 mg / kg) subcutaneously for anesthesia 10 Perfusion of the animal and collecting tissue

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Mice were perfused with cardiac perfusion with phosphate buffered saline (PBS) to remove all blood. For experiment 1, the brains were sectioned along the coronary plane, and after fixation in 4% para-formaldehyde followed by 30% sucrose. 20 μm coronary sections were sectioned using a cryostat. For experiment 2, the brains were sectioned sagittally along the midline , The left hemisphere was dimensionally fixed in 4% para-formaldehyde followed by 0/30 of sucrose and then divided into sections of 20 μm using a cryostat. The right hemisphere (used for biochemical tests) was sectioned along the coronary axis using Matrix Mouse Brain MA), Holliston, (Harvard Apparatus), and the ganglion regions of striated and cortical were dissected using a 3 mm bio-specimen drill bit. This was prepared by flash freezing of brain tissue in liquid nitrogen and stored at -80 ° C during use.

TaqMan (Quantitative Real-Time PCR)

[0307] RNA levels were carried out by quantitative real-time RT-PCR. Node bits were used to plot for all RT-PCR analysis. Total RNA was extracted using a small QIAGEN RNEasy kit and then were reverse transcribed and amplified with TaqMan® One- kit. Step RT-PCR Master Mix Kit (Applied Biosystems) according to the manufacturer's instructions. The quantitative RT-PCR reactions of TaqMan were performed and analyzed on an ABI PRISM® 7500 Real Time PCR System (Applied Biosystems). Expression levels of Ηπ mRNA were calibrated according to mRNA levels of phosphorylene guanine hypoxanthin (fAHPRTl). Standard curves were generated using enzymatic series dilutions. The expression of the relative gene was determined using the standard curve or the Basha method and calibrated according to levels 18 L * / 14447. For detection of human HTT, the following prefixes were used: '5

G

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-183 from HTT 5. To detect 'CCATTTTGAGGGTTCTGATTTCC 3' and CTCCGTCCGGTAGACATGCT 3 '

In mouse, the following prefixes were used: '3 _______________' 5 and '5' ATCCCTTGCGGATCCTATCA 3 'Western spotting.

[0308] Protein levels were determined by means of Western blot analysis. 5 straw bits were used for all Westin blot analyzes. Tissues were homogenized, at a concentration of 50 mg / mL in Pierce (Τ-Per) buffer buffer solution containing total protease inhibitor method (Roche). The homogen was purified by centrifugation at 10,000 mg for 6 minutes at 4 ° C. The protein concentration was measured by using Pierce's BSA test. Twenty to thirty micrograms of the metabolites were dissolved with 3-8%. Of Tris-acetate gel in it and was reduced by counting 10 To a nitrocellulose membrane; the membranes were probed as a monoclonal antibody to two antigens from a mouse (Mab2166; 1: 000, 2 dilution, Millipore) and a rabbit polyclonal antibody against beta-tubulin (1: 750 dilution, Santa Cruz Biotechnology). Membranes were then infrared with secondary antibodies (1: 20,000 dilution, Rockland), and the proteins were photographed by quantitative fluorescence (LI-C0R) Biosciences (Odyssey). To control 15 loading variations, Htt protein was titrated according to-tubulin and expressed in the intestinal fraction form of saline-treated and untreated animals. Molecular weight markers were used to verify the identity of the proteins.

Immune tissue chemistry

10309 frozen brain sections were tracked with either a significant antibody of rabbit GM cell spotting 20 (1: 2,500; Germany, GlostrDAKO) or an Y-balance antibody for visualizing glial cells

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Small (1: 500; VA, RichmodWAKO Chemicals USA). Secondary antibodies used were FITC-conjugated antibody-specific antibodies of donkey genera or FITC-conjugated 3-sections visualization using a fluorescent microscope NY Melville, Nikon Nikon Eclipse 800.

statistics

A 10310 average values ​​were used for the statistical analyzes. Data were expressed as mean ± SEM. For studies using two groups, Student'st test was used for statistical comparison. For comparisons of more than two groups, a one-way ANOVA was used followed by a custom comparison test for general purpose For Tukey's Prism GraphPad, a statistically significant difference of 005&gt; was considered.

Results

[0311] As described previously,-eGFP vectors and AAV2HBKO-GFP were injected into an untreated mouse in Experiment 1 (Table 2). All animals were slaughtered 30 days after injection. Expression of GFP was restricted to the injection pathway for AAV2, with the lowest spread beyond the injection site (Fig. 9B). However, when compared to conventional AAV2, AAV2HBKO directed more expression. Density and strength of GFP, so that expression was observed beyond locus Fully injection (Fig. 9a) .These results show the robust and diffuse expression of the transgenes transferred to the CNS using the AAV2HBKO vector.

Example 4 comparing the expression of AAVJ-mediated AAVJ AAV2HBKO after injection into cells in YAC128 mouse brains

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10312 As described previously, AAVlj AAV2HBKO serotype vectors were injected into YAC128 mice in Experiment 2 (Table 3). These vectors directed the expression of a synthetic miRNA targeting human Htt and the GFP transmitter. Both AAV2HBO AAVl vectors showed a robust GFP distribution 30 days after injection into the striated node (Figs 10a and 10b). However, the expression pattern of GFP was markedly different between the two species. The worshipers for the carrier 8871. Expression of GFP (Fig. 10a) was more incomplete and less uniform than that of AAV2HBKO GFP (Fig. 10b). Transduction appeared to be exceptional in AAV2HBKO brains compared to serotypes for 8871 that transformed both neurons and glial cells, primarily cells. The results indicated that AAV2HBKO exhibits a very different expression and transfer properties when compared to [887 conventional].

Example 5: Injection of AAV2HBKO-miRNA-Htt into YAC128 mice resulted in a reduction of

HTT mRNA

[0313] Then, the ability of the two serotypes of the AAV2HBK vector AAVl to express miRNA suppression of HTT-expressing in the striated node of 28 7801 mice was evaluated. YAC128 adult pheromone received two injections into the striated antibodies of AAV2 /! - mNARNA-Htt or -AAV2HBK0. miRNA-Htt, and brains were analyzed after 30 days-treatment. The levels of striated node L for the human mutant HTT mRNA were significantly reduced in mice injected with AA72 / l-mïRNA-Htt AAV2HBKO-miRNA-Htj when compared to untreated comparison groups. (Figure A 1). Western blot analysis is explained Samples from cerebral cortex showed a trend towards lowering of Htt in both treatment groups; However, the diversity in the untreated comparison samples prevented the data from reaching statistical significance (Fig.11b). These results demonstrate the efficacy of gene domination by RNAi using AAVl. AAV2HBK vectors

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-186 Example # 6: AAV2HBKO-miRNAHtt injection: inside YAC128 mice does not cause neuritis

[0314] In order to determine whether injections of AAV confer levels of neuritis from the neuroinflammatory markers of glial fibrous acid protein (GFAP, glial cell marker) and Ibal (microglial cell marker) at 30 days post-treatment. No increase in Ibal levels was observed after treatment with AAV2HKO compared to untreated brains (see Figures 12a and 12b). However, AAV2 / 1 treatment did not cause an increase in Ibal levels at the injection site (12c) that was observed below. Previously. No increases in GFAP levels were observed in any of the brains treated at 30 days post-injection compared to the untreated comparison samples (Figs 13a-13c). These results indicated that AAV2HBKO was able to derive the expression of miRNA directed toward human HTT and reductase. HTT in the absence of microglial activation when compared with the serotype. Conclusion

10315 Vector serum subtypes demonstrated a limited distribution in the brain following administration to a single site (eg, cw. Et Neurology; Mandel,

240-247.-2010 (Curr.Opin.Mol.Ther.l2) .L. 8) .As discussed in detail here, vectors for AAV administration have been detected with special low binding to heparan and proteoglycan sulfate (HSPG) on surfaces. Cell that improves the transmission of infection to AAV at 05. These results indicated that the use of AAV has a modified HSPG correlation specific to CNS gene therapy as a broad vector distribution is required. In addition, the inventors have indicated that AAV2 vectors have HSPG correlation reduced and demonstrated in an exemplary safety scheme (as they are targeted exclusively at

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Neurotransmitter) while achieving broad spectrum activity and infection-specific efficacy. These vectors are thus useful for inducing CNS that requires extensive neuronal infection from access to the organism example # 7: In vitro and subretinal transmission by mutagenic vectors AAVrhSR. Methods lead to the creation of AAVrhSR-modified plasmids of arginine.

10316 The AAVrhSR rep / cap plasmid was mutated using a special multi-position illuminated Quikchange from a direct mutagenesis kit (Agilent Technologies) and a PCR mutagen initiator designed to introduce changes of alanine 586 to arginine (AAVrh8R-A586R) or arginine 533 to alanine (AAVrh8R-R533A). The products of the positive mutation were confirmed by the following. Create rAAV vectors

[0317] The vectors expressing AAV recombinant M were either an enhanced fluorescent fluorescence protein (EGFP) or a soluble VEGF receptor complex (SFLT02) produced by triple transmission of 293 cells using PAAVrhSR-AAVrhSR plasmids A586R or pAAVrh8R-R533A. PAdHelper and pAdHelper. Tests for transmitting infection under the control of the chicken-specific β-a-actin promoter (CBA).

[0318] HeLa cells, HeLaRC32, or NSI cells were placed in 24-eye dishes (1,102 cells per) p. After 24 hours of leeching, cells were infected with 1 10 x 72 / cell -1 735 108 / cell (+) Ad5tsl49. Infection efficacy was measured at 48 hr after transmission by either EGFP fluorescence with ELISA in order to calculate the bulb

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SFLT02 in human soluble VEGF RI ELISA by VEGF RI systems. (ELISA animals)

[0319] Adult mice obtained from Jackson Laboratories (Bar MHarbor) were purchased and kept in dry jams to allow animals access to food and drink for the duration of the study.All procedures were carried out under protocols approved by the Institutional Animal Care and Committee. Use Committee Subretinal Syringes

[0320] Mydriasis and cilia were induced by topical administration of topocamide (1, FortWorthcAlcon). Hibernation was induced and maintained with 305% isoflurane carried in 800 mL min of oxygen delivered to the animal through the nose cone. The eye was fixed using oblique forceps (FL, SarasotacWorld Précision Instruments) and an incision was placed about 2 mm below the limbus on the sclera using a 30 gauge needle. A sharp oblique gauge needle 33 was guided through the incision and was subsequently advanced to the end penetrating the posterior part of the The retina is for the sensory neuron retina. Conduction of one μL of material onto a second substance. The needle was held in position for approximately five seconds before being withdrawn The needle was held in position for approximately five seconds before withdrawal The animals were allowed to recover from the anesthesia state before returning to the cage.

SFLTO2,4-specific retinal lysis products

[0321] The SFLT02 in retinal analysis products was measured using a VEGF RI ELISA kit and R&amp;D systems.

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180 results

[0322] In order to examine the role of the subunits of arginine at the capsid surface in ligating HSPG in the transmission of infection to AAVrh8, the AAVrh8 vectors were generated using PROSATCAPSHD carrying mutations in the building blocks that correspond to the common arginine subunits at the ?8 to 0 LNLN14 comparison of five of the subunits. The structural characteristics of the task are to link AAV2 5 with HSPG to the corresponding building blocks in “A” * 887. As shown in Figure No., two mutations were generated, each carrying amino acid substitutions in these building blocks: A586R and R533A (numbering is based on the VP1 amino acid sequence).

10323 In order to evaluate the effect of adding arginine subunits to the AAVrhSR envelope in vitro in vitro infection was carried out to HeLa cells using either AAVrh8R-sFLTO2 or 10 transporter 1102 ** 8586 T3M8! 8871 modifier in which arginine was added at the site of Α586 AAVrhSR A586R) each. Repel 1 x 10 x DRP / cell. After transmission for 48 h, the efficacy of transmission was tested by measuring SFLT02 in cell culture media. AAVrh8R-A586R exhibited several higher doubled transmissions compared to untreated TM 8871108 (Fig. 16). 15th

[0324] In order to evaluate the effect of arginine sheath removal on in vitro infectivity, the HeLaR032 GLY was infected with either AAVrhSR or AAVrh8R-R533A vector (each at 1 DRP 4 10 X synthesis) AAV1-R533A and AAV1-R533A and alpha have transmission of infection to a reductant when compared. With AAVrhSR (Fig. 16b).

10325 Similar trials were accessed using the expression [0] 1] as a measure of efficacy of 20 in transmitting infection. AAVrh8R-A586R-EGFP displayed improved infection transfer from NSI cells by comparison

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With AAVrhSR (by comparing Figs. 17b and Fig. 17a). Conversely, the AAVrh8R-R533A-EGFP vector had no reduced infection transmission on HeLa cells as compared to AAVrhSR (by comparing Fig. 17d to Fig. 7A).

10326 These experiments suggest that adding arginine to the AAVrhSR shell improves 887788, transmitting the laboratory infection, while the arginine compounds are removed from the AAVrhSR 5 coat that induces the inhibition of infection transmission in the laboratory. These results indicated that the transmission of infection in the laboratory through AAVrhSR has been affected. Largely through the LARs on the capsid.

[0327] In order to determine the effect of compound Arginine on the transmission of infection for AAVrhSR, an injection was performed

C57B16 mice using 1B810nM 0.2 of AAVrhSR and AAVrh8R-A586R, or 10AAVrh8R-R533A expressing CBA promoter ^ mice were slaughtered 30 days after administration of the vector and SFLT02 was measured in the retinal analysis product.

[0328] The transmission of AAVrh8R-A586R was reduced from the pony's retina to mycelia compared to AAVrhSR and indeed to AAV2 Which has arginine at the same site as R585 (Fig. 18). Improved transmission of 338 K 8 * 88708 from retina of mice assimilated with 15 AAVrhSR (Fig. 18B).

10329 This data demonstrates the effect of arginine envelope on the transmission of subretinal infection from AAVrhSR and the suggestion of subretinal transmission that improves the removal of arginine compounds from the envelope 98 A0887. These results indicated that subretinal infection transmission by AAVrhSR is strongly influenced by the ARBUs on the capsid. 20

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Proverbs 8: The intravitreal conversion of AAVrhSR is improved by adding arginine at position 586

Roads

Vitreous injection

[0330] The fluff was induced and maintained with 3.5% isoflurane carried in 800 mL min-1 of oxygen conveyed to the animal via a snout. One μL of the test product was injected into the vitreous humoral using a Hamilton syringe fitted with a needle with a 33-inch tip tip. From NV, Reno, (Hamilton Co.). The needle was guided through the sclera below the limbus by approximately 22 and was carefully inserted into the vitreous chamber to avoid contact with the lenses. The test product was transferred over a 1-2 second period. After the injection, the needle was held in place for approximately five seconds before being withdrawn. The animal was left to recover from anesthesia before being returned to its cage.

Quantification of SFLT02 in retinal degeneration products

SFLT02 was measured in mouse retinal degeneration products using a human soluble VEGF RI ELISA kit by R&amp;D Systems.

Results

[0332] To evaluate the effect of adding arginine to the AAV capsid on the intravitreal conversion, C57B16 mice were injected with 1 9 10 X of DRP of AAVrhSR, AAV2, or AAVrh8R-A586R, each carrying a structure expressing SFLT02 of the CBA enhancer. The mice were slaughtered at 30 days post-vector administration and SFLT02 was measured in retinal degradation products. The conversion of AAVrh8R-A586R to the mouse retina was improved compared to AAVrhSR and, in fact, was comparable to AAV2, including arginine in the same position, R585 (Fig. 19). This results to

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That the conversion within the vitreous of the retina could be improved by adding arginine to the capsid 0887. Based on these results and the homogeneity of the sequence between AAV capsids (Fig. 20), the conversion within the vitreous of the retina could likewise be improved by AAV particles bearing AAV9, AAV8! AAV6, AAV, and AAVrhlO capsid, mesh sequences

All polypeptide sequences were trimmed from the N-terminus to the • terminus unless otherwise noted.

All nuclear sequences (5 to 3) are presented unless otherwise noted.

The amino acid sequence AAV2VP1

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMATGSGAPMADNNEGADGVGESSGNWHCDSTWMGDRVITTS TRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFFIRFHCHFSPRDWQRLrNNNWGFRPKRLNFKLFNIQVKEVT QNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPA

MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQ AATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVP ANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY

SEPRPIGTRYLTRNL

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(Sequence ID number: 1) sequence of the amino acid AAV2 VPl HBKO ^ GYLPDMEDTLSEGIRQWWKLKPGPPPPKPAEMKDDSRGLVLPGYKYLGPraGLDKGEPV NEADAAAEEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMA.TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRV1TTSTRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPINFKLFNIQVKEVT QNDGTTTIAUTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADfSEYSWTGATKYfGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQAGNA QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPV PANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR1VNPEIQYTSNYNKSYN7DFTVDTNGV

YSEPRPIGTRYLTRNL

(SEO ID No. 2) The AAV2 VP2 amino acid sequence

MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNT MATGSGAPMADNNEGADG7GNSSGNWEICDSTWMGDRVITTSTR'2WALPTYNNHLYKQISSQSG ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLIMWGFRPKRLNFKLFNIQVKEVTQNDGTT TIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLE YFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS

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D 194 QAGASDIRDQSRTIPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHK DDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATAD VNTQGVLPGMVWQD ^ VYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILKUTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIG

TRYLTRNL

(Sequence ID No. 3)

The amino acid sequence ____

MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNT MATGSGAPMADNNEGAEGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNSQKQISS

ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFJGNIQVKEVTQNDGTT

TIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLE YFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS QAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRESLVNPGPAMASFK

DDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQAGNAQAATAD

VNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT

FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIG

TRYLTRNL

(Sequence ID number: 4)

The AAV2 VP3 amino acid sequence

MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG

ASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLIMWGFRPKRLNFKLFNIQVKEVTQNDGTT

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-195TIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLE YFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS QAGASDIREJQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHK DDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATAD

VNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT

FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIG

TRYLTRNL

(Sequence ID number: 5)

The AAV2 VP3 HBKO amino acid sequence

MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG

ASNDNHYFGYSTPWGYFDFNILFHCHFSPRDWQRLINWIWGFRPKRLNFKLFNIQVKEVTQNDGTT TIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLE YFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS

QAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNESWTGATKYHLNGRDSLVNPGPAMASHK

DDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQAGNAQAATAD

VNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTT FSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVVDTVDIG

TRYLTRNL

(Sequence ID number: 6)

The amino acid sequence 20 AAV3 VPl

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MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGE PVNEADAAALEETOKAYDQQLKAGEiNPYLKYNHADAEFQERLQEETFGGNLGRAVFQAKKRILE PLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPA APTSLGSNTMASGGGAPMADNFIEGADGVGNSSGNWHCDSQ3VLGDRVITTSTRTWALPTYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCEfFSPRDWQRLIFJNNWGFRPKKLSFKLFNIQVKE VTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLfPLIDQYLYYLNRTQGTTSG ttnqsrllfsqagpqsmslqarnwlpgpcyrqqrlsktandnnnsnfpwtaaskyhlngrdslv NPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNIDNVMITDEEEIRTTNPVATEQYGTVANNL QSSNTAPTTRTIQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIK NTPVPANPPÏÏFSPAKEASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDT

NGVYSEPRPIGTRYLTRNL

(Sequence ID No. 7)

ITR mutagenic to no scAAV

CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCC

GGGCTTTGCCCGGGCG

(Sequence ID number: 8)

The AAVrhSR VPl amino acid sequence

MAADGYLPDWLEDNLSEGIREWVVDLKPGAPKPKANQQKQEiDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLG

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3943932

-197EPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTY NNHLYKQISNGTSGGSTDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLn'WNWGFRPKM-NFK LFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLT LNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLfPLIDQYLYY LVRTQTTGTGG'FQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKF KLNGRDSLMNPGVAMASHKDDEDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVA TEEYGAVAfQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMG GFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIE1VELQKENSKR1VNPEIQYTS NYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

(Sequence ID number: 9)

The VPl amino acid sequence of the AAVrhSR A586R mutant

MAADGYLPD1VLEDNLSEGIREW1VDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLG EPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTY NNHLYKQISNGTSGGSTNEiNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLIFINNWGFRPKRLNFK LFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLT LNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY LVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNEINSNFAWTGAAKF KLNGRDSLMNPGVAMASHKDDEDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVA TEEYGAVAfQRANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMG

١٨٨

3943932

-198GFGLKHPPPQILIKNTPVPADPPLTFNQAkLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTS

NYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

(Serial ID No. 10)

The VPl amino acid sequence of the AAVrhSR mutant R533A

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQK.QDDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLG EPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTY NNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFK LFNIQVKEVTTNEGTKANLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLT LNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY LVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKF KLNGRDSLMNPGVAMASHKDDEDAFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVA TEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMG GFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTS NYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

(Serial ID No. 11)

The AAVl 1 amino acid sequence

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSE SVPDPQPLGEPPATPAAVGPÏÏlSGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVI TTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRL INNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYYLGSAHQ GCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEyP FHSSYAHSQSLDRLMNPLIDQ1TjïïLNRTQNQSGSAQ'NKI) LLFSRGSPAGMSyQPKNWLP GPCYRQQRVSKTKTDNNNSNFT1VTGASK12NLNGRESIINPGTAMASÜpEDKFFPMSGV MIFGKESAGASNTALDNVMITDEEEIKATNPVATERFG'rVAVNFQSSSTDPATGDVG

fill

3943932

-199-

(Wim ID sequence: 12) YTEPRPIGTRYLTRPL

The AAV6 VP1 amino acid sequence

(Serial ID number: 13) YTEPRPIGTRYLTRPL

The Laney acid sequence AAV8VP1

GVYSEPRPIGTRYLTRNL (Serial ID Number: 4A)

The AAV9 VP1 amino acid sequence

(Sequence MI number: 15) YSEPRPIGTRYLTRNL

3943932

-200-

The amino acid sequence AAVrhlO VPl

GTYSEPRPIGTRYLTRNL (16: RPI sequence)

١٨٨

3943932

-2013043 T Sequence Listing translation

Slate sequences

Genzyme Foundation&gt; 1

Sakarya, Abraham

Sullivan, Jennifer - Μ Stanik, Lisa

For retinal and in vitro gytotherapy of the neurotransmitter central AAV vector &lt;120

159792010440 &lt;130&gt;

Not yet set &lt;140&gt; HAH 141&gt;

&lt;150&gt; US 62 / 114,575 &lt;151 2015-02-10 &lt;150&gt; US 61 / 988,131 &lt;151&gt; 2014-05-02 &lt;160&gt; 16

For Windows 0-4-170&gt; FastSEQ&gt;

1 &lt;210&gt;

&lt;211 735 &lt;212&gt; PRT

Glandular viruses &lt;213&gt;

<td>Met</td><td>Al a</td><td>Al a</td><td>Asp</td><td>Gly</td><td>Tyr</td><td>leu</td><td>Pro</td><td>Asp</td><td>Trp 10</td><td>leu</td><td>Glu</td><td>Asp</td><td>Thr</td><td>leu 15</td><td>Ser</td>

<td>Glu</td><td>Gly</td><td>Ile</td><td>Arg</td><td>Gin</td><td>Trp</td><td>Trp</td><td>Lys</td><td>leu</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Pro</td><td>Pro</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>NS</td><td>Pro</td><td>Al a</td><td>Glu</td><td>Arg</td><td>His</td><td>lys</td><td>Asp</td><td>Asp</td><td>ser</td><td>Arg</td><td>Gly</td><td>leu</td><td>val</td><td>leu</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gly</td><td>Tyr</td><td>Lys</td><td>Tyr</td><td>Leu</td><td>Gly</td><td>Pro</td><td>Phe</td><td>Asn</td><td>Gly</td><td>Leu</td><td>Asp</td><td>Ns</td><td>Gly</td><td>Glu</td><td>Pro</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Asn</td><td>Glu</td><td>Al a</td><td>Asp</td><td>Al a</td><td>Al a</td><td>Al a</td><td>leu</td><td>for any</td><td>His</td><td>Asp</td><td>Lys</td><td>Al a</td><td>Tyr</td><td>Asp</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>Gin</td><td>leu</td><td>Asp</td><td>Ser</td><td>Gly</td><td>Asp</td><td>Asn</td><td>Pro</td><td>Tyr</td><td>Leu</td><td>Lys</td><td>Tyr</td><td>Asn</td><td>His</td><td>Al a</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asp</td><td>Al a</td><td>Glu</td><td>Phe</td><td>Gin</td><td>Glu</td><td>Arg</td><td>Leu</td><td>Lys</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Ser</td><td>Phe</td><td>Gly</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Asn</td><td>leu</td><td>Gly</td><td>Arg</td><td>Al a</td><td>Val</td><td>Phe</td><td>Gin</td><td>Al a</td><td>Ns</td><td>Lys</td><td>R</td><td>val</td><td>Leu</td><td>Glu</td><td>Pro</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>leu</td><td>Gly</td><td>leu</td><td>6 C1</td><td>Glu</td><td>Glu</td><td>Pro</td><td>val</td><td>Lys</td><td>Thr</td><td>Al a</td><td>Pro</td><td>Gly</td><td>lys</td><td>Ns</td><td>Arg</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>ρ3</td><td></td><td>Glu</td><td>His</td><td>Ser</td><td>Pro</td><td>Val</td><td>Glu</td><td>Pro</td><td>Asp</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Gly</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>No 5</td><td>Al a</td><td>Gly</td><td>Gin</td><td>Gin</td><td>Pro</td><td>Al a</td><td>Arg</td><td>Lys</td><td>R</td><td>leu</td><td>Asn</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gly</td><td>Asp</td><td>Al a</td><td>Asp</td><td>Ser</td><td>Val</td><td>Pro</td><td>Asp</td><td>Pro</td><td>Gin</td><td>Pro</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Pro</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Al a</td><td>Al a</td><td>Pro</td><td>Ser</td><td>Gly</td><td>leu</td><td>Gly</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Met</td><td>Al a</td><td>Thr</td><td>Gly</td><td>Ser</td><td>Gly</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Al a</td><td>Pro</td><td>Met</td><td>Al a</td><td>Asp</td><td>Asn</td><td>Asn</td><td>Glu</td><td>Gly</td><td>Al a</td><td>Asp</td><td>Gly</td><td>Val</td><td>Gly</td><td>Asn</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Gly</td><td>Asn</td><td>Trp</td><td>His</td><td>Cys</td><td>Asp</td><td>Ser</td><td>Thr</td><td>Trp</td><td>Met</td><td>Gly</td><td>Asp</td><td>Arg</td><td></td><td>Ile</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>2hr</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Arg</td><td>Thr</td><td>Trp</td><td>Al a</td><td>leu</td><td>Pro</td><td>Thr</td><td>Tyr</td><td>Asn</td><td>Asn</td><td>His</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Tyr</td><td>lys</td><td>Gin</td><td>Ile</td><td>Ser</td><td>Ser</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Al a</td><td>Ser</td><td>Asn</td><td>Asp</td><td>Asn</td><td>His</td><td>Tyr</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Phe</td><td>Gly</td><td>Tyr</td><td>Ser</td><td>Thr</td><td>Pro</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Phe</td><td>His</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Cys</td><td>His</td><td>Phe</td><td>ser</td><td>Pro</td><td>R</td><td>Asp</td><td>Trp</td><td>Gin</td><td>Arg</td><td>leu</td><td>Ile</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Trp</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Phe</td><td></td><td>Pro</td><td>Lys</td><td>Arg</td><td>leu</td><td>Asn</td><td>Phe</td><td>Ns</td><td>Leu</td><td>Phe</td><td>Asn</td><td>Ile</td><td>Gin</td><td>Val</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

*١٦

3943932

-202] 3043 Sequence Listing translation

Lys

Thr val val

Gin 385 Gin

Asp leu

Asn

Ala 465 Pro

Asn

Arg

Asp

Gin 545 Asp

Gly

Ala

Arg

Asp 625 His

Pro

Tyr Glu

Asn 705 Ser

Glu Val Thr Gin

325 ser Thr Val Gin

340

Leu Gly Ser Ai a

355

Phe Met Val Pro

370

Al a val Gly Arg

Met Leu Arg Thr 405

Val Pro Phe His 420

Met Asn Pro leu

435

Thr Pro Ser Gly 450

Gly Ala Ser Asp

Cys Tyr Arg Gin 485 ser Glu Tyr Ser 500

Asp Ser Leu Val 515

Glu Glu Lys Phe 530

Gly Ser Glu Lys

Glu Glu Glu Ile

565

Ser Val Ser Thr

580

Asp Val Asn Thr

595

Asp Val Tyr Leu 610

Gly His Phe His

Pro Pro Gin

649

Ser Thr Thr Phe

660

Ser Thr Gly Gin

675

Asn Ser Lys Arg 690

Lys Ser Val Asn

Glu Pro Arg Pro

<td>Asn</td><td>Asp</td><td>Gly</td><td>Thr</td><td>Thr</td><td>Thr</td><td>Ile</td><td>Ala</td><td>Asn</td><td>Asn</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Val</td><td>Phe</td><td>Thr</td><td>Asp</td><td>Ser</td><td>Glu</td><td>Tyr</td><td>Gin</td><td>leu</td><td>Pro</td><td>Tyr</td>

<td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>His</td><td>Gin</td><td>Gly</td><td>Cys</td><td>leu</td><td>Pro</td><td>Pro</td><td>Php</td><td>Pro</td><td>Ala</td><td>Asp</td>

<td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Gin</td><td>Tyr</td><td>Gly</td><td>Tyr</td><td>leu</td><td>Thr</td><td>Leu</td><td>Asn</td><td>Asn</td><td>Gly</td><td>Ser</td>

<td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>ser</td><td>ser</td><td>Php</td><td>Tyr</td><td>Cys</td><td>Leu</td><td>Glu</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Ser</td>

<td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Gly</td><td>Asn</td><td>Asn</td><td>Phe</td><td>Thr</td><td>Php</td><td>Ser</td><td>Tyr</td><td>Thr</td><td>Php</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Ser</td><td>Ser</td><td>Tyr</td><td>Ala</td><td>His</td><td>Ser</td><td>Gin</td><td>Ser</td><td>Leu</td><td>Asp</td><td>Arg</td>

<td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Ile</td><td>Asp</td><td>Gin</td><td>Tyr</td><td>Leu</td><td>Tyr</td><td>Tyr</td><td>leu</td><td>Ser</td><td>Arg</td><td>Thr</td>

<td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Thr</td><td>Thr</td><td>Thr</td><td>Gin</td><td>Ser</td><td>Arg</td><td>leu</td><td>Gin</td><td>Php</td><td>Ser</td><td>Gin</td>

<td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Ile</td><td>Arg</td><td>Asp</td><td>Gin</td><td>Ser</td><td>And sold</td><td>Asn</td><td>Trp</td><td>leu</td><td>Pro</td><td>Gly</td>

<td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>Gin</td><td>Arg</td><td>Val</td><td>Ser</td><td>5 to 1</td><td>Thr</td><td>Ser</td><td>Ala</td><td>Asp</td><td>Asn</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td>Trp</td><td>Thr</td><td>Gly</td><td>Ala</td><td>Thr</td><td>Lys</td><td>Tyr</td><td>His</td><td>leu</td><td>Asn</td><td>Gly</td>

<td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td></td><td></td>

<td>Asn</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Ala</td><td>Met</td><td>Ala</td><td>Ser</td><td>His</td><td>Lys</td><td>Asp</td>

<td></td><td></td><td> 920</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Val</td><td>leu</td><td>Ile</td><td>Phe</td><td>Gly</td><td>Lys</td>

<td></td><td> 535</td><td></td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Asn</td><td>No</td><td>Asp</td><td>Ile</td><td>Glu</td><td>Lys</td><td>vai</td><td>Met</td><td>Tl P</td><td>Thr</td>

<td> 550</td><td></td><td></td><td></td><td></td><td> 555</td><td></td><td></td><td></td><td></td><td> 560</td>

<td>Arg</td><td>Thr</td><td>Thr</td><td>Asn</td><td>Pro</td><td>Val</td><td>Ala</td><td>Thr</td><td>Glu</td><td>Gin</td><td>Tyr</td>

<td></td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td></td><td></td><td> 575</td><td></td>

<td>Asn</td><td>leu</td><td>Gin</td><td>And sold</td><td>Gly</td><td>Asn</td><td>And the</td><td>Gin</td><td>Ala</td><td>Ala</td><td>Thr</td>

<td></td><td></td><td></td><td> 585</td><td></td><td></td><td></td><td></td><td> 590</td><td></td><td></td>

<td>Gin</td><td>Gly</td><td>vai</td><td>leu</td><td>Pro</td><td>Gly</td><td>Met</td><td>Val</td><td>Trp</td><td>Gin</td><td>Asp</td>

<td></td><td></td><td> 600</td><td></td><td></td><td></td><td></td><td> 605</td><td></td><td></td><td></td>

<td>Gin</td><td>Gly</td><td>Pro</td><td>Tl P</td><td>Trp</td><td>Ala</td><td>Lys</td><td>Ile</td><td>Pro</td><td>His</td><td>Thr</td>

<td></td><td> 615</td><td></td><td></td><td></td><td></td><td> 620</td><td></td><td></td><td></td><td></td>

<td>? Γ0</td><td>Ser</td><td>Pro</td><td>leu</td><td>Met</td><td>Gly</td><td>Gly</td><td>Phe</td><td>Gly</td><td>Leu</td><td>L7s</td>

<td> 630</td><td></td><td></td><td></td><td></td><td> 635</td><td></td><td></td><td></td><td></td><td> 640</td>

<td>Ile</td><td>leu</td><td>Ile</td><td>lys</td><td>Asn</td><td>Thr</td><td>Pro</td><td>1la1</td><td>Pro</td><td>Ai a</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 650</td><td></td><td></td><td></td><td></td><td> 655</td><td></td>

<td>Ser</td><td>Ala</td><td>Ala</td><td>Lys</td><td>Php</td><td>Ala</td><td>Ser</td><td>Phe</td><td>Tl P</td><td>Thr</td><td>Gin</td>

<td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td></td>

<td>Val</td><td>Ser</td><td>Val</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Leu</td><td>Gin</td><td>Lys</td>

<td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td></td><td></td><td></td>

<td>Trp</td><td>Asn</td><td>Pro</td><td>Glu</td><td>Ile</td><td>Gin</td><td>Tyr</td><td>Thr</td><td>Ser</td><td>Asn</td><td>Tyr</td>

<td></td><td> 695</td><td></td><td></td><td></td><td></td><td> 700</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Asp</td><td>Php</td><td>Thr</td><td>Val</td><td>Asp</td><td>Thr</td><td>Asn</td><td>Gly</td><td>Val</td><td>Tyr</td>

<td> 710</td><td></td><td></td><td></td><td></td><td> 715</td><td></td><td></td><td></td><td></td><td> 720</td>

<td>Ile</td><td>Gly</td><td>Thr</td><td></td><td>Tyr</td><td>leu</td><td>Thr</td><td>P</td><td>Asn</td><td>leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 730</td><td></td><td></td><td></td><td></td><td> 735</td><td></td>

&lt;20 2 &lt;211 735 &lt;212&gt; PRT

Syntax &lt;213&gt;

&lt;220&gt;

Synthetic structure &lt;223&gt;

&lt;400&gt; 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp 1 5

Glu Gly Ile Arg Gin Trp Trp Lys Leu

25

Lys Pro Ala Glu Arg His Lys Asp Asp

Trp leu Glu Asp Thr leu Ser 10 15

Lys Pro Gly Pro Pro Pro 30

Ser Arg Gly Leu Val leu Pro

-

fill

3943932

<td>dsv</td><td>UIP</td><td>dj 1</td><td>IBA</td><td>69W</td><td>A LP</td><td>OJd</td><td>nai</td><td>LBA</td><td>Aid</td><td>19 when</td><td>For the</td><td>For 5 l</td><td>98 a</td><td>dsv</td><td>BLV</td>

<td></td><td></td><td> 065</td><td></td><td></td><td></td><td></td><td>SSS</td><td></td><td></td><td></td><td></td><td>OSS</td><td></td><td></td><td></td>

<td>Did not sell</td><td colspan="2">BIV BIV</td><td>UID</td><td> ٥1</td><td>usv</td><td>A LP</td><td>BIV</td><td></td><td>na.</td><td>57 did not</td><td>For 1 l</td><td>95 did not</td><td></td><td>Z 0 did not</td><td>Aid</td>

<td></td><td> 5/٤</td><td></td><td></td><td></td><td></td><td>A / 0</td><td></td><td></td><td></td><td></td><td>S9S</td><td></td><td></td><td></td><td></td>

<td>Enner l</td><td>9 a</td><td>niD</td><td>For the</td><td>BIV</td><td> 1٥٨</td><td>H did 0</td><td>usv</td><td>For the</td><td>For the pain</td><td>6jv</td><td>ai</td><td>niD</td><td>niP</td><td>ΠΙΡ</td><td>dsv</td>

<td> 095</td><td></td><td></td><td></td><td></td><td>sss</td><td></td><td></td><td></td><td></td><td>oss</td><td></td><td></td><td></td><td></td><td>SFS</td>

<td>For the</td><td>9 II</td><td>aaw</td><td>LPA</td><td>sAi</td><td>niP</td><td>No 9</td><td>dsv</td><td>1ΒΛ</td><td>For 5 lm</td><td>For the pain</td><td>sA6</td><td>niD</td><td>95 did not</td><td>2 OMR negotiable Ald</td><td>UID</td>

<td></td><td></td><td></td><td></td><td>OFS</td><td></td><td></td><td></td><td></td><td>SSS</td><td></td><td></td><td></td><td></td><td>P 0</td><td></td>

<td>sA,</td><td>A LP</td><td>aqd</td><td>an</td><td>na6</td><td>IBA</td><td>A LP</td><td>jas</td><td>UID</td><td>OJd</td><td>aqd</td><td>aqd</td><td>sA6</td><td>niP</td><td>niD</td><td>dsv</td>

<td></td><td></td><td></td><td>SES</td><td></td><td></td><td></td><td></td><td>ozs</td><td></td><td></td><td></td><td></td><td>SIS</td><td></td><td></td>

<td>dsv</td><td>sA6</td><td>SLH</td><td>JBS</td><td>BIV</td><td>19W</td><td>2 0</td><td>OJd</td><td>Aid</td><td>OJd</td><td>usv</td><td>IBA</td><td>ΠΒ1</td><td>JBS</td><td>dsv</td><td>6jv</td>

<td></td><td></td><td>OIS</td><td></td><td></td><td></td><td></td><td>SOS</td><td></td><td></td><td></td><td></td><td>oos</td><td></td><td></td><td></td>

<td>A LD</td><td>UN</td><td>nai</td><td>s .LH</td><td>jAi</td><td>sAi</td><td>For the</td><td>BIV</td><td>Ald</td><td>For the</td><td>dj 1</td><td>jas</td><td>jAi</td><td>niP</td><td>JBS</td><td>usv</td>

<td></td><td> ٤61</td><td></td><td></td><td></td><td></td><td>06F</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>usv</td><td>57 did not</td><td>dsv</td><td>B IV</td><td>22 no</td><td>For the pain</td><td>sAi</td><td>jas</td><td>IBA</td><td>Bjv</td><td>uld</td><td>UID</td><td>Bjv</td><td>jAj.</td><td>sAp</td><td>OJd</td>

<td></td><td></td><td></td><td></td><td></td><td>SZF</td><td></td><td></td><td></td><td></td><td>OZF</td><td></td><td></td><td></td><td></td><td>59 F</td>

<td>A LD</td><td>No 0</td><td>nai</td><td>dj 1</td><td>For 5 l</td><td>Bjv</td><td>For as</td><td>UID</td><td>dsv</td><td>6jv</td><td>ail</td><td>dsv</td><td>JBS</td><td>BLV</td><td>Ald</td><td>BLV</td>

<td></td><td></td><td></td><td></td><td>09k</td><td></td><td></td><td></td><td></td><td>SSE</td><td></td><td></td><td></td><td></td><td>OSF</td><td></td>

<td>19 when</td><td>25 m</td><td>9qd</td><td>UID</td><td></td><td>Bjv</td><td>95 did not</td><td>No 15</td><td>For the pain</td><td>For the</td><td>jqi</td><td>Ald</td><td>J 2 lm</td><td>OJd</td><td>For the pain</td><td>usv</td>

<td></td><td></td><td></td><td>SW</td><td></td><td></td><td></td><td></td><td>OH</td><td></td><td></td><td></td><td></td><td>S8F</td><td></td><td></td>

<td>For the pain</td><td>6 IW</td><td>J 2 lm</td><td> 12</td><td>For Al</td><td>jAi</td><td>na6</td><td>jAi</td><td>UID</td><td>dsv</td><td>Ta 5</td><td>nai</td><td>OJd</td><td>usv</td><td>I</td><td>nai</td>

<td></td><td></td><td>08F</td><td></td><td></td><td></td><td></td><td>SZF</td><td></td><td></td><td></td><td></td><td> 0•1</td><td></td><td></td><td></td>

<td>6jv</td><td>dsv</td><td>na.</td><td>S</td><td>U LD</td><td>95 did not</td><td>SLH</td><td>BIV</td><td>For Al</td><td>J 3 m</td><td>JBS</td><td>s .LH</td><td>aqd</td><td>OJd</td><td>IBA</td><td>Why dsv</td>

<td></td><td>SIF</td><td></td><td></td><td></td><td></td><td>OIF</td><td></td><td></td><td></td><td></td><td>SOF</td><td></td><td></td><td></td><td></td>

<td>n LD</td><td>Bqd</td><td>For the</td><td>U Ai</td><td>JBS</td><td>9L | d</td><td>For pal</td><td>aqd</td><td>For 5 l</td><td>For 5 lm</td><td>Ald</td><td>For the</td><td>Bjv</td><td>ΠΒ1</td><td>19W</td><td>UID</td>

<td>OOP</td><td></td><td></td><td></td><td></td><td> 568</td><td></td><td></td><td></td><td></td><td> 068</td><td></td><td></td><td></td><td></td><td> 588</td>

<td>9mm</td><td>OJd</td><td>aqd</td><td>For Al</td><td>niD</td><td>nai</td><td>sAp</td><td>For Al</td><td>aqd</td><td>J 9 lm</td><td>2 h</td><td>ftiv</td><td>AlP</td><td>IBA</td><td>BLV</td><td>UID</td>

<td></td><td></td><td></td><td></td><td> 088</td><td></td><td></td><td></td><td></td><td>P / 4</td><td></td><td></td><td></td><td></td><td>ozs</td><td></td>

<td>JBS</td><td>Ald</td><td>usv</td><td>5 for what</td><td>nai</td><td>For 4 th</td><td></td><td>jAi</td><td>A LP</td><td>jAi</td><td>UID</td><td>May 0</td><td>IBA</td><td>iaw</td><td>aqd</td><td>LBA</td>

<td></td><td></td><td></td><td> 598</td><td></td><td></td><td></td><td></td><td>P 09</td><td></td><td></td><td></td><td></td><td>P. 44</td><td></td><td></td>

<td>dsv</td><td>2 a 0</td><td>M did 0</td><td>aqd</td><td>Tomorrow 0</td><td>OJd</td><td></td><td>sAp</td><td>A LP</td><td>UID</td><td>SLH</td><td>BIV</td><td>JBS</td><td>AlP</td><td>ΙΊΒ1</td><td>LBA</td>

<td></td><td></td><td>P 05</td><td></td><td></td><td></td><td></td><td>SH</td><td></td><td></td><td></td><td></td><td>OFS</td><td></td><td></td><td></td>

<td>jAi</td><td>Dream 0</td><td>nai</td><td>UID</td><td>j Al</td><td>D.</td><td>jas</td><td>dsv</td><td>For the pain</td><td>aqd</td><td>LBA</td><td>UID</td><td>LBA</td><td>For the</td><td>JBS</td><td>For the mother</td>

<td></td><td>P 5</td><td></td><td></td><td></td><td></td><td>P 0</td><td></td><td></td><td></td><td></td><td>SZS</td><td></td><td></td><td></td><td></td>

<td> 109</td><td>For 5 lm</td><td>For 15</td><td> 81</td><td>SLI</td><td>For the mother</td><td>For 1 th</td><td>For the</td><td>A LP</td><td>dsv</td><td>57 Why</td><td>UIP</td><td>For the pain</td><td>IBA</td><td>nLD</td><td>s sAi</td>

<td>ΟΖ8</td><td></td><td></td><td></td><td></td><td>SIS</td><td></td><td></td><td></td><td></td><td>OIS</td><td></td><td></td><td></td><td></td><td> 508</td>

<td>LPA</td><td>UID</td><td>9LI</td><td>For 5 l</td><td>aqd</td><td>nai</td><td>sA6</td><td>aqd</td><td>For 5 lm</td><td>nai</td><td>6jv</td><td>sAi</td><td>OJd</td><td>6JV</td><td>Bqd</td><td>Aid</td>

<td></td><td></td><td></td><td></td><td>H 00</td><td></td><td></td><td></td><td></td><td>S6Z</td><td></td><td></td><td></td><td></td><td>P 06</td><td></td>

<td>dj 1</td><td>For 52 when</td><td>usv</td><td>usv</td><td> 9 11</td><td></td><td>6jv</td><td>9 either</td><td>du 1</td><td>dsv</td><td>Bjv</td><td>• No 0</td><td>25 no</td><td>aqd</td><td>SLH</td><td>sAp</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 08•</td><td></td><td></td><td></td><td></td><td> -/٤</td><td></td><td></td>

<td>s LH</td><td>Bqd</td><td>6jv</td><td>usv</td><td>9L | d</td><td>dsv</td><td>aqd</td><td>jAi</td><td>A LP</td><td>dJi</td><td>OJd</td><td>For the</td><td>JBS</td><td>For Al</td><td>AlP</td><td>aqd</td>

<td></td><td></td><td>OZZ</td><td></td><td></td><td></td><td></td><td>S9Z</td><td></td><td></td><td></td><td></td><td> 09•</td><td></td><td></td><td></td>

<td>jAi</td><td>SLH</td><td>usv</td><td>dsv</td><td>USV</td><td>jas</td><td>BLV</td><td>Aid</td><td>S</td><td>UID</td><td>JBS</td><td>Science</td><td>BU</td><td>UIP</td><td>sAi</td><td>jAi</td>

<td></td><td>P</td><td></td><td></td><td></td><td></td><td>osz</td><td></td><td></td><td></td><td></td><td>SFZ</td><td></td><td></td><td></td><td></td>

<td></td><td>SLH</td><td>usv</td><td>usv</td><td>For Al</td><td>For the mother</td><td>D0</td><td>nai</td><td>BIV</td><td>dJi</td><td>For 1 th</td><td>Bjv</td><td>For the pain</td><td>JBS</td><td>For 1 th</td><td>For 1 th</td>

<td>Hh 0</td><td></td><td></td><td></td><td></td><td>58Ζ</td><td></td><td></td><td></td><td></td><td>08Ζ</td><td></td><td></td><td></td><td></td><td></td>

<td>9LI</td><td> 1٨</td><td>Bjv</td><td>dsv</td><td>A LP</td><td>69W</td><td>dJi</td><td>For the mother</td><td>jas</td><td>dsv</td><td>sAp</td><td>SLH</td><td>dJi</td><td>USV</td><td>AlP</td><td>95 did not</td>

<td></td><td></td><td></td><td></td><td>OZZ</td><td></td><td></td><td></td><td></td><td>SIZ</td><td></td><td></td><td></td><td></td><td> 01</td><td></td>

<td>J 3 m</td><td>5 l</td><td>Aid</td><td>LBA</td><td>AlP</td><td>dsv</td><td> 61</td><td>Aid</td><td>niP</td><td>usv</td><td>5 no</td><td>dsv</td><td>BIV</td><td>69W</td><td>Did not do 0</td><td>BLV</td>

<td></td><td></td><td></td><td>P 0</td><td></td><td></td><td></td><td></td><td> 00•</td><td></td><td></td><td></td><td></td><td>S6I</td><td></td><td></td>

<td>Aid</td><td>54 lm</td><td>Aid</td><td>For the</td><td>BIV</td><td> 1</td><td>For the</td><td>usv</td><td>For the mother</td><td>Aid</td><td>nai</td><td>A LP</td><td>2 no</td><td>OJd</td><td>BIV</td><td>BIV</td>

<td></td><td></td><td> 061</td><td></td><td></td><td></td><td></td><td>SSI</td><td></td><td></td><td></td><td></td><td> 08</td><td></td><td></td><td></td>

<td>OJd</td><td>D 0</td><td>UID</td><td>Ald</td><td>nai</td><td>OJd</td><td>19 when</td><td>OJd</td><td>dsv</td><td>OJd</td><td>IBA</td><td>JBS</td><td>dsv</td><td> 91٧</td><td>dsv</td><td>Aid</td>

<td></td><td>/a</td><td></td><td></td><td></td><td></td><td> 0/1</td><td></td><td></td><td></td><td></td><td>S 91</td><td></td><td></td><td></td><td></td>

<td>For the pain</td><td>9 a</td><td>Ald</td><td>aqd</td><td>For 9 lm</td><td></td><td>6jv</td><td>sAi</td><td>Did 6</td><td>BIV</td><td>OJd</td><td>9 a</td><td>UIP</td><td>AlP</td><td>BIV</td><td>sA6</td>

<td> 091</td><td></td><td></td><td></td><td></td><td>SSI</td><td></td><td></td><td></td><td></td><td> +0٤</td><td></td><td></td><td></td><td></td><td>SFI</td>

<td>A LD</td><td>For the</td><td>A LP</td><td>JBS</td><td>99 no</td><td>jas</td><td>dsv</td><td>OJd</td><td>niP</td><td>IBA</td><td>Old</td><td>JBS</td><td>SLH</td><td>niP</td><td>IBA</td><td>OJd</td>

<td></td><td></td><td></td><td></td><td>OH</td><td></td><td></td><td></td><td></td><td>SSI</td><td></td><td></td><td></td><td></td><td> 081</td><td></td>

<td>6jv</td><td>sAi</td><td>sAi</td><td>Ald</td><td>D 0</td><td> 0٨٧</td><td>For 1 th</td><td>sAi</td><td>LBA</td><td>OJd</td><td>niD</td><td>niD</td><td>IBA</td><td>ΠΒΊ</td><td>A LD</td><td>nai</td>

<td></td><td></td><td></td><td>s ZI</td><td></td><td></td><td></td><td></td><td> 021</td><td></td><td></td><td></td><td></td><td>SH</td><td></td><td></td>

<td>OJd</td><td>nLD</td><td>nai</td><td>LBA</td><td>6jv</td><td>sAi</td><td>sAi</td><td>BIV</td><td>UID</td><td>aqd</td><td>IBA</td><td> ٥1٧</td><td>6JV</td><td>AlP</td><td>ΠΘ1</td><td>usv</td>

<td></td><td></td><td>OII</td><td></td><td></td><td></td><td></td><td> 501</td><td></td><td></td><td></td><td></td><td> 00[</td><td></td><td></td><td></td>

<td colspan="2">Ald Aid</td><td>aqd</td><td>95 did not</td><td>For the pain</td><td>dsv</td><td>ΠΙΡ</td><td>sA6</td><td>nai</td><td>6jv</td><td>D.</td><td>9 a</td><td>Bqd</td><td>niP</td><td>BIV</td><td>Mine dsv</td>

<td></td><td> 06</td><td></td><td></td><td></td><td></td><td> 06</td><td></td><td></td><td></td><td></td><td>SS</td><td></td><td></td><td></td><td></td>

<td>BLV</td><td>SLH</td><td>usv</td><td>For Al</td><td>sA6</td><td></td><td>For Al</td><td>For • lm 0</td><td>usv</td><td>dsv</td><td>Ald</td><td>JBS</td><td>dsv</td><td>ΠΒ1</td><td>Alma</td><td>Bjv</td>

<td> 09</td><td></td><td></td><td></td><td></td><td>sz</td><td></td><td></td><td></td><td></td><td> 0/</td><td></td><td></td><td></td><td></td><td> 59</td>

<td>dsv</td><td>For Al</td><td> ٥1</td><td></td><td>dsv</td><td>s) H.</td><td>niP</td><td>nai</td><td>BIV</td><td>BIV</td><td>BIV</td><td>dsv</td><td>BLV</td><td>ΠΙΡ</td><td>USV</td><td>LBA</td>

<td></td><td></td><td></td><td></td><td> 09</td><td></td><td></td><td></td><td></td><td>SS</td><td></td><td></td><td></td><td></td><td>A 0</td><td></td>

<td>2 glands</td><td colspan="2">nLP Ald</td><td>ski</td><td>dsv</td><td>nai</td><td>Aid</td><td>usv</td><td>aqd</td><td>L did 0</td><td>Ald</td><td>ΠΒ1</td><td>For Al</td><td>sAi</td><td>jAi</td><td>Aid</td>

0 C p

UOLTELSUPJT 6U.L1S.11

H - £ 0 -

١٨٨

3943932

S. 204 Thr

Hi_\

640 Asn

Gin

Lys

Tyr

Tyr 720 ng translation 605

Lys Ile Pro His 620

Gly Phe Gly Leu

Pro Val Pro Al a

655

Ser Phe Ile Thr 670

Trp Glu Leu Gin

689

Tyr Thr Ser Asn 700

Thr Asn Gly val

Thr Arg Asn Leu

- 3043 sequence Listi 600

Gin Gly pro Ile Trp Ala 615

Pro Ser Pro Leu Met Gly

630 635

Ile leu Ile Lys Asn Thr 650

Ser Ala Ala Lys Phe Ala 665

Val Ser Val Glu Ile Glu 680

Trp Asn Pro Glu Ile Gin 695

Val Asp Phe Thr Val Asp 710 715

Ile Gly Thr Arg Tyr Leu leu

His

Gin 645 Phe

Gin

AO,

Asn

Pro 725

595

Val Tyr

His Phe

Pro Pro

Arg Asp

610

Asp Gly 625 His Pro

Pro Ser Thr Thr 660

Tyr Ser Thr Gly 675

Glu Asn Ser Lys

690

Asn Lys ser Val 705 ser Glu Pro Arg &lt;20 3 &lt;211 598 &lt;212&gt; PRT

Glandular viruses &lt;213&gt;

Pro

Lys

Glu

5 l

Arg

Val

Pro

Pro

Asp

Ala 30 Pro

Pro

Asn

Thr

Gly

Val 45 Leu

Gly 80 Thr

Glu

Asn

Asn leu

Ser 95 Ala

Asp

Cys

Gly

Ser

Trp 110 Gin

Thr

Tyr

Gly

Trp

Ser 125 Pro

Gin 160 phe

Trp

Asp

Arg

Thr

Asn 175 Gly

Leu

Au

Asp

Thr

Asp 190 phe

Asn

Gly

Gin val 205 His

Tyr 240 Tyr

Gly

Tyr

Gin

Phe

Phe 255 Asn

Ser

Ser

Ala

Tyr

Asn 270 Ser

Gly

Tyr

Gin

Asp

Ser 285 Ile

Gin 320 Gin

Thr

Thr

Thr

Ser

Asp 335 val

Arg

Ile

Au 350

Gin

<td>Met</td><td>Ala</td><td>Pro</td><td>Gly</td><td>lys</td><td>Lys</td><td>Arg</td><td>Pro</td><td>Val</td><td>Ra 1d</td><td>His</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td>

<td>Asp</td><td>Ser</td><td>Ser</td><td>ser</td><td>Gly</td><td>Thr</td><td>Gly</td><td>Lys</td><td>Ala</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td>

<td>Arg</td><td>Leu</td><td>Asn</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Asp</td><td>Ala</td><td>Asp</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td>

<td>Gin</td><td>Pro</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ala</td><td>Ala</td><td>Pro</td><td>Ser</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Met</td><td>Ala</td><td>Thr</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Ala</td><td>Pro</td><td>Met</td><td>Ala</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td>

<td>Ala</td><td>Asp</td><td>Gly</td><td>1 1</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Trp</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td>

<td>Trp</td><td>Met</td><td>Gly</td><td>Asp</td><td>Arg</td><td>Val</td><td>Ile</td><td>Thr</td><td>Thr</td><td>Ser</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td>

<td>Pro</td><td>Thr</td><td>Tyr</td><td>Asn</td><td>Asn</td><td>His</td><td>leu</td><td>Tyr</td><td>Lys</td><td>Gin</td><td>Ile</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td>

<td>Ala</td><td>Ser</td><td>Asn</td><td>Asp</td><td>Asn</td><td>His</td><td>Tyr</td><td>Phe</td><td>Gly</td><td>Tyr</td><td>Ser</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td>

<td>phe</td><td>Asp</td><td>phe</td><td>Asn</td><td></td><td>Phe</td><td>His</td><td>Cys</td><td>His</td><td>Phe</td><td>Ser</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td>

<td>Arg</td><td>leu</td><td>Ile</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Trp</td><td>Gly</td><td>Phe</td><td>Arg</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td>

<td>s for the pain</td><td>Leu</td><td>Phe</td><td>Asn</td><td>Ile</td><td>Gin</td><td>Val</td><td>lys</td><td>Glu</td><td>Val</td><td>Thr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td>

<td>Thr</td><td>Thr</td><td>Ile</td><td>Ala</td><td>Asn</td><td>Asn</td><td>leu</td><td>Thr</td><td>Ser</td><td>Thr</td><td>Val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td>

<td>Ser</td><td>Glu</td><td>Tyr</td><td>Gin</td><td>leu</td><td>Pro</td><td>Tyr</td><td>Val</td><td>leu</td><td>Gly</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td>

<td>leu</td><td>Pro</td><td>Z03</td><td>Phe</td><td>Pro</td><td>Ala</td><td>Asp</td><td>val</td><td>Phe</td><td>Met</td><td>Dla</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td>A 23</td>

<td>Leu</td><td>Thr</td><td>leu</td><td>Asn</td><td>Asn</td><td>Gly</td><td>Ser</td><td>Gin</td><td>Ala</td><td>val</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td>

<td>Cys</td><td>Leu</td><td>Glu</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Ser</td><td>Gin</td><td>Met</td><td>Leu</td><td>K2u</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 269</td><td></td><td></td>

<td>Thr</td><td>Phe</td><td>Ser</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>Glu</td><td>Asp</td><td>val</td><td>Pro</td><td>Phe</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td>

<td>His</td><td>Ser</td><td>Gin</td><td>Ser</td><td>leu</td><td>Asp</td><td></td><td>Leu</td><td>Met</td><td>Asn</td><td>Pro</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td>

<td>leu</td><td>Tyr</td><td>Tyr</td><td>leu</td><td>Ser</td><td>Arg</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Pro</td><td>Ser</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td>

<td>Ser</td><td>Arg</td><td>Leu</td><td>Gin</td><td>Phe</td><td>Ser</td><td>Gin</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Ser</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td>

<td>ser</td><td>Arg</td><td>Asn</td><td>Trp</td><td>leu</td><td>Pro</td><td>Gly</td><td>Pro</td><td>Cys</td><td>Tyr</td><td>Arg</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td>

Ser

Gin

Ser

His

Arg

Ser

S loom

Gin

Gin

Au

Thr

His

Asp

Gin

Ala 220 Pro leu 300 Gly

Gly 60

Asp

Thr 140 Pro

*١٦

3943932

205Lys Thr Ser Ala 355

Thr Lys Tyr His 370

Ala Met Ala Ser 385

Gly Val Leu Ile

Ile Glu Lys Val

420

Pro Val Ala Thr

435

Gly Asn Arg Gin 450

Pro Gly Met val 465

Trp Ala Lys Ile

Met Gly Gly Phe

500

Asn Thr Pro Val

515

Phe Ala Ser Phe

530

Ile Glu Trp Glu 545

Ile Gin Tyr Thr

Val Asp Thr Asn

580

Tyr leu Thr Arg

Asp leu

His

Phe 405 Met

Glu

Ala

Trp

Pro 485 Gly

Pro

Ile leu

Ser 565 Gly

Asn

Asn

Asn

Lys 390 Gly

Ile

Gin

Ala

Gin 470 His

Leu

Ala

Thr

Gin 550 Asn val

Leu

3043 Sequence Listing translation Asn Asn Ser Glu Tyr Ser Trp Thr Gly

Gly Arg Asp Ser 375

Asp Asp Glu Glu

Lys Gin Gly ser

410

Thr Asp Glu Glu 425

Tyr Gly Ser Val 440

Thr Ala Asp Val 455

Asp Arg Asp Val

Thr Asp Gly His

490

Lys His Pro Pro 505

Asn Pro ser Thr 520

Gin Tyr Ser Thr 535

Lys Glu Asn ser

Tyr Asn Lys Ser

570

Tyr Ser Glu Pro

Leu val Asn Pro Gly 380

Lys Phe Phe Pro Gin 395

Glu Lys Thr Asn val

415

Glu Ile Arg Thr Thr

430

Ser Thr Asn Leu Gin

445

Asn Thr Gin Gly Val 460

Tyr leu Gin Gly Pro 475

Phe His Pro Ser Pro

495

Pro Gin Ile leu Ile

510

Thr Phe ser Ala Ala

525

Gly Gin Val Ser Val 540

Lys Arg Trp Asn Pro 555

Val Asn Val Asp Phe

٢15

Arg Pro Ile Gly Thr

Ala

Pro

Ser 400 Asp

Asn leu

Ile 480 leu

Lys

Lys

Glu

Glu 560 Thr &lt;21 598 &lt;212&gt; PRT

Syntax &lt;213&gt;

&lt;220&gt;

Synthetic structure &lt;223&gt;

&lt;400&gt; 4

Met Ala Pro Gly lys lys Arg

Asp Ser Ser Gly Thr Gly 20

Arg Leu Asn Phe Gly Gin Thr 35

Gin Pro Leu Gly Gin Pro Pro 50 55

Thr Met Ala Thr Gly Ser Gly 65 70

Ala Asp Gly Val Gly Asn Ser 85

Trp Met Gly Asp Arg Val Ile 100

Pro Thr Tyr Asn Asn His Leu 115

Ala Ser Asn Asp Asn His Tyr

130 135

Phe Asp Phe Asn Arg Phe His

145 150

Arg leu Ile Asn Asn Asn Trp 165

Lys Leu Phe Asn Ile Gin Val 180

Thr Thr Ile Ala Asn Asn leu

Pro Val Glu His Ser Pro Val Glu Pro 1015

Lys Ala Gly Gin Gin Pro Ala Arg Lys 2530

Gly Asp Ala Asp Ser Val Pro Asp Pro

4045

Ala Ala Pro Ser Gly Leu Gly Thr Asn 60

Ala Pro Met Ala Asp Asn Asn Glu Gly 7580

Ser Gly Asn Trp His Cys Asp Ser Thr 9095

Thr Thr Ser Thr Arg Thr Trp Ala Leu 105 110

Tyr Lys Gin Ile Serum Gin Ser Gly

120125

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 140

Cys His Phe Ser Pro Arg Asp Trp Gin 155 160

Gly Phe Arg Pro Lys Arg Leu Asn phe 170 175

Lys Glu Val Thr Gin Asn Asp Gly Thr 185 190

Thr ser Thr Val Gin Val Phe Thr Asp

١٨٨

3943932

-206] 3043 sequence Listing translation

<td>Ser</td><td>Glu</td><td rowspan="2">Tyr</td><td>Gin</td><td>leu</td><td>Pro</td><td>Tyr</td><td>val</td><td>leu</td><td colspan="2">Gly Ser</td><td>Ala</td><td>His</td><td>Gin</td><td>Gly</td><td>Cys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 2 2 0</td><td rowspan="2">Gin</td><td></td><td rowspan="2">Gly</td><td></td>

<td>Leu</td><td rowspan="2">Pro</td><td>Pro</td><td>Phe</td><td>Pro</td><td>Ala</td><td rowspan="2">Asp</td><td>val</td><td>Phe</td><td>Met</td><td>val</td><td>Pro</td><td>Tyr</td><td>Tyr</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td rowspan="2">Phe</td><td> 240</td>

<td rowspan="2">Leu</td><td rowspan="2">Thr</td><td>leu</td><td>Asn</td><td>Asn</td><td rowspan="2">Gly</td><td>Ser</td><td>Gin</td><td>Ala</td><td>Val</td><td>Gly</td><td></td><td>Ser</td><td>Ser</td><td>Tyr</td>

<td></td><td></td><td> 245</td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td rowspan="2">Gly</td><td></td><td> 255</td><td rowspan="2">Phe</td>

<td>Cys</td><td>leu</td><td>Glu</td><td>Tyr 260</td><td>Phe</td><td>Pro</td><td>Ser</td><td>Gin</td><td>Met 265</td><td>LU</td><td>Ara</td><td>Thr</td><td>Asn 270</td><td>Asn</td>

<td rowspan="2">Thr</td><td>Phe</td><td>Ser</td><td rowspan="2">Tyr</td><td>Thr</td><td>Phe</td><td>Glu</td><td>Asp</td><td>Val</td><td>pro</td><td>Phe</td><td>His</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>1 Ala</td>

<td></td><td> 275</td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td>yen</td><td></td><td rowspan="2">Gin</td><td></td>

<td rowspan="2">His</td><td>Ser</td><td>Gin</td><td>ser</td><td>leu</td><td rowspan="2">Asp</td><td>Ara</td><td>leu</td><td>Met</td><td>Asn</td><td>Pro</td><td>leu</td><td>Ile</td><td>Asp</td><td>Tyr</td>

<td> 290</td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td rowspan="2">Thr</td><td rowspan="2">Thr</td><td rowspan="2">Gin 320</td>

<td>c 305</td><td>Tyr</td><td>Tyr</td><td>LU</td><td>Ser</td><td>Ntu 310</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Pro</td><td>Ser 315</td><td>Gly</td><td>Thr</td>

<td rowspan="2">Ser</td><td rowspan="2">Arg</td><td>leu</td><td>Gin</td><td>Phe</td><td>Ser</td><td>Gin</td><td>Ala</td><td>Gly</td><td>Ala</td><td>Ser</td><td>Asp</td><td>1 Ile</td><td>Arg</td><td>Asp</td><td>Gin</td>

<td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td rowspan="2">Gin</td><td></td><td> 335</td><td></td>

<td>Ser</td><td rowspan="2">Ntu</td><td>Asn</td><td>Trp</td><td>LU</td><td>Pro</td><td>Gly</td><td>Pro</td><td>cys</td><td>Tyr</td><td>Ara</td><td>Gin</td><td>Arg</td><td>Val</td><td>Ser</td>

<td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td rowspan="2">Gly</td><td rowspan="2">Ala</td>

<td rowspan="2">lys</td><td>Thr</td><td>Ser</td><td>Ala</td><td rowspan="2">Asp</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Ser</td><td>Glu</td><td>Tyr</td><td>Ser</td><td>Trp</td><td>Thr</td>

<td></td><td> 355</td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 369</td><td></td><td rowspan="2">Gly</td><td></td>

<td>Thr</td><td>Lys</td><td rowspan="2">Tyr</td><td>His</td><td>Lu</td><td>Asn</td><td>Gly</td><td>Arg</td><td>Asp</td><td>ser</td><td>LU</td><td>val</td><td>Asn</td><td>Pro</td><td>Pro</td>

<td></td><td> 370</td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 3 8 0</td><td rowspan="2">Phe</td><td></td><td rowspan="2">Gin</td><td></td>

<td>Ala</td><td>Met</td><td>Ala</td><td>ser</td><td>His</td><td>Lys</td><td rowspan="2">Asp</td><td>Asp</td><td>Glu</td><td>Glu</td><td>lys</td><td>phe</td><td>Pro</td><td>Ser</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td> 395</td><td></td><td rowspan="2">Thr</td><td></td><td rowspan="2">Val</td><td> 400</td>

<td rowspan="2">Gly</td><td>Val</td><td>LU</td><td>Ile</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">lys</td><td>Gin</td><td>Gly</td><td>Ser</td><td>Glu</td><td>Lys</td><td>Asn</td><td>Asp</td>

<td></td><td></td><td></td><td> 405</td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td rowspan="2">Thr</td><td> 415</td><td></td>

<td>Ile</td><td>Glu</td><td rowspan="2"></td><td>C n</td><td>Met</td><td>Ile</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Ile</td><td>Arg</td><td>Thr</td><td>Asn</td>

<td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td rowspan="2">Gin</td><td rowspan="2">Ala</td>

<td rowspan="2">Pro</td><td>Val</td><td>Ala</td><td>Thr</td><td>Glu</td><td>Gin</td><td rowspan="2">Tyr</td><td>Gly</td><td>ser</td><td>Val</td><td>Ser</td><td>Thr</td><td>Asn</td><td>leu</td>

<td></td><td> 435</td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td rowspan="2">Gly</td><td rowspan="2">Val</td><td></td>

<td rowspan="2">Gly</td><td>Asn</td><td>Ala</td><td>Gin</td><td>Ala</td><td>Ala</td><td>Thr</td><td>Ala</td><td>Asp</td><td>Val</td><td>Asn</td><td>Thr</td><td>Gin</td><td>leu</td>

<td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td rowspan="2">Gin</td><td rowspan="2">Gly</td><td></td><td rowspan="2">Ile</td>

<td>Pro</td><td rowspan="2">Gly</td><td>Met</td><td>val</td><td rowspan="2">Trp</td><td>Gin</td><td rowspan="2">Asp</td><td></td><td>Asp</td><td>Val</td><td>Tyr</td><td>Lu</td><td>Pro</td>

<td> 465</td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td> 475</td><td rowspan="2">His</td><td></td><td></td><td></td><td> 480</td>

<td rowspan="2">Trp</td><td>Ala</td><td rowspan="2">Lys</td><td>Ile</td><td>Dr</td><td>His</td><td>Thr</td><td>Asp</td><td>Gly</td><td>His</td><td>Phe</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Lu</td>

<td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td rowspan="2">Ile</td><td></td><td> 495</td><td></td>

<td>Met</td><td colspan="2">Glv Gly</td><td>phe</td><td rowspan="2">Gly</td><td>leu</td><td>Lys</td><td>His</td><td></td><td>Pro</td><td>Pro</td><td>Gin</td><td>Lu</td><td>Ile</td><td>Cys</td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td rowspan="2">Ala</td><td></td>

<td rowspan="2">Asn</td><td>Thr</td><td>Pro</td><td>Val</td><td>Pro</td><td>Ala</td><td>Asn</td><td>Pro</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Phe</td><td>Ser</td><td>Ala</td><td>Lys</td>

<td></td><td> 515</td><td></td><td></td><td></td><td></td><td> 520</td><td></td><td></td><td></td><td></td><td> 525</td><td></td><td rowspan="2">Val</td><td rowspan="2">Glu</td>

<td>Phe</td><td>Ala</td><td>Ser</td><td>Phe</td><td>Ile</td><td>Thr</td><td>Gin</td><td rowspan="2">Tyr</td><td>ser</td><td>Thr</td><td>Gly</td><td>Gin</td><td>Val</td><td>Ser</td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td> 535</td><td></td><td></td><td></td><td> 540</td><td></td><td></td><td></td><td rowspan="2">Glu</td>

<td>Ile</td><td>Glu</td><td rowspan="2">Trp</td><td>Glu</td><td>leu</td><td>Gin</td><td rowspan="2"></td><td>Glu</td><td>Asn</td><td>Ser</td><td>Lys</td><td>Arg</td><td>Trp</td><td>Asn</td><td>Pro</td>

<td> 545</td><td></td><td></td><td></td><td> 550</td><td></td><td></td><td></td><td> 555</td><td></td><td rowspan="2">Val</td><td></td><td rowspan="2">Phe</td><td> 560</td>

<td>Ile</td><td>Gin</td><td rowspan="2">Tyr</td><td>Thr</td><td>ser</td><td>Asn</td><td rowspan="2">Tyr</td><td>Asn</td><td>lys</td><td>ser</td><td>Val</td><td>Asn</td><td>Asp</td><td>Thr</td>

<td></td><td></td><td></td><td> 565</td><td></td><td></td><td></td><td> 570</td><td></td><td></td><td rowspan="2">Ile</td><td rowspan="3">Gly 590</td><td> 575</td><td></td>

<td>val</td><td>Asp</td><td>Thr</td><td rowspan="2">Asn 580 Arg</td><td>Gly</td><td>Val</td><td>Tyr</td><td>Ser</td><td rowspan="2">Glu 585</td><td>Pro</td><td></td><td>Pro</td><td>Thr</td><td>Arg</td>

<td>Tyr</td><td>LU</td><td>Thr</td><td>Asn</td><td>leu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;20 5 &lt;21 533 &lt;212&gt; PRT

Glandular viruses &lt;213&gt;

&lt;400&gt; 5

Met Ala Thr Gly Ser Gly Ala Pro Met

Asp Gly val Gly Asn Ser Gly Asn 2025

Met Gly Asp Arg val Ile Thr Thr Ser 3540

Thr Tyr Asn Asn His Leu Tyr Lys Gin 5055

Ser Asn Asp Asn His Tyr phe Gly Tyr 6570

Asp Phe Asn Arg phe His Cys His Phe

Al a Asp Asn Asn 1 What Gly Al a 10 15

Trp His cys Asp Ser Thr Trp 30

Thr Arg Thr Trp Ala Leu Pro 45

Ile Ser Ser Gin Ser Gly Ala 60 ser Thr Pro Trp Gly Tyr Phe

80

Ser Pro Arg Asp Trp Gin Arg

fill

3943932

-207] 3043 sequence Listing translation

<td rowspan="2">leu</td><td rowspan="2">Ile,</td><td>Asn</td><td>Asn,</td><td>Asn</td><td rowspan="2">Trp</td><td rowspan="2">Gly</td><td>Phe</td><td>Arg</td><td>Pto</td><td>lys</td><td>AtQ</td><td>leu</td><td>Asn</td><td>Phe</td><td>lys</td>

<td></td><td> 100</td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td rowspan="2">Thr</td><td rowspan="2">Thr</td>

<td rowspan="2">leu</td><td rowspan="2">Phe</td><td>Asn</td><td>Ile</td><td>Gin</td><td>Val</td><td rowspan="2">lys</td><td>Glu</td><td></td><td>Thr</td><td>Gin</td><td>Asn</td><td>Asp</td><td>Gly</td>

<td> 115</td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td rowspan="2">Thr</td><td></td><td></td>

<td rowspan="2">Thr</td><td>Ile</td><td>Al a</td><td>Asn</td><td>Asn</td><td>leu</td><td>Thr</td><td>ser</td><td>Thr</td><td>Val</td><td>Gin</td><td>Val</td><td>Phe</td><td>Asp</td><td>Ser</td>

<td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td rowspan="2">Gin</td><td rowspan="2">Gly</td><td></td><td></td>

<td>Glu 145</td><td>Tyr</td><td>Gin</td><td>leu</td><td>Pro</td><td>Tyr 150</td><td>val</td><td>Leu</td><td>Gly</td><td>Ser</td><td>Al a 155</td><td>His</td><td>Cys</td><td>leu 160</td>

<td rowspan="2">Pro</td><td rowspan="2">Pro</td><td>Phe</td><td>Pro</td><td>Al a</td><td rowspan="2">Asp</td><td>Val</td><td>Phe</td><td>Met</td><td>Val</td><td>Pro</td><td>Gin</td><td>Tyr</td><td>1 Gly</td><td>Tyr</td><td>Leu</td>

<td></td><td></td><td> 165</td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td rowspan="2">Phe</td><td> 175</td><td></td>

<td rowspan="2">Thr</td><td>leu</td><td>Asn</td><td>Asn</td><td rowspan="2">Gly</td><td>ser</td><td>Gin</td><td>Al a</td><td>val</td><td>Gly</td><td>RtQ</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>cys</td>

<td></td><td></td><td> 180</td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td rowspan="2">Phe</td><td rowspan="2">Thr</td>

<td rowspan="2">leu</td><td>Glu</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Ser</td><td>Gin</td><td>Met</td><td>leu</td><td>Arg</td><td>Thr</td><td>Gly</td><td>Asn</td><td>Asn</td>

<td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td rowspan="2">Al a</td><td rowspan="2">His</td>

<td rowspan="2">Phe</td><td>Ser</td><td rowspan="2">Tyr</td><td>Thr</td><td>Phe</td><td>Glu</td><td>Asp</td><td>val</td><td>Pro</td><td>Phe</td><td>His</td><td>Ser</td><td>Ser</td><td>Tyr</td>

<td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td rowspan="2">Gin</td><td></td><td></td>

<td>Ser</td><td>Gin</td><td>Ser</td><td>Leu</td><td rowspan="2">Asp</td><td>Arg</td><td>Leu</td><td>Met</td><td>Asn</td><td>Pro</td><td>leu</td><td>Ile</td><td>Asp</td><td>Tyr</td><td>leu</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 239</td><td></td><td rowspan="2">Thr</td><td rowspan="2">Thr</td><td rowspan="2">Gin 255</td><td> 240</td>

<td>Tyr</td><td>Tyr</td><td>leu</td><td>ser</td><td>Arg 245</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Pro</td><td>Ser 250</td><td>Gly</td><td>Thr</td><td>Ser</td>

<td rowspan="2">Arg</td><td rowspan="2">leu</td><td>Gin</td><td>Phe</td><td>Ser</td><td>Gin</td><td>Al a</td><td rowspan="2">Gly</td><td>Al a</td><td>Ser</td><td>Asp</td><td>Ile</td><td>Arg</td><td>Asp</td><td>Gin</td><td>ser</td>

<td></td><td> 260</td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td rowspan="2">Arg</td><td>Asn</td><td>Trp</td><td>Leu</td><td>Pro</td><td>Gly</td><td>Pro</td><td>lys</td><td>Tyr</td><td>Amlog</td><td>Gin</td><td>Gin</td><td>Arg</td><td>val</td><td>Ser</td><td>Lys</td>

<td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td rowspan="2">Gly</td><td rowspan="2">Al a</td><td rowspan="2">Thr</td>

<td>Thr</td><td>Ser</td><td>Al a</td><td rowspan="2">Asp</td><td>Asn</td><td>Asn</td><td>Asn</td><td>Ser</td><td>Glu</td><td>Tyr</td><td>Ser</td><td>Trp</td><td>Thr</td>

<td></td><td> 290</td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td rowspan="2">Gly</td><td></td><td rowspan="2">Al a 320</td>

<td> 305</td><td>Tyr</td><td>His</td><td>leu</td><td>Asn</td><td>Gly 310</td><td>A39</td><td>Asp</td><td>Ser</td><td>leu</td><td>val 319</td><td>Asn</td><td>Pro</td><td>Pro</td>

<td>Met</td><td>Al a</td><td>Ser</td><td>His</td><td>Lys</td><td rowspan="2">Asp</td><td>Asp</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Phe</td><td>Phe</td><td>Pro</td><td>1 Gin</td><td>Ser</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td rowspan="2">Val</td><td> 335</td><td rowspan="2">Ile</td>

<td>val</td><td>leu</td><td>Ile</td><td>Phe</td><td rowspan="2">Gly</td><td rowspan="2">lys</td><td>Gin</td><td>Gly</td><td>Ser</td><td>Glu</td><td>Lys</td><td>Thr</td><td>Asn</td><td>Asp</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td rowspan="2">Ihr</td><td> 3 5 0</td><td></td><td></td>

<td rowspan="2">Glu</td><td rowspan="2">lys</td><td>Val</td><td>Met</td><td>Ile</td><td>Thr</td><td>Asp</td><td>Glu</td><td>Glu</td><td>Glu</td><td>Ile</td><td>Arg</td><td>Thr</td><td>Asn</td><td>Pro</td>

<td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td rowspan="2">Gin</td><td></td><td rowspan="2">Gly</td>

<td rowspan="2">val</td><td>Al a</td><td>Thr</td><td>Glu</td><td>Gin</td><td rowspan="2">Tyr</td><td>Gly</td><td>Ser</td><td>Val</td><td>Ser</td><td>Thr</td><td>Asn</td><td>Leu</td><td>Arg</td>

<td> 370</td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td rowspan="2">Gly</td><td rowspan="2">Val</td><td></td><td></td>

<td>Asn</td><td rowspan="2">Arg</td><td>Gin</td><td>Al a</td><td>Al a</td><td>Thr</td><td>Al a</td><td>Asp</td><td>Val</td><td>Asn</td><td>Thr</td><td>Gin</td><td>leu</td><td>Pro</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td rowspan="2">Gin</td><td rowspan="2">Gly</td><td></td><td rowspan="2">Ile</td><td> 400</td>

<td rowspan="2">Gly</td><td>Met</td><td>Val</td><td rowspan="2">Trp</td><td>Gin</td><td rowspan="2">Asp</td><td>Arg</td><td>Asp</td><td>Val</td><td>Tyr</td><td>leu</td><td>Pro</td><td>Trp</td>

<td></td><td></td><td> 405</td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> ٦ ٠٦ 415</td><td></td>

<td>Al a</td><td rowspan="2">L 1</td><td>Ile</td><td>Pro</td><td>His</td><td>Thr</td><td rowspan="2">Asp</td><td>Gly</td><td>His</td><td>Phe</td><td>His</td><td>Pro</td><td>Ser</td><td>Pro</td><td>leu</td><td>Met</td>

<td></td><td></td><td> 420</td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td rowspan="2">Ile</td><td></td><td> 430</td><td></td><td></td>

<td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Phe</td><td rowspan="2">Gly</td><td>leu</td><td>lys</td><td>His</td><td>Pro</td><td> 000</td><td>Pro</td><td>Gin</td><td>Leu</td><td>Ile</td><td>lys</td><td>Asn</td>

<td> 435</td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td rowspan="2">Al a</td><td></td><td rowspan="2">Phe</td>

<td rowspan="2">Thr</td><td>Pro</td><td>ya6</td><td>Pro</td><td>Al a</td><td>Asn</td><td>Pro</td><td>ser</td><td>Thr</td><td>Thr</td><td>Phe</td><td>Set</td><td>Al a</td><td>lys</td>

<td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td rowspan="2"></td><td rowspan="2">Glu</td><td rowspan="2">Ile</td>

<td>Al a</td><td rowspan="2">Ser</td><td>Phe</td><td>Ile</td><td>Thr</td><td>Gin</td><td rowspan="2">Tyr</td><td>ser</td><td>Thr</td><td>Gly</td><td>Gin</td><td>val</td><td>Ser</td>

<td> 465</td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td rowspan="2">Glu</td><td>0 8 ah</td>

<td rowspan="2">Glu</td><td rowspan="2">Trp</td><td>Glu</td><td>leu</td><td>Gin</td><td rowspan="2">Lys</td><td>Glu</td><td>Asn</td><td>ser</td><td>lys</td><td>Arg</td><td>Trp</td><td>Asn</td><td>Pto</td><td>Ile</td>

<td></td><td></td><td> 485</td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td rowspan="2">Val</td>

<td>Gin</td><td rowspan="2">Tyr</td><td>Thr</td><td>Ser</td><td>Asn</td><td rowspan="2">Tyr</td><td>Asn</td><td>lys</td><td>Ser</td><td>Val</td><td>Asn</td><td>Val</td><td>Asp</td><td>Phe</td><td>Thr</td>

<td></td><td></td><td> 500</td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td> 510</td><td> ١</td><td></td>

<td rowspan="2">Asp</td><td>Thr</td><td>Asn</td><td rowspan="2">Gly</td><td>Val</td><td>Tyr</td><td>Ser</td><td>Glu</td><td>Pro</td><td>Arg</td><td>Pro</td><td colspan="2">Ile Gly</td><td>Thr</td><td>Arg</td><td>Tyr</td>

<td></td><td> 515</td><td></td><td></td><td></td><td> 520</td><td> ١</td><td></td><td></td><td></td><td>Ded</td><td></td><td></td><td></td>

<td>leu</td><td>Thr</td><td rowspan="2">Arg</td><td>Asn</td><td>Leu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 530</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

533 PRT

Industrial progression &lt;210&gt;

&lt;211 &lt;212&gt;

&lt;213&gt;

Asp Asn Asn Glu Gly Al a

Met Al a

Al a Pro

Synthetic structure &lt;220&gt;

&lt;223&gt;

* -an

Met Al a Thr Gly Ser Gly

What

fill

3943932

-208] 3043 sequence Listing translation

Asp

Gly

Solution

Met

Gly

Thr

Ser 65 Asp

Tyr 50 Asn

Asp 35 Asn

Asp leu leu

Thr

Glu 145 Pro

Thr leu

Phe

Ser 225 Tyr

Arg

Arg

Thr

Lys 305 Met

Val

What my

Phe

Ile

Phe

Ile 130 Tyr

Pro

Leu

Glu ser 210 Gin

Tyr leu

Asn

Asn

Asn

Ser 290 Tyr

Asn 115 Al a

Al a leu

Lys

Gin

Phe

Asn

Tyr 195 Tyr

Ser leu

Asn 385 Gly

Al a 370 Al a

Met

Al a

Lys

Gly Gly

Thr

Al a 465 Glu

Pro 450 Ser

Trp

Gin

Tyr

Asp

Thr

Leu

Gly 20

Arg

Asn

Asn

Arg

Asn 100 Ile

Asn leu

Pro

Asn 180 Phe

Thr

Leu

Gin

Trp 275 Al a

Hi s

Ser

Ile

Val 355 Thr

Gin val

Ile

Phe 435 val

Phe

Glu

Thr

Asn ser ser ser Gly Asn

Val

H, s

Hic

Phe 85 Asn

Gin

Asn

Pro

Al a 165 Gly

Pro

Phe

Asp

Ile

Tyr 70 His

Trp

Val eu

Tyr 150

Asp

Ser

Ser

Glu

Thr Thr

Tyr 55 Phe

Cys

Gly

Lys

Thr 135 val

Val

Gin

Gin

Lys

Ser

Gin

Gly

Tyr ms

Phe

Glu 120 Ser leu

Phe

Al a

Phe

١٣٩

105 val

Thr

Gly

Met

Trp His

Thr Arg

Ile

Ser

Ser

Ser 90 Pro

Thr val

Ser

Cys

Thr

Asp

Thr

530

Asn 515 Ara

Thr 75 Pro

Ser 60 Pro

Arg lys

Gin

Gin

Arg

Asn

Trp 45 Gin

Trp

Asp leu

Ser 30 Al a

Ser

Gly

Trp

Thr Trp leu

Pro

Gly

Tyr

Al a

Phe

260 leu

Asp

Leu

Hi S

Phe 340 Met

Glu

Al a

Trp

Pro 420 Gly

Pro

Ile leu

Ara 245 ser

١٣٩

230

Thr

Asp 215 leu

Met 200 Val val 185 Leu

Pro

Pro

Asn

Asn

Lys 325 Gly

Ile

Gin

Al a

Gin 405 His leu

Al a

Thr

Met

Asn

Gin

Gly

Asn

Gly 310

Asp

Lys

Thr

Tyr

Thr 390 Asp

Thr lys

Asn

Asn

Thr

Pro

Al a

Gly

Pro

Asn 295 Arg

Asp

Gin

Asp

Gly 375 Al a

Ara

Asp ms

Cys 280 ser

Al a 265 Tyr

Glu

Asp

Glu

Gly

Glu 360 ser

Asp

Asp

Gly

Ser

Glu

Ser 345 Glu val val

Val

Ser 500 Gly

Gin 485 Asn

Gin 470 Lys

Pro 455 Tyr

Pro

440 ser

Ser

Glu

Asn

Asn val leu

Tyr

Tyr

Asn

Ser

Lys

Glu

520 val 170 Gly

Al a 155 Pro

140 His

Asp 125 phe

Asn 110 Gly

Gin 95 Phe

Thr

Gin

Thr

Gly

Gin

Tyr

Gly

Arg

Phe

Pro

Ser 250 Ser

١٣٩

Tyr leu

Lys 330 Glu

Glu ser

Asn

Asp cys

١٣٩

Ser

Ser

Thr

Gly

HTS leu 235 Gly

Asp

Gin

Ser

Dl

315 phe

Lys

Ile

Thr

Ser

220 Ile

Asn 205 Ser

Phe 190 Asn

Tyr

Thr

Ile

Gin

Trp 300 Asn

Phe

Thr

Arg

Asp

Thr

Ara 285 Thr

Pro

Pro

Asn

Gin

Thr

Asp 270 \ 1T

Gly

Gly

Gin

Tyr 175 Tyr

Phe

Al a

Tyr

Gin 255 Gin ser ms 425 Pro

Thr

Thr

Ser

Ser 505 Pro

Tyr 410 Phe

Thr 395 leu

Asn 380 Gin

Thr 365 leu

Val 350 Thr

Gin

Gin

Pro

Thr

Gly

Lys 490 Val

١٣٩

His

Gin

Phe

Gin 475 Arg

Asn

Pro

Pro

Ile

Ser 460 val

Trp

Val

Ile

Gly

Gly

Ser leu 445 Al a

Ser

Asn

Asp

Gly 525 pro

٢٣0

430

Ile

Al a

Val

Pro

Phe 510 Thr

Phe 80 Arg

Al a

Pro

Lys

Thr

Ser 335 Asp

Asn ser

Al a leu

Ile

415 Leu lys leu 160 Leu cys

Thr

Hl S

Leu 240 Ser

Ser

Lys

Thr

Al a 320 Gly

Ile

Pro

Gly

Pro 400 Trp

Met

Asn lys

Phe

Glu

Glu 495 Thr

Arg

Ile 480 Ile

Val

Tyr

fill

3943932

-209] 3043 sequence Listing translation &lt;20 7 &lt;211 736 &lt;212&gt; PRT

Glandular viruses &lt;213&gt;

leu ser 15 Gin Pro leu Pro

Glu Pro

Tyr Asp

His Ala 95 Gly Gly

Glu Pro

39 no

7al Gly

160 Gin Thr 175 Pro

Gly Gly

Asn Ser

Val Ile 240 His Leu 255 His Tyr

Phe His

Asn Trp

Gin val 320 Asn leu 335 Pro Tyr

Ala Asp

Gly Ser

Pro Ser 400 Phe Glu 415 Asp Arg

Arg Thr

Phe Ser

Pro her

480 Asp Asn 495

Glu Asp Asn

Gly val Pro 30

Gly Leu val 45

Asp Lys Gly 60

Asp Lys Ala

Lys Tyr Asn

Phe 110 leu

Lys

Gly

Thr Ser

Arg Ile

A 12

Pro Gly

140

Ser Ser

Asn Phe Gly leu Gly Glu 190 Ala Ser Gly 205

Gly Val Gly 220

Gly Asp Arg

Tyr Asn Asn

Asn Asp Asn

270

Phe Asn Arg

285

Ile Asn Asn

300

Phe Asn Ile

Ile Ala Asn

Tyr Gin Leu

350

Pro phe pro lah Asn Asn 380

Glu Tyr Phe

Ser Tyr Thr

Gin Ser Leu

430

Tyr leu Asn 445

Arg leu leu 460

Arg Asn Trp

Thr Ala Asn

Lys Tyr His

Pro Asp Trp leu 10

Ala leu Lys pro 25

Asp Asn Arg Arg 40

Gly Asn Gly leu

Ala Leu Glu His

Asn Pro Tyr leu 90 leu Gin Glu Asp

105

Gin Ala Lys Lys 120

Ala Lys Thr Ala

Glu Pro Asp Ser

155

Arg Lys Arg leu

170

Asp Pro Gin Pro

185

Ser Asn Thr Met

200

Glu Gly Ala Asp

Ser Gin Trp leu 235

Ala Leu Pro Thr

250

Ser Gly Ala Ser

265

Gly Tyr phe Asp

280

Trp Gin Arg Leu

Ser Phe Lys leu

315

Gly Thr Thr Thr

330

Thr Asp Ser Glu

345

Gly Cys Leu Pro 360

Gly Tyr Leu Thr

Phe Tyr Cys Leu 395 Asn Phe Gin Phe 410

Tyr Ala His Ser 425

Gin Tyr Leu Tyr 440

Thr Asn Gin Ser

Ser Leu Gin Ala

475

Arg leu Ser Lys 490

Thr Ala Ala Ser

Gly Tyr leu Glu Trp Trp Gin His Gin leu Gly Pro 55

Asp Ala Ala 70

Ala Gly Asp 85

Gin Glu Arg

Ala Val Phe &lt;400&gt; 7

Met Ala Ala Asp 1

Glu Gly Ile Arg

Lys Ala Asn Gin

Gly Tyr Lys Tyr 50 val Asn Glu Ala 65

Gin Gin leu Lys

Glu Ala 135

Pro Gin 150

Pro Ala val Pro

Glu ser

Gin 165 Ser

Ser leu Gly

Asn 215 Asp

Trp

Gin

Asp Asn

His Cys

230

Arg Thr 245

Ser Ser

Thr Pro Trp

Pro Arg Asp

295

Lys Lys leu 310

Gin Asn Asp 325

Gin Val Phe

Ala His Gin

Gin Tyr

Pro

375

Arg Ser Ser 390

Thr Gly Asn 405

His ser Ser

Leu Ile Asp

Ser Gly Thr

455

Gin Ser Met

470

Arg Gin Gin 485

Phe Pro Trp

Asp Ala Glu Phe

100

Asn leu Gly Arg 115

Leu Gly Leu Val

130 pro val Asp Gin 145

Lys Ser Gly Lys

Gly Asp Ser Glu

180

Ala Ala Pro Thr

195

Ala Pro Met Ala

210

Ser Gly Asn Trp 225

Thr Thr Ser Thr

Ile

260 ser

Dr

Thr

6 D1

340 Ser val

Gly

Arg

Phe 420 Pro

Thr

Pro

Tyr

Tyr Lys Gin

Phe Gly Tyr

275

Cys His Phe

290

Gly Phe Arg 305

Lys Glu Val

Thr ser Thr

Gly

355

Met val

Leu val Leu

Val Phe

370

Gin Ala 385

Gin Met

Asp Val Pro Lamha Met Asn 435

Gin Gly Thr

450

Gin Ala Gly 465

Gly Pro Cys

Asn Asn Ser Asn

*١٦

3943932

-210] 3043 sequence Listing translation

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td rowspan="2">Ala 525</td><td> 510</td><td rowspan="2">His</td><td rowspan="2">Lys</td>

<td>Gly</td><td colspan="2">Arg Asp ? 1?</td><td>Ser</td><td>Leu</td><td>Val</td><td>Asn</td><td>Pro 520</td><td>Gly</td><td>Pro</td><td>Ala</td><td>Met</td><td>ser</td>

<td>Asp</td><td>Asp 530</td><td>Glu</td><td>Glu</td><td>Lys</td><td>Phe</td><td>Phe 535</td><td>Pro</td><td>Met</td><td>His</td><td colspan="2">Gly Asn 540</td><td>Leu</td><td>1 Ile</td><td>Phe</td><td>1 Gly</td>

<td>Lys 545 Thr</td><td>Glu Asp</td><td>Gly Glu</td><td>Thr Glu</td><td>2hr Glu 565</td><td>Ala 550</td><td>Ser AtQ</td><td>Asn Thr</td><td>Ala Thr</td><td>Glu Asn 570</td><td>Lu 555 Pro</td><td>Asp val</td><td>Asn Ala</td><td>1 val Thr</td><td>Met Glu 575</td><td>6 Ile 560 Gin</td>

<td>Tyr</td><td>Gly</td><td>Thr</td><td>val 580</td><td>Ala</td><td>Asn</td><td>Asn</td><td>leu</td><td>Gin 585</td><td>ser</td><td>Ser</td><td>Asn</td><td>Thr</td><td>6 Ala 590</td><td>Pro</td><td>h Thr</td>

<td>Thr</td><td></td><td>Thr 595</td><td>1 1</td><td>Asn</td><td>Asp</td><td>Gin</td><td>Gly 600</td><td>Ala</td><td>leu</td><td>Pro</td><td>Gly</td><td>Met 605</td><td>Val</td><td>Trp</td><td>1 Gin</td>

<td>Asp</td><td>Kvq 610</td><td>Asp</td><td>YaT</td><td>Tyr</td><td>leu</td><td>Gin 615</td><td>Gly</td><td>Pro</td><td>Ile</td><td>Trp</td><td>Ala 620</td><td>Lys</td><td>1 Ile</td><td>Pro</td><td>His</td>

<td>Thr 625</td><td>Asp</td><td>Gly</td><td>His</td><td>Phe</td><td>His 630</td><td>Pro</td><td>Ser</td><td>Pro</td><td>Lu</td><td>Met 635</td><td>Gly</td><td>Gly</td><td>M Phe</td><td>6 Gly</td><td>leu 640</td>

<td>Lys</td><td>His</td><td>Pro</td><td>Pro</td><td>Pro 645</td><td>Gin</td><td>Ile</td><td>Met</td><td>Ile</td><td>Lys 650</td><td>Asn</td><td>Thr</td><td>Pro</td><td>6 val</td><td>Pro 655</td><td>Ala</td>

<td rowspan="2">Asn</td><td rowspan="2">Pro</td><td>Pro</td><td>Thr</td><td>Thr</td><td>Phe</td><td>Ser</td><td>Pro</td><td>Ala</td><td>Lys</td><td>Phe</td><td>Ala</td><td>Ser</td><td>Phe</td><td>Ile</td><td>Thr</td>

<td></td><td> 660</td><td></td><td></td><td></td><td></td><td> 665</td><td></td><td></td><td></td><td></td><td> 670</td><td></td><td rowspan="2">Gin</td>

<td rowspan="2">Gin</td><td rowspan="2">Tyr</td><td>Ser</td><td>Thr</td><td rowspan="2">Gly</td><td>Gin</td><td>Val</td><td>Ser</td><td>Val</td><td>Glu</td><td>Ile</td><td>Glu</td><td>Trp</td><td>Glu</td><td>leu</td>

<td> 675</td><td></td><td></td><td></td><td> 680</td><td></td><td></td><td></td><td></td><td> 685</td><td rowspan="2">Thr</td><td></td><td></td>

<td>lys</td><td>Why?</td><td>Asn</td><td></td><td>No 1</td><td>Arg</td><td>Trp</td><td>Asn</td><td>Pro</td><td>Glu</td><td>Ile</td><td>Gin</td><td>Tyr</td><td>Ser</td><td>Asn</td>

<td>Tyr 705</td><td>690 Asn</td><td>lys</td><td>Ser</td><td>Val</td><td>Asn 710</td><td>695 val</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Val 715</td><td>7 0 0 Asp</td><td>Thr</td><td>Asn</td><td>Gly</td><td>val 720</td>

<td>Tyr</td><td>Ser</td><td>Glu</td><td>Pro</td><td>ktu 725</td><td>Pro</td><td>Ile</td><td>Gly</td><td>Thr</td><td>Arg 730</td><td>Tyr</td><td>leu</td><td>Thr</td><td></td><td>Asn 735</td><td>leu</td>

&lt;20 8 &lt;21 78 &lt;212&gt; DNA

Syntax &lt;213&gt;

Synthetic structure &lt;223&gt;

cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccacgc 60 ccgggctttg cccgggcg 78 &lt;21 9 &lt;211&gt; 736 &lt;212&gt; PRT

Syntax &lt;213&gt;

&lt;220&gt;

Synthetic structure &lt;223&gt;

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp leu Glu Asp Asn Leu Ser Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro Lys Ala Asn Gin Gin Lys Gin Asp Gly Arg Gly Leu val leu Pro Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly leu Asp Lys Gly Glu Pro Val Asn Ala Ala Asp Ala Ala Ala leu Glu His Asp Lys Ala Tyr Asp Gin Gin Leu Lys Ala Gly Asp Asn Pro Tyr leu Arg Tyr Asn His Ala Asp Ala Glu Phe Gin Glu Arg Leu Gin Glu Asp Thr Ser Phe Gly Gly Asn Leu Gly Arg Ala Val Phe Gin Ala Lys Lys Arg Val leu Glu Pro leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

*١٦

3943932

-211130

Pro Val

Glu

Gin

Ser

3043 T sequence Listing translation

140

145

Lys

Thr

Gly

Gin

Gly

Asp

Ser

Al a

Al a

Al a

Ser 225 Thr

Pro

210 Gly

Thr

Pro 195 Met

Asn

Ser

Tyr

Thr

NS

Gin

Phe

Asn 305 Gin

Asn

Pro

Al a

Gly 385 Pro

Phe

Asp

Tyr

His 290 Trp

Phe 275 Cys

Gly

Val leu