Lysosomal storage disease enzyme

The present invention provides LIPASE COMPOSITIONS ACID lysosomal RECOMBINANT HUMAN HAVING glycosylation patterns HOUSEHOLDS FOR INTERNALISATION CELL TARGET, A CONTAINING VECTOR ACID NUCLEIC ENCODING A LIPASE ACID lysosomal HUMAN, A host cell transformed with VECTOR, Pharmaceutical Compositions UNDERSTANDING LIPASE ACID RECOMBINANT HUMAN LYSOSSOMAL AND METHOD OF TREATMENT OF STATES ASSOCIATED WITH LYSOSSOMAL ACID LIPASE.

RESUME

LYSOSOMAL STORAGE DISEASE ENZYME

The present invention provides recombinant human lysosomal acid lipase compositions having particular glycosylation patterns for internalization in target cells, a vector containing the nucleic acid encoding human lysosomal acid lipase, a host cell transformed with the vector, pharmaceutical compositions comprising acid lipase. recombinant human lysosomal and method of treating conditions associated with lysosomal acid lipase deficiency.

DESCRIPTION

LYSOSOMAL STORAGE DISEASE ENZYME

RELATED ORDERS

This claim claims priority from Provisional US Order No.² 61/343177 filed April 23, 2010, Provisional US Application No.² 61/396376, filed May 26, 2010, Provisional US Application No.² 61/403011 filed September 9, 2010, Provisional US Application No.² 61/456014 filed October 29, 2010, Provisional US Application No.² 61/432372 filed January 13, 2011.

BACKGROUND OF THE INVENTION

Lysosomal Acid Lipase Deficiency (LAL) is a very rare lysosomal storage disease (LSD) characterized by an inability to degrade cholesterol (EC) and triglyceride (TAG) esters in lysosomes due to an enzyme deficiency. LAL deficiency is similar to other lysosomal storage diseases, with substrate accumulation in various tissues and cell types. In LAL deficiency, substrate accumulation is most marked in reticuloendothelial system cells, including Kupffer cells in the liver, histiocytes in the spleen and in the small intestine lamina propria. Reticuloendothelial cells express the macrophage mannose / N-acetylglucosamine receptor (also known as macrophage mannose receptor, MMR, CD206), which mediates protein binding, cell uptake and lysosomal internalization with GlcNAc-terminated or N-glycans. mannose and provides a pathway for the potential correction of enzyme deficiency in these key cell types.

LAL deficiency is a multisystem disease that is often quite a massive accumulation of one manifests with gastrointestinal, liver and cardiovascular complications and is associated with significant morbidity and mortality. The clinical effects of LAL deficiency are due to lipid material in lysosomes in various tissues and profound disturbance of the mechanisms of lipid homeostasis and cholesterol, including substantial increases in hepatic cholesterol synthesis. LAL deficiency has at least two phenotypes: Wolman's Disease (WD) and Cholesteryl Ester Storage Disease (CESD).

Wolman's disease is LAL deficiency. This most aggressive presentation characterized by phenotype is gastrointestinal and hepatic manifestations including growth failure, malabsorption, steatorrhea, profound weight loss and hepatomegaly. Wolman's disease is rapidly progressive and usually fatal, usually during the first year of life. Review of case reports indicate that survival beyond 12 months of age is extremely rare for patients with growth failure due to LAL deficiency in the first year of life. In this more aggressive form, failure to thrive is the predominant clinical feature and is a key contributor to early mortality. Liver involvement as evidenced by enlarged liver and elevated transaminases is also common in infants.

Physical manifestations include abdominal distension with hepatomegaly and splenomegaly, and radiographic examination often reveals calcification of the adrenal glands. Laboratory evaluations usually reveal elevated levels of serum transaminases and absent or greatly reduced endogenous enzyme activity. High blood levels of cholesterol and triglycerides are identified in patients.

Current treatment options for Wolman's disease are very limited. Antibiotics are given to children with fever and / or evidence of infection. Steroid replacement therapy for adrenal insufficiency and specialized nutritional support may be prescribed, and while there is no evidence that these interventions prevent death, it is as yet unknown whether they have an impact on short-term survival. In a series of four LAL-deficient patients treated with bone marrow transplantation, all four patients died within months of complications of the procedure after transplantation.

Patients with LAL deficiency may also, later in life, have predominant cardiovascular and hepatic involvement and this is often referred to as Cholesteryl Ester Storage Disease (CESD). In CESD, the liver is severely affected with severe hepatomegaly, hepatocyte necrosis, elevated transaminases, cirrhosis and liver fibrosis. Due to the increased levels of EC and TAG, hyperlipidemia and atherosclerosis in LAL deficiency are also observed. In particular, an accumulation of fat deposits on artery walls is described early in life. Deposits narrow the arterial lumen and may lead to vessel occlusion, increasing the risk of significant cardiovascular events, including myocardial infarction and strokes. The presentation of CESD is highly variable with some patients not being diagnosed until complications manifest in late adulthood, while others may have liver dysfunction evident in early childhood. CESD is associated with a short lifespan and significant health problems. The life expectancy of those with CESD depends on the severity of the associated complications.

Current treatment options for the CESD phenotype are focused on controlling lipid accumulation through a diet that excludes cholesterol- and triglyceride-rich foods and suppression of cholesterol synthesis and apolipoproterna B production by administering cholesterol-lowering drugs.

<td>Although it can be observed</td><td>any improvement</td><td>clinic,</td><td>at</td>

<td>underlying manifestations of</td><td>disease persist and</td><td colspan="2">still occurs</td>

<td>disease progression.</td><td></td><td></td><td></td>

<td>Compositions containing LAL</td><td>recombinant human</td><td>derivative</td><td>in</td>

different sources are disclosed, for example, in PCT publication WO 2012/112681 A1 (purified from cultured human cells); US Patent Publication 2009/0297496 A1 (produced in Pichia pastoris); PCT publication WO 01/56596 A1 (produced in E. coli); and in Ikeda et al., J. Biosci. Bioeng., 2004, 98 (5): 366-373 (produced in S. pombe). None of these documents disclose a recombinant human LAL-containing composition having a pH within the range claimed herein.

Production of glycosylated proteins in transgenic birds is disclosed, for example, in US patent publication 2010/0062982 A1 and US patent publication 2009/0178147 A1. None of these documents disclose a pharmaceutical formulation of a recombinant human LAL as claimed herein.

In most cases, therapy for LAL deficiencies requires lifelong treatment. In addition, due to the high cost of protein therapies, it is desirable to administer a minimal effective amount of therapy to treat LAL deficiency. However, to date, there is no effective therapy to treat LAL deficiency, particularly patients suffering from Wolman's Disease and CESD. Thus, there is a strong need for effective therapy with a minimized frequency of administration in order to improve the quality of life for patients. There is also a need for a robust, high-expression protein production platform that can produce LAL proteins that are stable and effectively targeted to the lysosomal compartment in affected tissue cells in patients.

SUMMARY OF THE INVENTION

Disclosed herein are LAL compositions which are particularly suitable for use in therapy, for example for the treatment of conditions associated with LAL deficiency. The LAL molecules described herein contain particular glycan structures which offer effective and rapid absorption into cell lysosomes when administered to an individual, for example a human individual.

The present invention provides pharmaceutical formulations as set forth in the appended claims.

In one embodiment, the compositions disclosed herein comprise human LAL, wherein a substantial percentage of human LAL contains at least one mannose-6-phosphate glycan unit, which may serve as a ligand for internalization by the mannose receptor. Phosphate on the surface of cells found, for example, in hepatocytes. In one embodiment, 30% or more, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, 90% at least 95% at least 97% or at least 99% of the LAL contained in the composition contains at least one mannose 6-phosphate unit. The mannose-6-phosphate moiety may be found in an N-glycan structure located at one or more residues selected from the group consisting of Asn¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵² and Asn³⁰⁰ of SEQ ID NO: 2.

In another embodiment, the compositions disclosed herein comprise human LAL, wherein a substantial percentage of human LAL does not contain a sialic acid moiety in any of its N-glycan structures, which may sometimes interfere with the internalization of the LAL. enzyme in cells. In one embodiment, 15% or less, for example 10% or less, 5% or less, 2% or less, 1% or less, or essentially none, of the LAL contained in the composition contains one unit of sialic acid in any of its N-glycan structures.

In another embodiment, the compositions disclosed herein comprise human LAL, wherein a substantial percentage of human LAL does not contain a fucose moiety in any of its N-glycan structures. In one embodiment, 50% or less, for example 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less or essentially none of the LAL contained in the composition contains a fucose unit in any of its N-glycan structures.

Described herein are suitable vectors, host cells, expression systems, and associated methods for producing LAL-containing compositions.

The LAL of the present disclosure is human LAL. In one embodiment, the composition comprising LAL includes mature LAL having the amino acid sequence of:

SGGKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGL LADSSNWVTRLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPAS INFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIRMFFALGPVASVAFCTSPMAKLGRLPDH EIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTS VQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNIL LTQITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 2).

Also disclosed is mature LAL, which has the amino acid sequence of:

GKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFTjQHGIJjA DSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASIN FILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVAS \ rAFCTSPMAKLGRLPDHLI KDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQ NMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLT QITNLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 3).

Also disclosed is mature LAL, which has the amino acid sequence of:

TAVDPETNMNVSEIFSYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPWFLQHGLLADSS NWVTNLANSSLGFILADAGFDVWMGNSRGNTW8RKHKTLSVSQDEFWAFSYDEMAKYDLPASINFIL NKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDL FGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNML HWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPIYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQIT NLVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 4).

Also disclosed is mature LAL, which has the amino acid sequence of:

AVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSN WVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILN KTGQEQVYYVGHSOGTTIGFIAFSQIPELAKRIKMFFALGPVASVAFCTSPMAKLGRLPDHLIKDLF GDKEFLPQSAFLKWLGTHVCTHVILKSLCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLH WSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITN LVFHESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO: 19).

In another embodiment, the mature LAL is a mixture of at least two polypeptides selected from the group consisting of: SEQ ID N²: 2, SEQ ID NO:²: 3, SEQ ID NO:²: 4 and SEQ ID N² : 19.

The disclosure also provides compositions containing isolated mixtures of an individual type of useful protein molecule, such as those proteins disclosed herein, wherein one or more of the protein molecules contained in the mixture has a fixed specific oligosaccharide structure, in particular an oligosaccharide structure disclosed herein. . For example, the disclosure provides isolated mixtures of LAL molecules, for example, human LAL molecules, which contain a glycosylated LAL molecule with one or more of the following 0 to n An structures:

<img file="PT2561069T_D0001.tif" />

Square = N-Acetyl Glucosamine Full Square = Mannose-6-Phosphate Circle = Mannose

Full circle = Galactose

Filled Triangle = Fucose

According to one aspect of the present disclosure, a composition comprises any single isolated polypeptide or combination of the above described polypeptides. In one case, the composition may be a pharmaceutical composition, for example a formulation which further comprises pharmaceutically acceptable carriers, such that the composition is, for example, suitable for administration to an individual (eg, a human, particularly a patient suffering or diagnosed with a condition). The composition may be administered in various ways, including by intravenous administration. In another case, the composition may further comprise a second agent. Such an agent may be an influencing or modifying medicament or one or an agent which may biological process when administered to an individual. For example, the second agent may be an immunomodulatory agent. Such immunomodulatory agents may include any agent which, when administered together (i.e. e., administered at the same time as, or shortly before or after) with any of the LAL compositions described herein, may have the effect of reducing the immunogenicity of the LAL composition in the subject (eg, Rituximab, or any other depletion antibody). cell B).

Also disclosed are methods and compositions for treating symptoms associated with LAL deficiency.

Further objects and aspects of the present invention will become more apparent upon review of the detailed description presented below when taken in conjunction with the accompanying figures and sequences.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 represents the amino acid sequences of human LAL. The amino acid sequence of recombinant hLAL shows 100% homology to that of natural human LAL. The mature form of hLAL is underlined.

Fig. 2 depicts the nucleotide sequence of recombinant hLAL, the pALVIN-OVR1-I-hLAL-dSA rhLAL transgene.

Figures 3A and 3B depict diagrams of pALVIN-OVR1-I-hLALdSA and their pro-viral region. Fig. 3A is a diagram of the human LAL retrovirus vector used in the production of transduction particles (plasmid DNA sequence is located in Appendix A). Fig. 3A depicts the proviral region of pALVIN-OVR1-I-hLAL-dSA proviral that has been integrated into the genome. SIN LTR, self-inactivating long terminal repeat; OV enhancer DHSIII, hypersensitive site III ovalbumin gene DNase; OV intron; 5 'untranslated region of ovalbumin and intron 1; hLAL, human LAL cDNA; OV 3 'UTR, untranslated region 3' of the ovalbumin gene; partial gag, partial gag gene; LTR, long terminal repeat.

Fig. 4 represents a nucleotide sequence of pALVIN-OVR1-I-hLAL-dSA.

Fig. 5 represents a nucleotide sequence of the pro-viral region of pALVIN-OVR1-I-hLAL-dSA that has been integrated into the genome.

<td>Fig.</td><td>6 represents a nucleotide sequence of the vector of</td>

<td>pALVIN-OV-1,</td><td> . 1-1.</td>

<td>Fig.</td><td>7 represents a nucleotide sequence of the adapter</td>

<td>from rhLAL.</td><td></td>

<td>Fig.</td><td>8 represents a rhLAL nucleotide sequence</td>

<td>including the</td><td>partial ovalbumin promoter.</td>

<td>Fig.</td><td>9 represents a nucleotide sequence of the promoter</td>

<td>from 0VR1.</td><td></td>

<td>Fig.</td><td>10 represents schematics of the steps used to</td>

<td>build the</td><td>pALVIN-OVR1-I-hLAL-dSA vector.</td>

<td>Fig.</td><td>11 represents a real time PCR analysis of</td>

blood DNA samples from a transgenic G1 haemotypic progeny of XLL109. Signals of duplicate DNA samples from hemizygous G1 progeny, 1LL7466, are indicated by curves that initiate an increase in Delta Rn before cycle 22. Curves for two non-transgenic progeny are shown;

These curves remain at or near the baseline for at least 34 cycles.

Fig. 12A-D depicts Southern analysis of G1 chickens carrying the ALVIN-OVR1-I-hLAL-dSA transgene. Fig. 12A illustrates integrated transgene schematics and flanking genomic regions are shown with the known position of the transgene BlpI site and predicted position of the flanking genomic BlpI sites. The position of the OV promoter probe and hLAL coding sequence probe (hLAL probe) are indicated by black bars. The positions of the 4.3 kb and 10.6 kb bands detected in the Southern analysis are shown, as well as the predicted genomic and transgene portions of the 4.3 kb and 10.6 kb bands. Fig. 12B illustrates a Southern blot of BlpI digested genomic DNA probed with the OV probe. WT CTRL is genomic DNA isolated from a non-transgenic chicken. ID numbers of G1 transgenics are indicated above the lanes. The position and size (kb) of the molecular weight markers are shown to the left of the transfer. The position and size of the detected transgene fragment (4.3 kb) and endogenous ovalbumin gene (4.1 kb) are shown to the right of the transfer. Fig. 12C represents a Southern blot that was probed with the hLAL probe. The position and size of the detected transgene fragment (10.6 kb) is shown to the right of the transfer. Fig. 12D represents a section of the image shown in Fig. 12B on a larger scale to demonstrate the presence of the 4.1 and 4.3 kb bands.

Fig. 13A depicts schemes of the ALVIN-OVR1-IhLAL-dSA transgene. The size of predicted ApaLI bands without detection by the OV probe and hLAL probe is also shown. Fig. 13B depicts schematics of a data transfer analysis.

Southern ALVIN-OVR1-I-hLAL-dSA transgene for confirmation of transgene size. Southern blot of ApaLI digested genomic DNA and probing with the OV probe (squared panel) or hLAL probe (right panel). WT CTRL is genomic DNA isolated from a non-transgenic chicken. The G1 ID number is indicated above each lane. The position and size (kb) of the molecular weight markers are shown on the edge of the transfers. The position and size of the detected transgene fragments (OV promoter probe, 3.6 kb; hLAL probe, 3.8 kb) and endogenous ovalbumin gene (7.7 kb) are shown to the right of the transfers.

Fig. 14 represents a strain of transgenic chickens. Shown for each chicken are the generation number (GO, G1 or G2), identification number, gender and incubation date. Other G1 chickens are watercolors of other strains.

Fig. 15 represents the hLAL purification steps of egg white.

Fig. 16 represents N-glycans found as an N-linked glycosylation structure in LAL produced according to the present disclosure. Square, N-Acetyl glucosamine; Solid square, mannose-6-phosphate; circle, mannose; full circle; galactose; and triangle filled, fucose.

Fig. 17 represents the relative position of N-glycan sites indicated on the LAL polypeptide (arrow) shown in SEQ ID NO:² 1. Shown are the N -glycans which are structurally representative of the dagueles detected at each site. Square, N-Acetyl glucosamine; Solid square, mannose-6-phosphate phosphate;

circle, mannose; full circle; galactose; and triangle to full fucose.

Fig. 18 depicts phosphorylated N-glycans released by PNGase and analyzed by MALDI-TOF. The structures are shown.

Fig. 19 represents the effect of LAL dephosphorylation on HPAEC-PAD retention time of N-glycans. The LAL produced according to the present disclosure was dephosphorylated with bacterial alkaline phosphatase (upper panel) or untreated (lower panel). The released N -glycans were analyzed by HPAEC-PAD.

Fig. 20 depicts the co-location of recombinant human LAL (SBC-102) and lysosomal marker in the lysosomes of these cells examined by confocal fluorescence microscopy using a sequential scanning mode.

Fig. 21 represents the binding specificity of recombinant human LAL (SBC-102) for the GlcNAc / mannose receptor assessed by competitive binding assays using the macrophage cell line, NR8383.

Fig. 22 depicts recombinant human LAL activity in normal and LAL deficient cells in vitro.

Fig. 23 represents the effect of treatment of recombinant human LAL (SBC-102) on the internal organ mass of LAL-deficient mice. Organ size is represented as percent body weight, determined at 8 weeks of age, in LAL rats.^_/_ and LAL mice^+/+ after weekly administration of vehicle or SBC-102 at 5 mg / kg for 4 weeks.

Wild and vehicle or dose Fig is week 4.

. 24 represents body weight in LAL-deficient mice following weekly administration of

SBC-102 at 5 mg-kg^-1 for 4 weeks. Highlighted X-axis administration by lozenges beginning in the

Cholesteryl Fig. 25 in WT and vehicle or LAL week rats.

shows LAL-deficient liver, ester and triglyceride cholesterol levels determined at 8 weeks of age following weekly administration of recombinant human (SBC-102) at 5 mg · kg^-1 during

Fig. 26 represents an increase in percent body weight in LAL deficient mice after 4 weeks of administration of recombinant human LAL (SBC-102) at the indicated levels and schedules, determined at 8 weeks of age.

Fig. 27 shows liver weight as a percentage of body weight in LAL-deficient mice after 4 weeks of administration of SBC-102 at the indicated levels and schedules, determined at 8 weeks of age.

Fig. 28 shows in deficient mice

SBC-102 at levels and weeks of age.

tissue cholesteryl ester levels in LAL after 4 weeks of indicated schedule administration, determined at 8

<td>Fig. 29</td><td>show progress</td><td>daily</td><td>at the</td><td>weight gain</td><td>in</td>

<td>mice that were</td><td>administered with 1</td><td>mg / kg</td><td>in</td><td>LAL per week</td><td>or</td>

<td colspan="2">5 mg / kg LAL per week or 5 mg / kg</td><td>from LAL</td><td>per</td><td>two weeks.</td><td></td>

Fig. 30 depicts the general pathological examination of treated animals showing substantial normalization in liver size and color as seen in upper panel dissection and LAL liver tissue histopathology of treated rats showing marked contrast-enhanced normal liver histology substantial accumulation of foamy macrophages in the placebo treated animals in the lower panels.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Certain definitions are given herein to illustrate and define the meaning and scope of the various terms and expressions used to describe the invention.

As used herein, the term acceptable with respect to a formulation, composition or ingredient as used herein means having no detrimental effect on the overall health of the subject being treated.

As used herein, the term administration or administration refers to providing a recombinant human lysosomal acid lipase of the invention to an individual in need of treatment.

A nucleic acid or polynucleotide sequence includes, but is not limited to, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, comprising the natural nucleoside bases adenine, guanine, cytosine, thymidine, and uracil. The term also encompasses sequences having one or more modified bases.

Avian, as used herein, refers to any species, subspecies or breed of organism of the taxonomic class of birds, such as, but not limited to chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and runners including ostrich, rhea and cassowary. 0 The term includes the various known strains of Gallus gallus or chickens (eg White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray), as well as strains of turkeys, pheasants. , quails, duck, ostriches and other poultry, commonly reared in commercial quantities. It also includes an individual avian organism at all stages of development, including embryonic and fetal stages.

Therapeutic proteins or pharmaceutical proteins include an amino acid sequence which, in whole or in part, constitutes a drug.

An open reading frame or coding sequence refers to a polynucleotide or nucleic acid sequence that can be transcribed and translated (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the heading. control of appropriate regulatory sequences. The limits of the coding sequence are determined by a 5 '(amino) translation initiation codon and a 3' (carboxyl) translation termination codon. A transcription termination sequence is usually located 3 'from the coding sequence. A coding sequence may be flanked at the 5 'and / or 3' ends by untranslated regions.

Exon refers to that part of a gene that, when transcribed into a nuclear transcript, is expressed in cytoplasmic mRNA after removal of introns or intervention sequences by nuclear excision union.

Control sequences or nucleic acid regulatory sequences refer to promoter sequences, translation initiation and termination codons, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like. as necessary and sufficient for transcription and translation of a given coding sequence into a defined host cell. Examples of suitable control sequences for eukaryotic cells are promoters, polyadenylation signals, and enhancers. All of these control sequences need not be present in a recombinant vector as long as those necessary and sufficient for transcription and translation of the desired gene are present.

Operative or operably linked refers to the configuration of coding and control sequences to perform the desired function. Thus, control sequences linked to a coding sequence are capable of expressing the coding sequence. A coding sequence is operably linked to or under the control of transcriptional regulatory regions in a cell when DNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA that can be translated into the encoded protein. Control sequences need not be contiguous with the coding sequence as long as they function to direct their expression. Thus, for example, untranslated but transcribed intervening sequences may be present between a promoter sequence and the coding sequence and the promoter sequence may still be considered operably linked to the coding sequence.

The terms heterologous and exogenous when referring to nucleic acid sequences, such as coding sequences and control sequences, denote sequences that are not normally associated with a region of a recombinant construct or a particular chromosomal locus, and / or are not normally associated with a particular cell. Thus, an exogenous region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not in association with the other molecule in nature. For example, an exogenous region of a construct could include a sequence flanked coding sequence that is not in association with the coding sequence in nature. Another example of an exogenous coding sequence is a construct where the coding sequence itself is not in nature (eg, synthetic sequences having codons other than the native gene). Similarly, a host cell transformed with a construct or nucleic acid, which is not normally present in the host cell, would be considered exogenous for purposes of this invention.

As used herein, the terms N-glycan, oligosaccharide and the terms standard glycosylation oligosaccharide structure, glycosylation profile and glycosylation structure are essentially the same meaning and refer to one or more structures, which are formed by from sugar residues and are linked to glycosylated proteins.

Exogenous protein, as used herein, refers to a protein that is not naturally present in a particular tissue or cell, a protein that is the expression product of an exogenous expression construct or transgene, or a protein that is not naturally present in a given amount in a particular tissue or cell. A protein that is exogenous to an egg is a protein not normally found in the egg. For example, a protein exogenous to an egg may be a protein that is present in the egg as a result of expression of a coding sequence present in an egg-laying animal transgene.

Endogenous gene refers to a naturally occurring or fragment thereof normally associated with a particular cell.

LAL stands for human lysosomal acid lipase, SBC-102 or human lysosomal acid lipase molecule and these terms and expressions are used interchangeably throughout the description.

The expression products described herein may consist of proteinaceous material having a defined chemical structure. However, the precise structure depends on several factors, particularly chemical modifications common to proteins. For example, since all proteins contain ionizable amino and carboxyl groups, the protein may be obtained in acidic or basic salt form, or in neutral form. The primary amino acid sequence may be derivatized using sugar molecules (glycosylation) or other chemical derivatisations involving covalent or ionic bonding with, for example, lipids, phosphate, acetyl groups and the like, often occurring through association with saccharides. These modifications may occur in vitro or in vivo, the latter being performed by a host cell through post-translational processing systems. Such modifications may increase or decrease the biological activity of the molecule, and it is intended that such chemically modified molecules also fall within the scope of the invention.

Alternative cloning, amplification, expression and purification methods will be apparent to the skilled artisan. Representative methods are disclosed in Sambrook, Fritsch, and Maniatis, Molecular Cloning, Laboratory Manual, 2^The Ed., Cold Spring Harbor Laboratory (1989).

bookmark sites

Vector means a polynucleotide composed of linear, double stranded, circular, or overcoiled spiral DNA or RNA. A typical vector may be composed of the following elements operably linked at appropriate distances to allow functional gene expression: origin of replication, promoter, enhancer, 5 'mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyadenylation, and selectable sequences. One or more of these elements may be omitted in specific applications. The nucleic acid cassette may include a restriction site for insertion of the nucleic acid sequence to be expressed. In a functional vector, the nucleic acid cassette contains the nucleic acid sequence to be expressed including translation initiation and termination sites. An intron may optionally be included in the construct, for example, 5 'of the coding sequence. A vector is constructed such that the particular coding sequence is situated in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences such that the coding sequence is transcribed under the control of the coding sequences. control or regulatory authorities. Modification of the sequences encoding the particular protein of interest may be desirable to achieve this purpose. For example, in some cases, it may be necessary to modify the sequence so that it can be linked to control sequences in the appropriate orientation; or to keep the reading grid. Control sequences and other regulatory sequences may be linked to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence may be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site that is in frame reading with and under regulatory control of the control sequences.

A promoter is a site in DNA to which RNA polymerase binds to initiate transcription of a gene. In some embodiments, the promoter may be modified by the addition or deletion of sequences, or substituted with alternative sequences, including natural and synthetic sequences, as well as sequences that may be a combination of natural and synthetic sequences. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter elements. The former, upstream of the transcription initiation site, is involved toward RNA polymerase to initiate transcription at the correct site, while the latter appears to determine the transcription rate and is upstream of the TATA drop. Enhancer elements may also stimulate transcription from linked promoters, but many function exclusively on a particular cell type. Many virus derived enhancer / promoters, eg the SV40 promoter, cytomegalovirus promoter (CMV), rous sarcoma virus promoter (RSV) and murine leukemia promoter (MLV) are all active in a wide range. variety of cell types and are called ubiquitous. Alternatively, non-constitutive promoters such as the rat mammary tumor virus (MMTV) promoter may also be used in the present invention. The nucleic acid sequence inserted at the cloning site may have any open reading frame which encodes a polypeptide of interest, provided that where the coding sequence encodes a polypeptide of interest, this lacks cryptic excision-binding sites that may blog the production. of appropriate mRNA molecules and / or to produce aberrantly abnormal or excised-bound mRNA molecules.

As used herein, the term pharmaceutical composition refers to a mixture of a compound described above with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents and / or excipients.

The term poultry-derived or poultry-derived refers to a composition or substance produced or obtained from poultry. Poultry refers to birds that may be kept as domestic animals, including, but not limited to chickens, ducks, turkeys, quails and runners. For example, poultry derivative may refer to chicken derivative, turkey derivative and / or quail derivative.

A retroviral particle, transducer particle or transduction particle refers to a replication competent or replication defective virus capable of transducing

DNA or RNA does not turn into a cell. In a particularly useful embodiment, the retroviral particles used to produce transgenic birds according to the invention are prepared as disclosed in US Pat.² 7524626, published April 28, 2009.

The terms transformation, transduction and transfection all denote the introduction of a polynucleotide into a bird blastodermal cell. Magno is that part of the oviduct between the infundibulum and the isthmus containing tubular gland cells that synthesize and secrete egg white proteins.

The term transgene refers to the heterologous nucleotide sequence inserted into an avian genome according to the invention. Transgene may specifically refer to an exogenous coding sequence, an exogenous coding sequence linked to an exogenous promoter or other regulatory sequences, the entire nucleotide sequence between two retroviral LTRs and / or retroviral LTRs and nucleotide sequence between LTRs, where LTRs are from a retrovirus used to introduce the transgene.

The term optimized is used in the context of optimized coding sequence, where the codons most often used for each amino acid found in egg white proteins ovalbumin, lysozyme, ovomucoid, and ovotransferrin are used in the design of the human interferon-α 2b polynucleotide sequence. (IFN-α 2b) which is inserted into vectors of the present invention. More specifically, the DNA sequence for human optimized IFN-α 2b is based on codon optimized use for chicken oviduct and is created using the Wisconsin Package Version 9.1 BACKTRANSLATE program (Genetics Computer Group Inc., Madison, Wis. ) with a codon usage table compiled from the proteins ovalbumin, lysozyme, ovomucoid and ovotransferrin (Gallus gallus). For example, the percent utilization for the four alanine amino acid codons in the four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG. Thus, GCU is used as the codon for most alanines in an optimized coding sequence. Vectors containing the gene for optimized human protein are used to produce transgenic birds expressing transgenic poultry protein derived protein in their tissues and eggs.

As used herein, the term individual encompasses mammals and non-mammals. Examples of mammals include, but are not limited to humans, chimpanzees, anthropoid monkeys, cattle, horses, sheep, goats, swine; rabbits, dogs, cats, rats, mice, guinea pigs, and the like.

As used herein, the term therapeutically effective amount refers to any amount of a compound which, when compared to a corresponding individual who has not received such an amount, results in improved treatment, cure or prevention or amelioration of a disease, disorder or side effect, or a decrease in the rate of progression of a disease or disorder. The term also includes within its scope effective amounts to improve normal physiological function.

The terms treat, treat and treat refer to methods for alleviating, diminishing or ameliorating the symptoms of a condition, preventing additional symptoms, ameliorating or preventing the underlying causes of symptoms, inhibiting the disease or condition, stopping the development of the disease or condition, alleviate the disease or condition, cause regression of the disease or condition, alleviate a condition caused by the disease or condition or stop the symptoms of the disease or condition prophylactically and / or therapeutically.

COMPOSITIONS OF LAL disclosure refers to lysosomal as

The disclosure generally relates to compositions comprising enzymes useful for therapy, for example, in the treatment of lysosomal storage disorders. In one case, the LAL storage disease enzymes have a pattern of glycosylation pattern that makes the molecule internalizable by certain cell types. Also included in the disclosure are recombinant human proteins, including LAL in isolated or purified form. 0 Isolation of lysosomal storage disease enzymes (such as LAL) can be accomplished by methodologies readily apparent to one skilled in the art of protein purification.

In one embodiment, the disclosure is directed to lysosomal storage disease enzymes including, but not limited to LAL, having an N-linked glycosylation pattern described herein.

In one embodiment, the compositions disclosed herein comprise human LAL, wherein a substantial percentage of human LAL contains a mannose-6-phosphate glycan moiety which may serve as a ligand for internalization by the mannose-6-phosphate receptor. on the surface of cells found, for example, in hepatocytes. In one embodiment, 30% or more, for example at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least At least 90%, at least 95%, at least 97% or at least 99% of the LAL contained in the composition contains at least one mannose-6-phosphate unit. The mannose-6-phosphate moiety may be found as an N-glycan structure located on one or more residues selected from the group consisting of Asn¹⁵, Asn⁵¹, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵² and Asn³⁰⁰ from SEQ ID N² 2. The mannose-6-phosphate moiety containing glycan structures include, for example, Gn and Hn shown in Fig. 16.

Recombinant human LAL according to the present invention contains multiple N-linked carbohydrate chains (eg, 5 or 6 carbohydrate chains). N-linked glycosylation structures at each of the five or six sites can be selected from one of An, Bn, Cn, Dn, En, Fn, Gn, Hn, In, Jn, Kn, Ln, Mn, Nn and On. shown in Fig. 16.

Also described herein is a mixture of LAL molecules (eg, more than one LAL molecule may be present in a mixture such as the LAL molecules set forth in SEQ ID N²: 2, 3, 4 and 19) wherein some or all LAL molecules have one or more glycosylation structures selected from Structure An, Structure Bn, Structure Cn, Structure Dn, Structure En, Structure Fn, Structure Gn, Structure Hn , Structure In, Structure Jn, Structure Kn, Structure Ln, Structure Mn, Structure Nn and Structure On (Fig. 16). In one embodiment, the mixture of lysosomal acid lipase molecules is isolated from an egg or purified or isolated from egg white produced in a transgenic bird.

The disclosure also includes an individual LAL molecule comprising an An Structure. The disclosure also includes an individual LAL molecule comprising an Bn Structure. The disclosure also includes an individual LAL molecule comprising a Cn Structure. The disclosure also includes an individual LAL molecule comprising a Dn Structure. The disclosure also includes an individual LAL molecule comprising an En structure. The disclosure also includes an individual LAL molecule comprising an Fn Structure. The disclosure also includes an individual LAL molecule comprising a Gn Structure. The disclosure also includes an individual LAL molecule comprising an Hn Structure. The disclosure also includes an individual LAL molecule comprising an In Structure. The disclosure also includes an individual LAL molecule comprising a Jn Structure. The disclosure also includes an individual LAL molecule comprising a Kn structure. The disclosure also includes an individual LAL molecule comprising an Ln Structure. The disclosure also includes an individual lysosomal acid lipase molecule comprising a Mn Structure. The disclosure also includes an individual LAL molecule comprising an Nn Structure. The disclosure also includes an individual LAL molecule comprising a 0-n Structure.

N-linked oligosaccharides linked to a human LAL according to the present disclosure have a shortage of terminal sialic acid and galactose residues. That is, only minor amounts of the N-linked oligosaccharide structures are terminally sialylated and few galactose residues are also present. In addition, terminal N-Acetyl Glucosamine (GlcNAc) is present extensively in the N-linked oligosaccharide structures of the described LAL. As such, the LAL produced according to the disclosure may be directed to cells such as monocyte macrophages and Kupffer cells.

One embodiment of the invention provides LAL compositions having essentially no sialic acid. In another embodiment, the compositions disclosed herein comprise recombinant human LAL, wherein a substantial percentage of human LAL does not contain a sialic acid moiety in any of its N-glycan structures, which may interfere with the internalization of the enzyme in cells. . In one embodiment, 15% or less, for example 10% or less, 5% or less, 2% or less, 1% or less, or essentially none, of the LAL contained in the composition contains a sialic acid unit in any of its N-glycan structures.

In one case, about 95% or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure does not contain sialic acid. In another case, about 90% or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure does not contain sialic acid. In another case, about 80% or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure does not contain sialic acid. In another case, more than about 70% or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure does not contain sialic acid.

In yet another embodiment, essentially none of the types of N-linked oligosaccharide structure present in LAL molecules contain sialic acid. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present in the LAL molecules do not contain sialic acid. For example, if there are 20 oligosaccharide structure types, then 18 or more of the structure types do not contain sialic acid. In another embodiment, about 80% or more of the types of N-linked oligosaccharide structure present in LAL molecules do not contain sialic acid. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types found to be associated with the LAL molecules do not contain sialic acid. In another embodiment, about 60% or more of the N-linked oligosaccharide structure types found to be associated with the LAL molecules do not contain sialic acid. In another embodiment, about 50% or more of the N-linked oligosaccharide structure types found to be associated with the LAL molecules do not contain sialic acid.

According to one embodiment of the invention, LAL as described above contains high levels of terminal N-Acetyl Glucosamine. In one case, about 95% or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain a terminal N-Acetyl Glucosamine. In another case, about 90% or more or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain one. In another case, about 80% or more or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain one. In another case, about 70% or more or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain one. In another case, about 60% or more or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain one. In another case, about 50% or more or more of the N-linked oligosaccharides present in the individual LAL molecule of the disclosure contain one.

In one embodiment, all types of N-linked oligosaccharide structure present in LAL molecules contain a terminal N-Acetyl Glucosamine. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present in the LAL molecules contain a terminal N-Acetyl Glucosamine. For example, if there are 20 oligosaccharide structure types, then 18 or more of the structure types do not contain a terminal N-Acetyl Glucosamine. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present in the LAL molecules contain a terminal N-Acetyl Glucosamine. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present in the LAL molecules contain a terminal N-Acetyl Glucosamine. In another embodiment, about 60% or more of the N-linked oligosaccharide structure types present in the LAL molecules contain a terminal N-Acetyl Glucosamine. In another embodiment, about 50% or more of the N-linked oligosaccharide structure types present in the LAL molecules contain a terminal N-Acetyl Glucosamine.

In another embodiment of the invention, the compositions disclosed herein comprise human LAL, wherein a substantial percentage of human LAL does not contain a fucose moiety in any of its N-glycan structure. In one embodiment, 50% or less, for example 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less or essentially none of the LAL contained in the composition contains a fucose unit in any of its N-glycan structure.

In one case, fucose is essentially not present in L-N-linked oligosaccharide structures produced according to the disclosure. In another case, about 95% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 90% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 85% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 80% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 70% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 60% or more of the N-linked oligosaccharides present in the individual LAL disclosure molecule do not contain fucose. In another case, about 50% or more of the N-linked oligosaccharides present in the LAL of the disclosure do not contain fucose.

In one embodiment, essentially none of the types of N-linked oligosaccharide structure present in LAL molecules contain fucose. In another embodiment, about 95% or more of the N-linked oligosaccharide structure types present in the LAL molecules do not contain fucose. For example, if there are 20 types of oligosaccharide structure, then 19 or more of the structure types do not contain fucose. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present in the LAL molecules do not contain fucose. In another embodiment, about 85% or more of the N-linked oligosaccharide structure types present in LAL molecules do not contain fucose. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present in LAL molecules do not contain fucose. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present in the LAL molecules do not contain fucose.

As discussed above, certain monosaccharides are abundantly present in LAL molecules produced in accordance with the present disclosure. Total monosaccharide species analyzed include fucose, N-acetyl galactosamine, N-acetyl glucosamine, galactose, glucose, mannose, mannose 6-phosphate, N-acetyl neuraminic acid and N-glycololneuraminic acid. Fucose may be present from about 0% to about 1% of the total monosaccharide composition. N-acetyl galactosamine may be present between about 0% and about 1% of the total monosaccharide composition. N-acetyl glucosamine may be present between about 35% and about 50% of the total monosaccharide composition. Galactose may be present in about 1-10% of the total monosaccharide composition. Glucose may be present at 0% of the total monosaccharide composition. Mannose is present between about 32% and about 50% of the total monosaccharide composition. Mannose-6-phosphate is present between about 1% and about 11% of the total monosaccharide composition.

In one embodiment, the LAL produced according to the present disclosure does not contain any xylose. Furthermore, since there is essentially no N-acetylgalactosamine (GalNac) in the LAL produced according to the disclosure, an embodiment of the invention includes an LAL composition having no O-linked glycosylation.

LAL has 6 potential sites in its amino acid sequence q &lt;7 0 ί q Ί for N-linked glycosylation, for example Asn, Asn, Asn, Asn¹⁶¹, Asn²⁷³ and Asn³²¹ as in SEQ ID N²: 1. Five of these, Asn³⁶, ί q ί Ίί ^ Ί 7 7 7 7 7 1 7 7

Asn Asn, Asn and Asn are glycosylated, while Asn may be unglycosylated or substantially unglycosylated (substantially unglycosylated means that in a mixture of LAL molecules, they are glycosylated minus Asn⁷² than any of Asn³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³ and Asn³²¹) (see Fig. 17). Accordingly, one aspect of the disclosure is an LAL composition that is unglycosylated and / or substantially unglycosylated on Asn.⁷². LAL owning an Asn⁷² glycosylated is within the scope of the disclosure. The Asn positions described herein are based on the amino acid sequence of LAL shown in SEQ ID N²It will be apparent to those skilled in the art that Asn numbering (ie, asparagine position) may vary depending on the individual LAL molecule and can be readily determined on other LAL molecules, such as those whose amino acid sequences are shown. in SEQ ID N²: 2, 3, 4 and 19.

mixture of bi-, N-acetylglucosamine, mannose and

LAL molecules produced in accordance with the present disclosure contain N-glycan structures comprising a mannose-6-phosphate (M6P) tri- and tetra-antennas as the major sugars (Figs. 16 and 17). According to one aspect of the disclosure, M6P-modified N-glycans reside at least in Asn, Asn and Asn. Thus, a case of the present disclosure includes an LAL composition having M6P-modified N-glycans residing in either Asn, Asn or Asn In yet another case, the present disclosure includes an LAL composition having M6P-modified N-glycans residing in Asn²⁷³. In another case, the present disclosure includes an LAL composition having monophosphorylated N-glycans (M6P) residing

Em Ί ν Ί ο 7 q in any of Asn, Asn, or Asn. In yet another case, the present disclosure includes an LAL composition having monophosphorylated N-glycans residing in Asn¹⁶¹ and Asn²⁷³ . In yet another case, the present disclosure includes an LAL composition having monophosphorylated N-glycans residing in Asn¹⁰¹ and Asn²⁷³ . In a specific case, an LAL produced according to the present disclosure may contain bisphosphorylated mannose (bis-M6P) in Asn¹⁰¹.

LAL molecules produced according to the present disclosure contain reduced levels of galactose (eg, Gal). One aspect of the present disclosure includes an LAL composition having terminal galactose in any of Asn³⁶, Asn¹⁶¹ or asn³²¹. In yet another case, the present disclosure includes an LAL composition having Asn terminal galactose³⁶ and Asn¹⁶¹. In yet another case, the present disclosure includes an LAL composition having Asn terminal galactose¹⁶¹ and Asn³²¹. In yet another case, the present disclosure includes an LAL composition having Asn terminal galactose³⁶ and Asn³²¹. In yet another case, the present disclosure includes an LAL composition having

Ait ΑΟΊ £ \ Ί terminal terminal galactose in Asn, Asn and Asn. In yet another case, the present disclosure includes an LAL composition having no terminal galactose.

Several types of N -glycans have been found in LAL at different N-linked glycosylation sites. N-glycan structures include a mixture of bi-, tri- and tetra-antenary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as the major sugars.

Specifically, in one embodiment of the present invention, LAL contains an N-glycan structure selected from GlcNAc4Man3GlcNAc2 or GallGlcNAc4Man3GlcNAc2 at the first N-linked glycosylation site (Asn¹⁵ as in SEQ ID N² : 2) . In another

LAL does not contain glycosylated on the second N-linked glycosylation (Asn⁵¹ as in SEQ ID N²: 2) . In yet another embodiment, LAL contains Phos2Man7GlcNAc2 at its third N-linked glycosylation site (Asn⁸⁰ as in SEQ ID N²: 2) . In yet another embodiment, LAL contains one of N-glycan selected from PhoslMan6GlcNAc2,

Man3GlcNAc2, GlcNAc2Man3GlcNAc2,

GlcNAc4Man3GlcNAc2, or its fourth N-linked glycosylation site (Asn^{1, u} as in SEQ ID N²: 2) . In yet another embodiment, LAL contains an N-glycan structure selected from Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, PhoslMan8GlcNAc2, or PhoslMan9GlcNAc2 at its fifth N-linked glycosylation site or is GlycMan2NAcNAcNA3 , GallGlcNAc4Man3GlcNAc2 no. 140 (Asn

252 as in SEQ ID N²: 2) . In yet another embodiment,

LAL contains a GlcNAc2Man3GlcNAc2, GallGlcNAc4Man3GlcNAc2, GallGlcNAc5Man3GlcNAc2, N -glycan structure selected from

GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, GlcNAc5Man3 GlcNAc2,

GlcNAc6Man3GlcNAc2, or

GallGlcNAc6Man3GlcNAc2 in your sex N-linked glycosylation site (Asn³⁰⁰ as in SEQ ID N²: 2).

According to certain aspects of the disclosure, the LAL compositions include glycosylated LALs in Asn, Asn, Asn Asn.¹⁶¹, Asn²⁷³ and Asn³²¹ from SEQ ID N²: 1 (or corresponding Asparagine residues within SEQ ID N²: 2, 3, 4, and 19) with a

N-glycan in designated Asn position as shown below:

a) in Asn³⁶, GlcNAc4Man3GlcNAc2, or

Gall GlcNAc4Man3GlcNAc2;

without glycosylation;

b) in Asn⁷²,

c) in Asn¹⁰¹, Phos2Man7GlcNAc2;

d) in Asn¹⁶¹, phoslMan6GlcNAc2,

GlcNAclPhoslMan6GlcNAc2 Man3GlcNAc2; GlcNAc2Man3GlcNAc2; GlcNAc3Man3GlcNAc2;

GlcNAc4Man3GlcNAc2, or GallGlcNAc4Man3GlcNAc2;

e) in Asn²⁷³ , Man7GlcNAc2,

Man8GlcNAc2,

Man9GlcNAc2, PhoslMan8GlcNAc2, or PhoslMan9GlcNAc2; and

f) in Asn³²¹, GlcNAc2Man3GlcNAc2,

GlcNAc3Man3GlcNAc2, GlcNAc4Man3GlcNAc2, GallGlcNAc4Man3GlcNAc2, GlcNAc5Man3GlcNAc2, GallGlcNAc5Man3GlcNAc2, GlcNAc6Man3GlcNAc2, or GallGlcNAc6 = Man3, where2

GlcNAc = N-Acetyl Glucosamine,

Phos = phosphate, and Gal = galactose.

In one case, GallGlcNAc4Man3GlcNAc2 can be found as a

Glycan component in any one of Asn, Asn or Asn in LAL produced according to the disclosure. In a specific case, GallGlcNAc4Man3GlcNAc2 can be found as an Asn glycan component.³⁶, Asn¹⁶¹ and Asn³²¹.

In the LAL of the present disclosure, Asn101 and Asn273 typically monitor the high mannose type having about 6 to about 10 mannose molecules (MAN6-MAN10 as described above) as a major component. Accordingly, an aspect of the present disclosure includes an LAL composition having a high mannose structure in Asn.¹⁰¹ or asn²⁷³ . In another case, an LAL composition of the disclosure may comprise an N-glycan structure having at least 6 mannose in Asn¹⁰¹ or asn²⁷³ . In another case, an LAL composition contains an N-glycan having 7, 8 or 9 mannose in Asn.¹⁰¹ or

Asn. In another case, the present disclosure includes a composition of LAL having 7, 8 or 9 mannose in Asn.¹⁰¹ and Asn²⁷³ .

In yet another case, the present disclosure includes an LAL composition having 7, 8 or 9 mannose in Asn and / or Asn and at least one of the mannose is phosphorylated.

And to be understood that the glycosylation sites and numbers associated with Asn described above are based on the amino acid sequence of LAL shown in SEQ ID NO:^s: 1 and see the glycosylation profiles described above in the context of SEQ ID Ν²: 1 also apply to LAL molecules set forth in SEQ ID N²

For example,

<td> : 2, 3,</td><td>4 and</td><td>19 despite</td><td>gives</td><td>numbering</td><td>gives</td><td>Asn</td>

<td>power</td><td>vary</td><td>depending on</td><td>gives</td><td>molecule</td><td>in</td><td>LAL.</td>

<td colspan="2">Asn³⁶ at SEQ</td><td colspan="3">ID N²: 1 matches</td><td>Asn¹⁵</td><td>at</td>

<td>Asnl3 on</td><td>SEQ ID</td><td>N² : 3, Asn¹⁰</td><td>at</td><td>SEQ ID NO:²:</td><td>4 and</td><td>Asn⁹</td>

<td>at</td><td>SEQ</td><td>ID</td><td>N²</td><td>: 19. Asn⁷²</td><td>at SEQ</td><td>ID N²: 1</td><td>stands for</td><td>Asn⁵¹</td><td>at</td>

<td>SEQ</td><td>ID</td><td>N² :</td><td> 2,</td><td colspan="2">Asn⁴⁹ at SEQ ID N² :</td><td>3, Asn46</td><td>i at SEQ ID N²:</td><td colspan="2">4 and Asn⁴⁵</td>

<td>at</td><td>SEQ</td><td>ID</td><td>N²</td><td>: 19. Asn¹⁰¹</td><td>at SEQ</td><td>ID N²: 1</td><td>stands for</td><td>Asn⁸⁰</td><td>at</td>

<td>SEQ</td><td>ID</td><td>N² :</td><td> 2,</td><td colspan="2">Asn⁷⁸ at SEQ ID N² :</td><td>3, Asn⁷⁵</td><td>at SEQ ID N²:</td><td colspan="2">4 and Asn⁷⁴</td>

<td>at</td><td>SEQ</td><td>ID</td><td>N²</td><td>: 19. Asn¹⁶¹</td><td>at SEQ</td><td>ID N²: 1</td><td>stands for</td><td>Asn¹⁴⁰</td><td>at</td>

<td>SEQ</td><td>ID</td><td>N² :</td><td> 2,</td><td>Asn¹³⁸ at</td><td colspan="3">SEQ ID NO:² : 3, Asn¹³⁵ at SEQ ID</td><td>N² : 4</td><td>and</td>

<td>Asn</td><td> 134</td><td>at</td><td colspan="2">SEQ ID NO:²: 19.</td><td>Asn²⁷³</td><td>from SEQ</td><td colspan="2">ID N²: 1 matches</td><td>The</td>

<td>Asn</td><td> 252</td><td>at</td><td colspan="2">SEQ ID NO:²: 2,</td><td>Asn²⁵⁰</td><td colspan="3">at SEQ ID N²: 3, Asn247</td><td>at</td>

<td>SEQ</td><td>ID</td><td>N² :</td><td> 4</td><td colspan="3">and Asn²⁴⁶ at SEQ ID N² : 19.</td><td>Asn³²¹ from SEQ</td><td>ID N² :</td><td> 1</td>

<td colspan="4">match</td><td>to Asn³⁰⁰ at</td><td>SEQ ID</td><td>N² : 2,</td><td>Asn²⁹⁸ at SEQ</td><td>ID N² :</td><td> 3,</td>

<td>Asn</td><td colspan="3">²⁹⁵ at SEQ</td><td colspan="2">ID N²: 4 and Asn²⁹⁴ at</td><td>SEQ ID NO:</td><td>² : 19.</td><td></td><td></td>

In the present invention, N-linked glycosylated LAL in Asn¹⁵, Asn⁸⁰, Asn¹⁴⁰, Asn²⁵² and Asn³⁰⁰ from SEQ ID N²: 2. In one embodiment, the LAL N-glycan structures of SEQ ID N²: 2 do not have xylose, while less than 15%, 10%, 5%, or 1% of N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%, 90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one case, the LAL is N-linked glycosylated at least at a position selected from the group consisting of Asn¹³, Asn⁴⁹, Asn⁷⁸, Asn¹³⁸, Asn²⁵⁰ and Asn²⁹⁸ from SEQ ID N²: 3. In another case, LAL is N-linked glycosylated in Asn¹³, Asn⁷⁸, Asn¹³⁸, Asn²⁵⁰ and Asn²⁹⁸ from SEQ ID N²: 3. In yet another case, the LAL N-glycan structures of SEQ ID N:²: 3 do not have xylose while less than 15%, 10%, 5%, or 1% of N-glycan structures contain sialic acid;

less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of N-glycan structures contain fucose; and at least 30%, 50%, 60%, 70%, 80%,

90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one case, the LAL is N-linked glycosylated at least at a position selected from the group consisting of Asn¹⁰, Asn⁴⁶, Asn⁷⁵, Asn¹³⁵, Asn²⁴⁷ and Asn²⁹⁵ from SEQ ID N²: 4. In another case, LAL is N-linked glycosylated in Asn¹⁰, Asn⁷⁵, Asn¹³⁵, Asn²⁴⁷ and Asn²⁹⁵ from SEQ ID N²: 4. In yet another case, the LAL N-glycan structures of SEQ ID N:²: 4 do not have xylose, while less than 15%, 10%, 5%, or 1% of N-glycan structures contain sialic acid; less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of

N-glycan contain fucose; and at least 30%, 50%, 60%, 70%, 80%,

90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

In one case, the LAL is N-linked glycosylated at least at a position selected from the group consisting of Asn⁹, Asn⁴⁵, Asn⁷⁴, Asn¹³⁴, Asn²⁴⁶ and Asn²⁹⁴ from SEQ ID N²: 19. In another case, LAL is N-linked glycosylated in Asn⁹, Asn⁷⁴, Asn¹³⁴, Asn²⁴⁶ and Asn²⁹⁴ from SEQ ID N²: 19. In yet another case, the LAL N-glycan structures of SEQ ID N:²: 4 do not have xylose, while less than 15%, 10%, 5%, or 1% of N-glycan structures contain sialic acid;

less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of

N-glycan contain fucose; and at least 30%, 50%, 60%, 70%, 80%,

90% and 95% of N-glycan structures contain phosphorylated mannose (M6P).

The composition according to the present invention may be produced in a variety of ways, including by using transgenic birds, transgenic fish, transgenic mammals, for example transgenic goats or in transgenic plants such as tobacco and duckweed (Lemna minor ) and certain types of cell culture.

The present invention also contemplates compositions comprising pegylated LAL. The LAL enzyme as described herein may be PEGylated as disclosed, for example, in US Patent Publication No.² 20070092486, published April 26, 2007.

In one embodiment, the derived glycosylation pattern is obtained by expression of specialized expression systems, for example of bird oviduct cells, for example tubular gland cells. For example, the glycosylation patterns disclosed herein have been shown to be present in lysosomal storage disease enzymes produced in oviduct cells of a bird, such as a chicken, according to the present disclosure.

Proteins produced according to the disclosure may be purified from egg white by any useful procedure, such as those apparent to one skilled in the art of protein purification. For example, human LAL (hLAL) produced in transgenic birds according to the disclosure may be purified from egg white by methods apparent to those skilled in the art of protein purification. An example of a purification protocol for egg white LAL is described in the Examples.

The disclosure includes eggs and egg white and poultry (eg, chicken, turkey and quail) laying eggs and producing egg white containing lysosomal acid lipase molecules comprising one or more of the glycosylation structures disclosed herein.

LAL EXPRESSION IN BIRDS

Disclosed herein are vectors and methods for the stable introduction of exogenous nucleic acid sequences into the avian genome to express desired proteins, such as those that benefit (eg, obtain greater efficacy) from the addition of mannose-6-phosphate, such as enzymes. including without limitation lysosomal acid lipase (LAL) and other proteins such as those specifically disclosed herein. In particular, transgenic birds are produced which express exogenous sequences in their oviducts and deposit exogenous proteins, such as pharmaceutical proteins, in their eggs. Poultry eggs containing such exogenous proteins are also described herein. New forms of LAL that are effectively expressed in the oviduct of transgenic birds and deposited in poultry eggs are also disclosed.

One aspect of the disclosure relates to LAL-containing compositions, ie, LAL molecules produced according to the disclosure. In a particularly useful case, LAL is purified or isolated. For example, LAL has been removed from the contents of a hard shell egg laid by a transgenic bird. In a particularly useful case, LAL is human LAL. In one embodiment, LAL has a glycosylation pattern resulting from LAL being produced in a bird oviduct cell. For example, the compositions may contain a mixture of poultry-produced LAL molecules, for example chickens, according to the disclosure and isolated from egg white. According to the invention, LAL-containing compositions are pharmaceutical formulations.

In one embodiment, the invention relates to compositions containing isolated LAL molecules as claimed herein, wherein LAL is produced in a bird, which contains a transgene encoding LAL. In one embodiment, LAL is produced in an oviduct cell (eg, a tubular gland cell) of a

<td>bird</td><td>transgenic</td><td>(and.</td><td>g.,</td><td>transgenic chicken) and</td><td>LAL is isolated</td>

<td>in</td><td>egg white</td><td>gives</td><td>bird</td><td>transgenic. In a form</td><td>of realization,</td>

<td>LAL</td><td colspan="2">is glycosylated</td><td>at</td><td>oviduct cell (and</td><td>. g.</td>

tubular gland) of the bird, for example a chicken.

Methods for producing exogenous proteins, such as lysosomal storage disease enzymes, for example, LAL, in bird specific tissues are provided. Such exogenous proteins may be expressed in the oviduct, blood and / or other embryonic transgenes, produce one sound per bird, and the bird's cells and tissues. In one case, introduced into example blastodermal cells, near stage X, for transgenic, such that the protein of interest expressed in the lumen secreted tubular gland cells of the oviduct, and deposited in the egg white of a hard shell . Such a transgenic bird can carry the transgene in its germ line providing transmission of the exogenous transgene to the progeny of the bird stably in a Mendelian mode.

The present disclosure encompasses methods of producing exogenous protein, such as LAL, in a bird oviduct. The methods may include a first step of providing a vector containing a coding sequence and a promoter operably linked to the coding sequence, so that the promoter can perform nucleic acid expression in the bird oviduct. Transgenic cells and / or tissues may be produced, wherein the vector is introduced into newly isolated bird embryonic blastodermal cells in culture, or into an embryo, such that the vector sequence is inserted into the bird genome. The mature transgenic bird expressing the exogenous protein, such as LAL, in its oviduct may be derived from transgenic cells and / or tissue.

In one aspect of the disclosure, the production of a transgenic bird is accomplished by transducing embryonic blastodermal cells with replication defective or replication-competent retroviral particles carrying the transgene between the 5 'and 3' LTRs of the retroviral vector. For example, a retroviral avian leukemia virus (ALV) vector or a retroviral murine leukemia virus (MLV) vector can be used which comprises a modified pNLB plasmid containing an exogenous gene that is inserted downstream of a segment of a promoter region. An RNA copy of the modified retroviral vector, packaged in viral particles, can be used to infect embryonic blastoderms that develop in transgenic birds.

Another aspect of the disclosure provides a vector that includes a coding sequence and a promoter in operative and position relationship such that the coding sequence is expressed in a bird oviduct. Such vectors include, but are not limited to, a retroviral avian leukosis virus (ALV) vector, a retroviral murine leukemia virus (MLV) vector, and a lentivirus vector. In addition, the vector may be a nucleic acid sequence that includes an LTR of a retroviral avian leukemia virus (ALV) vector, a retroviral murine leukemia virus (MLV) vector, or a lentivirus vector. The promoter is sufficient to effect expression of the coding sequence in the bird oviduct. The coding sequence codes for an exogenous protein that deposits in the egg white of a hard shell. As such, the coding sequence codes for exogenous proteins, such as transgenic poultry-derived proteins, such as transgenic poultry-derived lysosomal acid lipase (TPD LAL).

In one case, the vectors used in the methods of the invention contain a promoter that is particularly suitable for expression of exogenous proteins in birds and their eggs. As such, expression of the exogenous coding sequence can occur in the oviduct and blood of the transgenic bird and in the egg white of your bird. Promoters include, but are not limited to, cytomegalovirus (CMV) promoter, rous sarcoma virus (RSV) promoter, β-actin promoter (eg , a chicken β-actin promoter), a murine leukemia virus (MLV) promoter, a rat mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, and an ovotransferrin promoter. Optionally, the promoter may be a segment of at least one promoter region, such as an ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucine, and ovotransferrin promoter region segment. In one case, the promoter is a combination or a fusion of one or more promoters or a fusion of a portion of one or more promoters, such as ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucine and ovotransferrin promoters.

In one case, the signal peptide vector which is coding, such that signal directs the secretion of human LAL, in the light of which one includes a coding sequence operatively linked to the sequence after translation into a cell, the exogenous peptide expressed by the vector, such as like hard shell egg.

One aspect of the disclosure provides sequence coding for exogenous proteins produced as disclosed herein wherein the coding sequence is codon optimized for expression in a bird, for example a chicken. Codon optimization can be determined from codon usage of at least one and preferably more than one protein expressed in a bird cell (eg, a chicken cell). For example, codon usage can be determined from the nucleic acid sequences encoding chicken ovalbumin, lysozyme, ovomucine and ovotransferrin proteins. For example, the DNA coding sequence for the exogenous protein can be codon optimized using the Wisconsin Package version 9.1 BACKTRANSLATE® program (Genetics Computer Group, Inc., Madison, WI) with a codon usage table compiled from of ovalbumin, lysozyme, ovomucoid, and chicken ovotransferrin (Gallus gallus) proteins.

An important aspect of the present disclosure relates to poultry hard shell eggs (eg, chicken hard shell eggs) which contain an exogenous peptide or protein including but not limited to human LAL. The exogenous peptide or protein, such as human LAL, may be encoded by a transgene of a transgenic bird. Often, the exogenous peptide or protein (eg, LAL) is glycosylated. The protein may be present in any useful amount. In one case, the protein is present in an amount ranging from about 0.01 pg per hard shell egg to about 1 gram per hard shell egg. In another case, the protein is present in an amount ranging from about 1 pg per hard shell egg to about 1 gram per hard shell egg. For example, the protein may be present in an amount ranging from about 10 pg per hard shell egg to about 1 gram per hard shell egg (eg , range from about 10 pg per hard shell egg to about 400 milligrams per hard shell egg).

In one case, the exogenous protein of the invention is present in egg white. In one case, the protein is present in an amount ranging from about 1 ng per milliliter of egg white to about 0.2 grams per milliliter of egg white. For example, the protein may be present in an amount ranging from about 0.1 pg per milliliter of egg white to about 0.2 grams per milliliter of egg white (eg. , the protein may be present in an amount ranging from about 1 pg per milliliter of egg white to about 100 milligrams per milliliter of egg white. In one case, the protein is present in an amount ranging from about 1 pg per milliliter of egg white to about 50 milligrams per milliliter of egg white. For example, the protein may be present in an amount in the range of about 1 pg per milliliter of egg white and about 10 milligrams per milliliter of egg white (eg, the protein may be present in an amount in the range of about 1 µg per milliliter). 1 pg per milliliter of egg white and about 1 milligrams per milliliter of egg white). In one case, the protein is present in an amount of more than 0.1 pg per milliliter of egg white. In one case, the protein is present in an amount of more than 0.5 pg per milliliter of egg white. In one case, the protein is present in an amount of more than 1 pg per milliliter of egg white. In one case, the protein is present in an amount of more than 1.5 pg per milliliter of egg white.

Birds producing exogenous proteins disclosed herein (eg, LAL), which are developed to parts of the blastodermal cells into which the vector was introduced, are generation G0 and may be referred to as founders. Founder birds are typically chimeric for each inserted transgene. That is, only some of the GO transgenic bird cells contain the transgene (s). The GO generation is typically also hemizygous for the transgene (s). Generation GO can be crossed for non-transgenic animals to give rise to G1 transgenic progeny which are also hemizygous for the transgene and contain the transgene (s) in essentially all bird cells. G1 hemizygous progeny may be crossed for non-transgenic animals to give rise to G2 hemizigotic progeny or may be cross-linked to give homozygous G2 progeny for the transgene. Substantially all bird cells that are transgene positive that are derived from the G1 progeny contain the transgene (s). In one case, the hemizygous G2 progeny of the same line may be crossed to produce the homozygous G3 progeny for the transgene. In one case, hemizygous GO or G1 animals, for example, are cross-bred to give homozygous G1 progeny containing two copies of the transgene (s) in each animal cell. These are merely examples of certain reproduction methods and the present disclosure contemplates the use of any useful reproduction method, such as those known to those skilled in the art.

In one case, the disclosure provides for LAL to be isolated. That is, the LAL contained in the composition may be an isolated LAL. For example, LAL may be isolated from egg white. Isolated LAL can be LAL molecules having a variety of glycosylation structures between LAL molecules.

By the methods of the present disclosure, transgenes can be introduced into poultry embryonic blastodermal cells to produce a transgenic chicken, transgenic turkey, transgenic quail and other bird species, which transgene the genetic material of their germ line tissue in a manner producing the proteins of the invention. Blastodermal cells are typically stage VII-XII cells or their equivalent and, in one embodiment, are close to stage X.

Some vectors useful in carrying out the methods of the present disclosure are described herein. In one case, the coding sequence and vector promoter are both positioned between the 5 'and 3' LTRs prior to introduction into blastodermal cells. In one case, the vector is retroviral and the coding sequence and promoter are both positioned between the 5 'and 3' LTRs of the retroviral vector. In a useful case, the LTR or retroviral vector is derived from avian leukosis virus (ALV), murine leukemia virus (MLV) or lentivirus.

In one case, the vectors are used to transfect blastodermal cells and the generation of stable integration into the avian genome contains a coding sequence and an operative and positional promoter to express the coding sequence in the bird oviduct magnum tubular gland cell, wherein the exogenous protein, such as a lysosomal enzyme (eg, LAL) is deposited in the egg white of a hard shell.

The promoter may optionally be a segment of the ovalbumin promoter region that is sufficiently broad to direct expression of the coding sequence in tubular gland cells. The truncation of the ovalbumin promoter and / or condensation of the critical regulator elements of the ovalbumin promoter so that it retains the sequences necessary for expression in oviduct magnum tubular gland cells, while it is small enough that it can be easily Embedded in vectors are included within the scope of the invention. In one case, an ovalbumin promoter region segment may be used. This segment comprises the 5 'flanking region of the ovalbumin gene.

The promoter may also be a promoter that is quite, but not fully, specific to the magnum, such as the lysozyme promoter. The promoter may also be a rat mammary tumor virus (MMTV) promoter. Alternatively, the promoter may be a constitutive promoter (eg, a cytomegalovirus (CMV) promoter, a rous sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, etc.). In one embodiment, the promoter is a cytomegalovirus (CMV) promoter, an MDOT promoter, a rous sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a tumor virus promoter. rat mammary gland (MMTV), an ovalbumin promoter, a lysozyme promoter, conalbumin promoter, ovomucoid promoter, ovomucin promoter and / or ovotransferrin promoter. Optionally, the promoter may be at least one segment of a promoter region, such as an ovalbumin, lysozyme, conalbumin, ovomucoid, ovomucine and ovotransferrin promoter region segment.

In a blastodermal cell transfection method, a packaged retroviral vector is used to distribute the vector to embryonic blastodermal cells so that the vector is integrated into the avian genome.

Retroviruses useful for randomly introducing a transgene into the bird genome are replication-deficient avian leukosis virus (ALV), replication-deficient murine leukemia virus (MLV), or lentivirus. In one case, a pNLB vector is modified by inserting an ovalbumin promoter region and one or more exogenous genes between the 5 'and 3' long terminal repeat (LTR) of the retrovirus genome. The present disclosure contemplates that any coding sequence downstream of a promoter that is active in tubular gland cells may be expressed in tubular gland cells. For example, the ovalbumin promoter may be expressed in oviduct magnum tubular gland cells, since the ovalbumin promoter drives expression of the ovalbumin protein and is active in oviduct tubular gland cells.

Any of the above described vectors may also optionally include a coding sequence which encodes a signal peptide that directs the secretion of the protein expressed by the coding sequence of the vector from oviduct tubular gland cells. This aspect effectively broadens the spectrum of exogenous proteins that can be deposited in poultry eggs using the above described methods. Where an exogenous protein would not otherwise be secreted, the vector containing the coding sequence is modified to comprise a DNA sequence comprising about 60 bp that encodes a lysozyme gene signal peptide. The DNA sequence encoding the signal peptide is inserted into the vector such that it is located at the N-terminus of the DNA encoded protein.

Another aspect of the disclosure involves the use of internal ribosomal entry site (IRES) elements in any of the vectors of the present invention to allow translation of two or more proteins from a dicistronic or polycistronic mRNA. IRES units are fused to 5 'ends of one or more additional coding sequences which are then inserted into the vectors at the end of the original coding sequence so that the coding sequences are separated from one another by an IRES.

In one case, when using an IRES, post-translational modification of the product is facilitated as one coding sequence may encode an enzyme capable of modifying the product of the other coding sequence. For example, the first coding sequence may encode collagen, which would be hydroxylated and made active by the enzyme encoded by the second coding sequence, wherein an IRES is employed as understood in the art.

In another aspect, the vector coding sequences used in any of the methods of the present disclosure are provided with a 3 'untranslated region (3' RTU) to impart stability to the RNA produced. When a 3 'UTR is added to a retroviral vector, the orientation of the promoter, gene X and 3' UTR must be reversed in the construct so that the addition of the 3 'UTR does not interfere with transcription of full length genomic RNA. . In one embodiment, the 3 'UTR

<td>Can be</td><td>that of the genes of</td><td>ovalbumin or</td><td>lysozyme,</td><td>or of</td>

<td>any</td><td colspan="2">3 'RTU that is functional in a cell</td><td>of magno,</td><td>1 and.,</td>

<td>the region</td><td>SV40.</td><td></td><td></td><td></td>

<td>on one</td><td>case a prosecutor</td><td>constitutive is</td><td>used</td><td>for</td>

express the coding sequence of a transgene in the bird. In this case, the expression is not limited to the magnum; expression also occurs in other tissues within the bird (eg, blood). The use of such a transgene, which includes a constitutive promoter and coding sequence, is particularly suitable for effecting or conducting expression of a protein in the oviduct and subsequent secretion of the protein in the egg.

Transducer particles (i.e. transduction particles) are produced for the vector and are titrated to determine the appropriate concentration that can be used to inject embryos. Bird eggs are drilled according to the Speksnijder procedure (US Pat.² 5897998), and the eggs are injected with transducer particles. Eggs hatch about 21 days after injection and male birds are selected for breeding. In order to screen for GO roosters containing the transgene in their sperm, DNA is extracted from rooster sperm samples. GO roosters with the highest transgene levels in their sperm samples are crossbred with non-transgenic chickens by artificial insemination. Blood DNA samples are screened for the presence of the transgene. Transgenic rooster serum is tested for the presence of exogenous protein. If the exogenous protein is confirmed, the sperm of transgenic roosters is used for artificial insemination of non-transgenic chickens. A certain percentage of progeny then contains the transgene (eg, more than 50%). When exogenous protein is present in eggs produced according to the present disclosure, the protein may be isolated. The protein can also be tested for biological activity.

Methods of the disclosure which provide for the production of exogenous protein in the bird oviduct and the production of eggs containing the exogenous protein involve a subsequent further step to provide a suitable vector and to insert the vector into embryonic blastodermal cells such that the vector is integrated into the bird's genome. The subsequent step involves deriving a mature transgenic bird from the transgenic blastodermal cells produced in the previous steps. Mature transgenic birds may be obtained from cells of a blastodermal embryo that has been transfected or transduced with the vector directly within the embryo. The resulting embryo is allowed to develop and the offspring allowed to mature.

The transgenic bird produced from blastodermal cells is known as a founder. Some founders will carry the transgene into tubular gland cells on the magnum of their oviducts. These birds will express the transgene-encoded exogenous protein in their oviducts. Exogenous protein may also be expressed in other tissues (eg, blood) besides the oviduct. If the exogenous protein contains the appropriate signal sequence (s), it will be secreted into the oviduct lumen and egg white.

Some founders are founders of germline. A germ line founder is a founder who transports the transgene into genetic material from her germ line tissue and can also carry the transgene into cells of the oviduct magnum tubular glands that express the exogenous protein. Thus, according to the disclosure, the transgenic bird may have tubular gland cells expressing the exogenous protein, and the offspring of the transgenic bird may also have oviduct magnum tubular gland cells expressing the exogenous protein. Alternatively, the offspring expresses a phenotype determined by the expression of the exogenous gene in bird specific tissue (s). In one embodiment, the transgenic bird is a chicken or turkey.

PHARMACEUTICAL COMPOSITIONS &amp; THERAPEUTIC METHODS

While it is possible that for therapeutic use the therapeutic proteins produced as described herein may be administered in crude form, it is preferred to administer the therapeutic proteins as part of a pharmaceutical formulation. Thus, there are further provided pharmaceutical formulations comprising poultry-derived glycosylated therapeutic proteins such as LAL or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and / or prophylactic ingredients and methods of administration. administration of such pharmaceutical formulations. The carrier (s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and not deleterious to their recipient. Methods of treating a patient (eg, amount of pharmaceutical protein administered, frequency of administration and duration of treatment period) using pharmaceutical compositions of the invention may be determined using standard methodologies known to those skilled in the art.

Compositions comprising carriers, including composite molecules, are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14).^The Ed., Mack Publishing Co., Easton, Pa.). The carrier may comprise a diluent. In one case, the pharmaceutical carrier may be a liquid and the recombinant human LAL may be in the form of a solution. The pharmaceutical carrier may be wax, fat or alcohol. In one case, the wax or fat-based vehicle contains no ester. In another case, the pharmaceutically acceptable carrier may be a solid in the form of a powder, a lyophilized powder or a tablet. In one case, the carrier may comprise a liposome or a microcapsule.

Pharmaceutical formulations include those suitable for injection administration, including intramuscular, subcutaneous and intravenous administration. Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral. Pharmaceutical formulations also include those for administration by inhalation or insufflation. The formulations may, where appropriate, conveniently be presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Pharmaceutical formulation production methods typically include the step of bringing the therapeutic protein into association with finely divided liquid carriers or solid carriers and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units, such as capsules, wafers or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution; as a suspension; or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients, such as binders, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

LAL may also be formulated for parenteral administration (eg, by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multidose containers. with added preservative. Therapeutic proteins may be injected by, for example, subcutaneous injections, intramuscular injections, and intravenous infusions or injections.

LAL may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. It is also contemplated that the therapeutic protein may be in powder form obtained by aseptic isolation of the sterile solid or by lyophilization from the solution for reconstitution with a suitable vehicle, eg sterile, water in pyrogens, prior to use.

For intravenous infusions or injection, the LAL produced according to the disclosure may be formulated as an aqueous suspension or solution. Suitable excipients for the formulation for infusion or intravenous injection may include one of the following: dehydrated trisodium citrate, citric acid and human serum albumin. The pharmaceutical formulation may also include other suitable excipients, well known in the art, used for other products for lysosomal storage disorders. The pH of LAL produced according to the disclosure is maintained between about 5.6 and about 6.2. Preferably, the pH of the LAL formulation is maintained at 5.9 ± 0.2.

For topical administration to the epidermis, the therapeutic proteins produced according to the disclosure may be formulated as ointments, creams or lotions or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base and may also contain one or emulsifiers, stabilizing agents, thickening agents, suspending agents or more dispersing agents, coloring agents

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably represented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art and the suppositories may conveniently be formed by a mixture of the active compound with the softened or melted carrier (s). followed by cooling and molding in molds.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

For intranasal administration, the therapeutic proteins of the disclosure may be used as a liquid spray or dispersible powder or in droplet form. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently distributed from pressurized packaging.

For administration by inhalation, the therapeutic proteins according to the disclosure may conveniently be delivered from an insufflator, nebulizer or pressurized package or other convenient means of dispensing an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to dispense a metered amount.

For administration by inhalation or insufflation, the therapeutic proteins according to the disclosure may take the form of a dry powder composition, for example, a powder mixture of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, eg, gelatin or blister packs, from which the powder may be administered with the aid of an inhaler or insufflator.

Where desired, the above-described formulations adapted to obtain sustained release of the active ingredient may be employed.

The pharmaceutical compositions described herein may also contain other active ingredients, such as antimicrobial agents or preservatives.

diphenhydramine) as herein technique.

In one case,

Further, it is contemplated that the therapeutic proteins disclosed herein may be used in combination with other therapeutic agents. For example, the disclosure contemplates methods for pretreatment with a pharmaceutically effective dose of an antihistamine to minimize or prevent any potential infusion-related anaphylactic reactions. For example, the antihistamine may be any pharmaceutically acceptable antihistamine (e.g. disclosed and as known in the antihistamine is administered at a dose between about 1 mg and about 10 mg per kilogram body weight. For example, the antihistamine may be administered at a dose of about 5 mg per kilogram. In one case, the antihistamine is administered from about 10 minutes to about 90 minutes, for example about 30 minutes to about 60 minutes, prior to administration of lysosomal acid lipase using an ambulatory system attached to the vascular access port. . In one case, the dose of diphenhydramine effectively counteracts potential anaphylactic infusion reactions.

Immunosuppressants such as antihistamines, corticosteroids, sirolimus, voclosporin, cyclosporine, methotrexate, IL-2 receptor-directed antibodies, T-cell-directed antibodies, TNF-α-directed antibodies or fusion proteins may also be administered ( infliximab, etanercept or adalimumab), CTLA4-Ig (e.g. abatacept), anti-OX-40 antibodies before, during or after administration of LAL if an anaphylactic reaction or adverse immune response is experienced by the patient.

The disclosure also contemplates therapy involving the administration of LAL-containing compositions in combination with one or more cholesterol lowering agents (eg, HMG-CoA reductase inhibitors). Non-limiting examples of such agents include: atorvastatin (Lipitor (Lescol), lovastatin (Mevacor,

Torvast), fluvastatin Altocor®, Altoprev®), pitavastatin (Livalo®, Pitava®), pravastatin (Pravachol®, Selektine®, Lipostar®), rosuvastatin (Crestor®) and simvastatin (Zocor, Lipex)

The compositions or proteins described herein may be used to treat various conditions. For example, there are conditions for which treatment therapies are known to those skilled in the art. The present disclosure contemplates that therapeutic proteins (eg, LAL) produced in a bird system containing a poultry-derived glycosylation pattern may be employed to treat such conditions. That is, also contemplated is the treatment of conditions known to be treatable by therapeutic proteins produced by conventionally using therapeutic proteins produced as described herein. For example, LAL produced as described herein may be used to treat conditions resulting from or associated with LAL deficiency or insufficiency (collectively, LAL deficiency), such as Wolman's disease and cholesteryl ester storage disease (CESD). As described herein, LAL deficiency also encompasses states in which LAL expression is reduced due to a state (eg, a genetic mutation), physiological or environmental factors leading to a reduction or deficiency of LAL produced in the body. LAL produced as described herein may also be used to treat other conditions such as atherosclerosis, fatty liver disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis (NASH) and cirrhosis. LAL produced as described herein may also be used to treat other conditions, such as those disclosed in US Patent No.² 6849257 published February 1, 2005, US publication N²-2009/0297496 published December 3, 2009; US publication N² 2004/0223960 published on November 11,

<td>2004; and</td><td>US publication</td><td>N²</td><td> 2007/0264249</td><td>published</td><td>on 15</td>

<td>November</td><td>of 2009.</td><td></td><td></td><td></td><td></td>

<td>it is</td><td colspan="2">also contemplated</td><td>that LAL</td><td>produced</td><td>like here</td>

<td>disclosed</td><td colspan="2">can be used</td><td>to treat</td><td colspan="2">certain states</td>

<td colspan="2">including</td><td colspan="2">pancreatitis, by</td><td>example,</td><td>pancreatitis</td>

chronic and / or acute pancreatitis, as well as alcohol-induced pancreatic injury, such as alcohol-induced pancreatitis.

LAL produced by any useful method, such as those disclosed herein, is contemplated for use to treat diseases due to alcohol-induced cell damage including, but not limited to, those alcohol-induced cell lesions that result in the accumulation of lipid esters in body tissue. such as but not limited to liver, spleen, intestinal and cardiovascular tissue. The disclosure also contemplates treatment of malabsorption by administration of LAL.

One aspect of the disclosure relates to methods of treating a patient comprising administering to a patient a therapeutically effective amount of a composition comprising recombinant human LAL as described herein. The patient may be suffering from or diagnosed with any number of conditions, including those associated with LAL deficiency. In one case, the therapeutically effective amount is a quantity which increases a patient's red blood cell count to a desired amount. It is contemplated that LAL, produced according to the disclosure, may be used to treat chronic kidney disease, for example, where tissues fail to sustain lysosomal acid lipase production.

It is also contemplated that LAL produced by any useful method may be useful for treating patients with Tangier disease and familial hypoalphalipoproteinemia. Familial Tangier / hypoalphalipoproteinemia disease is associated with accumulation of cholesterol esters in macrophages accompanied by hepatosplenomegaly and / or lymphadenopathy along with low levels of high density lipoproteins (HDL) which can be treated by administration of exogenous LAL. For example, without wishing to limit disclosure to any particular theory or mechanism of operation, it is thought that reduced LAL activity decreases ABCA1 expression and, conversely, an increase in LAL activity, obtained by administering exogenous LAL to A patient with familial Tangier / hypoalphalipoproteinemia disease will increase ABCA1 expression to overcome the effects of an ABCA1 gene with reduced functional activity as a result of polymorphism.

For the treatment of a condition, the dosage administered may generally vary depending on known factors such as age, health and weight of the recipient, concurrent treatment type, frequency of treatment, and the like. Typically, a dosage of active ingredient may be between about 0.0001 and about 10 milligrams per kilogram body weight. The precise dosage, frequency of administration and period of treatment may be determined by a physician skilled in the art of administering the respective therapeutic protein.

In addition, it has been found that dosages of 1 mg / kg and lower may be effective in treating LAL deficiencies. The present disclosure provides methods of treating conditions comprising administering to a mammal (eg, a patient, preferably a human patient) a therapeutically effective dose of lysosomal acid lipase between once every 5 days and once every 25 days. days, for example, between once every 7 days and once every 14 days. In one case, the dose of lysosomal acid lipase administered is between about 0.1 mg and about 50 mg per kilogram body weight, for example, the dose may be between about 1 mg and 5 mg per kilogram.

In a particularly useful case, the disclosure provides methods of treating a condition by administering a lysosomal acid lipase dose of from about 0.1 mg to 1.0 mg per kilogram body weight according to any therapeutically effective dosage regimen. , such as those described herein.

The disclosure provides methods for treating any complication of LAL deficiency that may benefit from administration of a therapeutically effective dose of LAL. In one case, malabsorption and growth failure may be treated according to the methods described herein. In another case, complications observed in patients with LAL deficiency including but not restricted to hepatomegaly and liver dysfunction may be treated using the methods provided herein.

The disclosure provides treatment with recombinant LAL (eg, recombinant human LAL) that can be produced by any useful protein expression system, for example, transgenic mammals and birds as is understood in the art. Other protein expression systems may include, but are not limited to cell culture, bacteria and plant systems.

The disclosure encompasses administration of recombinant LAL as a part of a pharmaceutically acceptable composition by any route that can achieve the intended therapeutic effect as determined by one of ordinary skill in the art. In one case, LAL may be administered by intravenous infusion over a period of about five hours. For example, infusion may be facilitated by an ambulatory infusion pump connected to a vascular access port (eg, a Port-a-Cath).

The disclosure also includes monitoring the clinical and pathological presentation of conditions, for example, Wolman's Disease and CESD, in the mammal (eg, the human patient). In one case the assessments consist of, but are not limited to: lipid analysis, chest x-ray, liver function tests, stool plot, plasma mevalonic acid, immunogenicity, lysosomal acid plasma lipase, chitotriosidase, PARC, portal hypertension, anthropometry, volume and characterization of the liver, spleen and gastrointestinal tract using, for example, imaging technology.

For example, the imaging technology mentioned above may consist of ultrasound, magnetic resonance imaging, and nuclear magnetic resonance spectroscopy.

EXAMPLES

The present invention will be further exemplified by the following examples. The examples are for illustrative purposes only and are not intended and should not be construed as limiting the invention in any way.

Example 1

Vector Construction (pALVIN-OVR1-I-hLAL-dSA) that Carries Recombinant Human Lysosomal Acid Lipase (rhLAL) Coding Sequence

The nucleotide sequence of the hLAL gene in the pALVIN-OVR1-I-hLAL-dSA vector encodes a protein that is identical to the amino acid sequence of the protein produced by the human lysosomal acid lipase gene (GenBank Accession, NP_000226) (FIG. 1). Transcription of this sequence and subsequent translation of the resulting mRNA yields a 399 amino acid precursor protein which is processed to a mature 378 amino acid protein identical to human LAL (GenBank Accession, NP_000226) (FIG. 1) as defined in SEQ ID NO:²1. Expression of the hLAL gene (see FIG. 2 for cDNA sequence) in this Example is controlled by non-coding elements derived from the ovalbumin gene, including untranslated enhancer, promoter, intronic, and 5 'sequences and 3 '. The ovalbumin gene produces ovalbumin, the main constituent protein of egg white. The activity of the chicken ovalbumin promoter is very specific to the cells within the chicken oviduct that produce egg white; Expression in other tissues is minimal.

pALVIN-OVR1-I-hLAL-dSA plasmid vector (FIG. 3A; the nucleotide sequence of which is shown in FIG. 4) was used to produce a replication deficient retrovirus (RDR) that stably integrated the hLAL transgene into the genome of the founder (XLL109). This plasmid vector includes retroviral nucleotide sequences required for RNA packaging, reverse transcription, and integration, but does not contain the intact sequences for the gag, pol, and env genes. Methods used to produce the retroviral vector and its use in subsequent transgenesis procedures are described herein.

The retroviral part of pALVIN-OVR1-I-hLAL-dSA is based on the ALV vector, pNLB. pNLB was modified such that LTR would be self-inactivating (SIN) (FIG. 3B). To accomplish this, 273 bp of the 3 'LTR were deleted, which includes the U3 region CAAT box and enhancer. Since the inactivated U3 region at the 3 'end of the retroviral sequence serves as a template for a new U3 region present at the 5' end of an integrated provirus, 5 'LTR is also normally inactivated. Deletion of LTR sequences in the SIN construct decreases promoter interference with the LTR internal promoter, and minimizes the possibility for sequence recombination to form a replication competent retrovirus. The new vector is called pALVIN for ALV inactivation vector.

Downstream of the 5 'LTR are the partial gag and env coding sequences which were carried from the pNLB vector. In pALVIN-OVR1-I-hLAL-dSA, a small part (12%) of the gag protein precursor sequence remains (55% of the mature p9 peptide sequence) and a small part (1.7%) of the env precursor sequence remains. RAV2 (GenBank Access, AF033808). These truncated gag and env regions are unable to produce functional proteins necessary to produce the replication competent retrovirus (Cosset, 1991).

Chicken ovalbumin gene transcription and translation control elements were inserted into pALVIN to produce pALVIN-OV-1.1-1 (whose sequence is shown in FIG. 6; SEQ ID N²: 8). The first section of pALVIN-OV-1.1-1 is composed of a contiguous section of the chicken ovalbumin gene that includes the 1.1 kb proximal promoter region, the first exon, first intron and part of the 2² exon. The following section is a stuffer insertion fragment that takes the place of the ovalbumin protein coding sequences. The stuffer is followed by the 3 'untranslated region (UTR) of the chicken ovalbumin gene, which includes sequences that facilitate proper mRNA processing, including polyadenylation. In general, the stuffer fragment is replaced by DNA fragments encoding the desired protein, in this case hLAL. 0 The result is a vector that has specific elements that promote expression of regulated transcription and translation of an mRNA in the oviduct of transgenic chickens, which closely mimics the regulation of endogenous ovalbumin mRNA and allows high expression of the protein of interest in egg white.

pALVIN-OV-1.1-1 vector includes the first intron of the ovalbumin gene. Since the intron is susceptible to excision-binding during retroviral RNA genome production and packaging, we have inserted the expression cassette in the opposite orientation relative to LTR. Thus, the intron is not recognizable in retroviral RNA and is packaged without splice excision. For convenience all maps in this document are drawn with the LTR in the opposite orientation and the expression cassette in the direct or clockwise orientation.

pALVIN-OV-1.1-1 is the base vector into which the hLAL coding sequence (CDS) has been inserted. Two DNA fragments, hLAL adapter and Syn hLAL, which constitute the hLAL CDS and sequences necessary for pALVIN-OV-1.1-1 compatibility, were synthesized in Integrated DNA Technologies, Coralville,

A fragment A fragment

Iowa, (see FIG. 7 and 8; SEQ ID NO:² : 9 and 10).

229 bp Hpal / BamHI from hLAL adapter and 1113 bp Syn hLAL BamHI / BstBI were inserted into the Hpal / BstBI 7882 fragment of pALVIN-OV-1.1-1, thereby replacing the stuffer region with the hLAL CDS and producing pALVIN-OV-1.1-1-hLAL.

It was found that there was a cryptic splice site in the hLAL CDS antisense strand that prevented the packaging of intact retroviral DNA. The cryptic splice site was removed by altering the DNA sequence without altering the amino acid sequence of hLAL. This change was made by polymerase chain amplification of region 232 to 534 of pALVIN-OV-1.1-I-hLAL with the 5'-AGAAACTGAGAGTGTCTTAT-3 'primer (SEQ ID N² : 12) and 5'-TGACAGCTGTGGATCCAGAAACAAACATG-3 'primer (SEQ ID NO:²: 13), yielding a 329 bp amplicon. This amplicon was digested with BamHI and SexAI and ligated with the 8940 bp BamHI / SexAI fragment from pALVIN-OV-1.1-I-hLAL to yield pALVIN-OV-1.1-I-hLAL-dSA.

hLAL-dSA for performed as follows DHSIII enhancer and

A putative promoter enhancer containing the chicken ovalbumin gene DNase hypersensitive site III (DHSIII) (-3819 to -2169 relative to the OV promoter initiation site) (Kaye, Bellard et al., 1984) was inserted. in pALVIN-OV-1.1-1produce pALVIN-OVR1-I-hLAL-dSA. This was a DNA fragment that included the 1.1 kb proximal OV promoter called the 0VR1 promoter (see FIG. 9; and SEQ ID NO:²: 11 for the sequence) were isolated by digestion with XhoI and BlpI. To facilitate subcloning, a fragment of the pSIN-OV-1.1-1 PCR adapter was produced by PCR amplification of the 6752 to 7974 region of pALVIN-OV-1.1-1 with 5'-GCCGCTCGAGCGAGGAATATAAAAAAATT-3 'primers (SEQ ID N²: 14) and

5'-TCCGCGCACATTTCCCCGAA-3 '(SEQ ID NO:²: 15) followed by digestion with NgoMI and Xhol. The 2772 bp XhoI / Blpl fragment of the 0VR1 promoter and 1067 bp NgoMI / XhoI PCR fragment from pSIN-OV-1.1-1 were inserted into the 7043 bp NgoMl / BlpI fragment of pALVIN-OV1.1-I-hLAL- dSA, thereby producing pALVIN-OVR1-I-hLAL-dSA (see FIG. 10 for the pALVIN-OVR1-IhLAL-dSA construction schemes). The construction of the retroviral vector segment of the vector, referred to as pALVIN (akapAVIJCR-A395.22.3.1-KM or pALV-SIN), is described in U.S. Patent Application No.² 2008/0064862.

In addition, LAL production according to the disclosure using a promoter and / or vector disclosed in US Patent Publication No.² 2008/0064862 published March 13, 2008.

Example 2

<td>Production</td><td colspan="4">of the viral particle</td>

<td>0 male</td><td>transgenic founder</td><td>G0,</td><td>XLL109, which</td><td>carries the</td>

<td>transgene of</td><td>hLAL in your genome,</td><td>was</td><td>produced</td><td>using a</td>

<td colspan="2">retroviral transgenesis method</td><td>how</td><td>follows.</td><td>The particles</td>

replication defective viruses carrying the pALVIN-OVR1-I-hLAL-dSA vector were produced by transient transfection of an immortalized chicken fibroblast cell line. These chicken fibroblast cells were transfected simultaneously with three plasmids, PALVIN-OVR1-I-hLAL-dSA, pCMV-gag-pol and pCMV-VSV-G. pCMV-gag-pol expresses the avian leukosis virus strain RAV1 gag and pol genes. pCMV-VSV-G expresses the vesicular stomatitis virus envelope protein. Four hours after transfection, the medium was replaced with DMEM supplemented with 10% fetal bovine serum, 100 units / mL penicillin and 100 pg / mL streptomycin. The medium was collected at 48 hours post transfection, filtered through a 0.45 micron filter (Millipore) and concentrated by ultracentrifugation. Concentrated retrovirus carrying the ALVIN-OVR1-I-hLAL-dSA transgene was collected and used for early stage embryo transduction. Note that because p is the notation for the plasmid form of the vector, p is absent from the transgene designations when the transgene is in packaged vector form or integrated transgene.

Example 3

Embryo Transgenesis

Integration of the ALVIN-OVR1-I-hLAL-dSA expression cassette into the genome of an embryo was achieved by early stage embryo transduction (Speksnijder and Ivarie, 2000). Freshly fertilized White Leghorn eggs were obtained from a breeding colony. An opening was made in the shell to provide access to the embryo. Seven microliters of concentrated replication deficient retrovirus particles carrying the ALVIN-OVR1-I-hLAL-dSA expression cassette described above were injected into the subgerminal cavity of the embryo. The eggs were sealed with hot glue and then incubated and hatched under standard conditions. The offspring produced from these injections were given individual identification markers at hatching for identification and traceability. Blood samples from progeny were transgene positive when analyzed by real time PCR for hLAL transgene using hLAL coding sequence specific PCR primers (as described below). This gave an indication that the transgenesis procedure was successful. The real time PCR assay for the hLAL transgene utilizes Taqman® chemistry (Applied Biosystems). The forward and reverse primers were 5'-ACGACTGGCTTGCAGATGTCT-3 '(SEQ ID N² : 16) and 5'-ACCOAAATGAAGTCAAGATGCT-3 '(SEQ ID NO:² : 17), respectively. The Taqman® probe sequence was 5'-CCGGAATGCTCTCATGGAACACCAA-3 '(SEQ ID NO:²: 18) and was tagged with FAM (as the emitter) at the 5 'end and Iowa Black (as the mitigator) at the 3' end. Primers, probe and 1 µl extracted DNA were added to 30 µl Taqman® Universal Master Mix (Applied Biosystems). Control reactions included various dilutions of a plasmid carrying the sequence of hLAL and wild-type chicken DNA (data not shown). Standard cycling parameters were used on a 7500 Fast Real-Time PCR System from Applied Biosystems.

Example 4

GO Founder Identification

Semen was collected from sexually mature males and DNA was extracted and assayed using the hLAL real time PCR assay. The number of transgene copies in each sample was estimated using known standards (a plasmid that will make up the hLAL gene) mixed with negative control semen DNA. The DNA content of the transgene cassette in male XLL109 was at a level that would allow transmission of the transgene to its progeny, as estimated by real time PCR. This male XLL109 was the GO transgenic founder and was mated with non-transgenic chickens to produce the G1 hemizygous transgenic chickens.

Example 5

Propagation and Characterization of G1 Hemizygotic Birds

The progeny of the transgenic XLL109 founder were tested for the presence of the transgene in blood cell DNA using the hLAL real time PCR assay. Blood was collected from 1-2 weeks of age and DNA was extracted using a high throughput technique (Harvey et al., 2002). DNA solutions were not quantified prior to the Taqman assay to facilitate high throughput screening. Typically, 1 µl of DNA solution contains 50 to 400 ng of DNA, which is sufficient to produce a positive amplification signal. A total of 1322 XLL109 standardized offspring were tested, and positive offspring were bled again and tested for confirmation. According to PCR results, 22 offspring were positive for the ALVIN-OVR1-I-hLAL-dSA transgene. An example of Taqman results is shown in FIG. 11

Example 6

High expression line identification and characterization

One of the Gl chickens, 1LL7466, laid eggs with significantly higher levels of rhLAL protein in egg white compared to the other Gl chickens. Southern blot analysis was performed on 1LL7466 and Gl brother males to identify which sibling males they had the same place of integration as the chicken under high pressure. Digestions were performed with a restriction enzyme that cut only once into the transgene (BlpI), and Southern blots were probed with a segment of the ovalbumin promoter or hLAL coding sequence (FIG. 12A-D). The position of 2² The restriction site, which resides in the flanking genomic region, varies depending on the integration site. Thus, the size of the BlpI band detected by the OV probe or hLAL probe is unique for each line produced.

The OV probe detected a single 4.1 kb band in BlpI digested DNA from wild-type chickens, which corresponded to the expected size of a BlpI segment within the endogenous ovalbumin gene of the chicken genome (FIG. 12B and 12D). A second 4.3 kb band was detected with chicken 1LL7466, which corresponded to the transgene band. Three additional female sisters, 1LL10409, 1LL10686 and 1LL12058 and three additional male sisters, 1LL8922, 1LL9330 and 1LL11217 had the 4.3 kb band, indicating that these siblings may be in the same line (FIG. 12B and 12D).

As expected, the hLAL probe did not detect a band in wild-type chicken DNA since the chicken lysosomal acid lipase gene DNA sequence and the coding sequence for recombinant human lysosomal acid lipase are sufficiently differentiated to not allow hybridization under the conditions used in these Southern assays (FIG. 12C). The hLAL probe detected a single -10.6 kb band in BlpI-digested genomic DNA from the same chickens that were positive for the 4.3 kb band detected by the OV probe, indicating that these 7 G1 chickens have the same site. integration and thus are in the same vein.

No other bands were detected, indicating that 1LL7466, 1LL10409, 1LL10686, 1LL12058, 1LL8922, 1LL9330 and 1LL11217 all had a single integration site.

Southern analysis also indicated that the transgene was integrated, since the bands detected by the OV and hLAL probes were of different sizes and larger in size than the transgene alone. In FIG. 12A is shown a map showing the predicted integrated transgene structure and position of the BlpI sites in the flanking genomic regions.

To confirm that the transgene is intact, two steps were taken. First, the hLAL coding sequence was isolated by PCR from 1LL7466. PCR products were tracked on both strands of the hLAL start codon to the stop codon. The DNA sequence was exactly as expected, not indicating changes in the DNA sequence of coding regions in the transgene. Second, Southern blot analysis was conducted using the restriction enzyme ApaLI, which digests the intact transgene into 2 segments, 3.6 and 3.8 kb (FIG. 13A). Love the bands of 3.6 and

3.8 kb were detected in Gls ApaLI digested genomic DNA, indicating that the transgene was integrated in a fully intact mode (FIG. 13B).

Example 7

G2 Propagation and Characterization

FIG. 14 shows the lineage of hLAL G2 single-descended G2 lineages GO, XLL109. At stage G1, the transgene was characterized in relation to copy number, integrity, hLAL sequence, and integration site - and seven G1 transgenics were identified and characterized (guatro hens and three roosters). G2 propagation was achieved by artificial insemination of non-transgenic chickens with semen collected from the Gl1LL8922, 1LL9330 and 1LL11217 progenitors (FIG. 14). Each inseminated chicken, its eggs and subsequent offspring were housed separately from the other offspring. Hatched offspring were tested for the presence of the hLAL transgene using the hLAL real time PCR assay. Since the G1 founders were hemizygous with respect to the transgene, half of the progeny were expected to be transgenic G2. Of 610 G2 descendants analyzed to date, 330 or 54% were transgenic.

Example 8

Genetic Analysis of Birds with hLAL

After identification of each G2 chicken by the hLAL blood DNA real-time PCR assay, the production line is subjected to the following genetic assays: the hLAL gene was PCR amplified from the blood DNA and followed to confirm 100%. of homology with human following; the transgene integration site was confirmed by PCR of the integration site as described above. PCR tracking and integration site analysis were performed on: each chicken in a production line with &lt;10 chickens; 10% chickens (minimum 10) for the 11-100 chicken production line; 5% chicken (minimum 10) for the 101-1000 chicken production line; 1% chickens (minimum 50) for the 1001-10000 chicken production line; 0.1% chickens (minimum 100) for production line with&gt; 10001 chickens. Detailed records were kept at each step of the growth and production phase.

Example 9

HLAL Purification from Egg White

The egg white (EW) containing LAL was solubilized at pH 6 overnight and clarified by centrifugation (or deep filtration) with 0.2 µm filtration. EW was adjusted with 1 M NaOAc buffer (pH 4) to pH 6.

Clarified EW was loaded onto a Phenyl-HIC column (EW: column size = 2: 1) equilibrated with 20 mM phosphate buffer / 137 mM NaCl (pH 6). Upon completion of loading, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). LAL was eluted with 30% propylene glycol with 5 mM Tris buffer (pH 7.2).

The eluted LAL fraction was adjusted to pH 5 with 1 M acid and then loaded onto a GigaCap S column (EW: column size = 10: 1). The column was equilibrated with 50 mM NaOAc buffer (pH 5). Upon completion of loading, the column was washed with equilibration buffer. LAL was eluted with 50 mM NaOAc / 60 mM NaCl (pH 5).

The LAL fraction outside the GigaCap S column was adjusted to pH 6 with 1 M Tris buffer and then loaded onto a Butyl-HIC column (EW: column size = 10: 1). The column was equilibrated with 20 mM phosphate buffer / 137 mM NaCl (pH 6). Upon completion of loading, the column was washed with equilibration buffer and 5 mM phosphate buffer (pH 6). Pure LAL was eluted with 50% propylene glycol with 5 mM Tris buffer (pH 7.2). FIG. 15 represents the steps of purifying hLAL from egg white.

Example 10

HLAL Carbohydrate Analysis Transgenic Bird Derivative

Oligosaccharide structures were determined for bird-derived human LAL using the following analysis techniques as are well known to those of ordinary skill in the art.

Two hundred micrograms were digested with trypsin and chymotrypsin for 18 h at 37 ° C. ²C, in 0.1 M Tris-HCl, pH 8.2, containing 1 mM CaCl 2. Digestion products were enriched and released from contaminants per Sep-Pak 08 cartridge column. After enrichment, glycopeptides were digested with 2 pL PNGaseF (7.5 units / ml) in 50 pL 20 mM sodium phosphate buffer. pH 7.5 for 18 h at 37 ° C²C. The released oligosaccharides were separated from the peptide and enzyme by passage through a Sep-Pak 08 cartridge column system.

The glycan fraction was dissolved in dimethyl sulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). The reaction was mitigated by the addition of water and the per-O-methylated carbohydrates extracted with dichloromethane. Per-O-methylated glycans were dried under a stream of nitrogen.

MALDI / TOF-MS (flight time ionization desorption matrix laser assisted mass spectrometry) was performed in the reflective positive ion mode using α-dihydroxybenzoic acid (DHBA, 20 mg / mL solution in 50% of methanol: water) as a matrix. All spectra were obtained using a Microflex LRF (Bruker).

Analyzes of MALDI-TOF-MS and ESI MS / MS (electrospray tandem mass spectrometry) were performed on oligosaccharides after release of the peptide structure and purification as understood in the art. Samples of the individual polysaccharide species were also digested with certain enzymes and digestion products were analyzed by HPLC as understood in the art.

There are believed to be about six N-linked glycosylation sites present in human LAL. See, Zschenker, et al. , (2005) J. Biochem., Vol 137, p 387-394. This reference also indicates that there may be an O-linked glycosylation site in the

LAL human.

The identified linked oligosaccharide structures are shown in FIG. 16

The data revealed that many or all of these structures were found as an N-linked glycosylation structure in the LAL produced according to the disclosure (FIG. 16). For example, An is found to be linked to LAL produced according to the disclosure. For example, On is found linked to LAL produced according to the disclosure. For example, at least one of Bn, Cn and Dn is found to be linked to LAL produced according to the disclosure. For example, at least one of En and Fn is found to be linked to LAL produced according to the disclosure. For example, at least one of In and Jn is found to be linked to LAL produced according to the disclosure. For example, at least one of Kn and Ln is found to be linked to LAL produced according to the disclosure. For example, at least one of Mn and Nn is found linked to LAL produced according to the disclosure.

For example, Gn is found linked to LAL produced according to the disclosure. For example, Hn is found linked to LAL produced according to the disclosure.

Example 11

Species of LAL N-glycan Transgenic Bird Derivative

Purified transgenic bird derived hLAL samples (600 pg / sample) were dialyzed using a Tube-O-Dialyzer (4.0 kDa cutoff membrane; G BioSciences) against 4 nanopure water ²C for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period.

After dialysis, each sample was divided into three aliquots: ~ 1/4 of the sample weight for neutral and amino sugars analysis, ~ 1/4 of the sample weight for mannose-6-phosphate analysis, and ~ 1/2 of the sample weight for oligosaccharide profile. The aliquot for the analysis of neutral and amino sugars was hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100 ° C.²C for 4 hours and the aliquot for mannose-6-phosphate analysis was hydrolyzed with 6.75 N TFA at 100 ° C. ²C for 1.5 hours. The hydrolysates were then dried under N₂, redissolved with 50 µL H 2 O, sonicated for 7 min on ice and transferred to a vial. However, neutral and amino sugar samples were diluted 2-fold since the peaks produced from the originally dissolved hydrolysates were too large.

A mixture of standards for neutral and amino sugars, and for mannose-6-phosphate with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentrations of the standard neutral sugar and amino mixture (Fuc &amp; GalNAc, 0.2, 0.4, 0.8, and 1.6 nmoles per 10 pL; GlcNAc, 0.5, 1.0, 2, O, and 4.0 nmoles per 10 µL, Gal &amp; Man, 0.3, 0.6, 1.2, and 2.4 nmoles per 10 µL, and Glc, 0.1, 0.2, 0.4 , and 0.8 nmoles per 10 µl) and mannose-6-phosphate (640, 1280, 2560, 5120 picomoles per 10 µl) to establish a calibration equation. The number of moles in each sugar in the sample was quantified by linear interpolation from the calibration equation.

Neutral and amino sugars and mannose-6-phosphate were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector and an autosampler. The individual neutral and amino sugars and mannose-6-phosphate were separated by a Dionex CarboPac PA20 analytical column (3 x 150 mm) with an amino trap. Gradient programs used eluents A, degassed nanopure water and B, 200 mM NaOH for neutral and amino sugars, and C, 100 mM NaOH and D, 1 M sodium acetate in 100 mM NaOH for mannose-6-phosphate. The injection (10 pL / injection) was performed every 40 minutes for the determination of neutral and amino sugar and every 35 minutes for the determination of mannose-6-phosphate. All methods were based on protocols described by Hardy and Townsend (Hardy, MR, and Townsend, R. R., High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates, 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition were obtained using Dionex chromeleon software. Results are shown in Table 1 below. The control sample is ovomucoid purified from EW.

Table 1. Control monosaccharide and LAL composition by HPAEC.

<td colspan="2">ID of</td><td colspan="3">nanomoles /</td>

<td>Sample</td><td>Analyte</td><td>nanomoles</td><td>ug</td><td>mol%</td>

<td>Control</td><td>Fucose</td><td>na</td><td> -</td><td> -</td>

<td></td><td>N-acetyl galactosamine</td><td> 5, 066</td><td> 0,020</td><td> 9, 6</td>

<td></td><td>N-acetyl glucosamine</td><td> 26,947</td><td> 0,108</td><td> 51,4</td>

<td></td><td>Galactose</td><td> 3, 876</td><td> 0,016</td><td> 7,4</td>

<td></td><td>Glucose</td><td>na</td><td> -</td><td> -</td>

<td></td><td>Manose</td><td> 16,565</td><td> 0,066</td><td> 31,6</td>

<td></td><td>Mannose-6-phosphate</td><td>na</td><td> -</td><td> -</td>

<td></td><td>N-acetyl neuraminic acid</td><td>ndm</td><td> -</td><td> -</td>

<td></td><td>N-glycolyl neuraminic acid</td><td>ndm</td><td> -</td><td> -</td>

<td>hLAL</td><td>Fucose</td><td>na</td><td> -</td><td> -</td>

<td>derived from</td><td>N-acetyl galactosamine</td><td> 17,932</td><td> 0,120</td><td> 37,6</td>

<td>bird</td><td>N-acetyl glucosamine</td><td> 0,879</td><td> 0,006</td><td> 1,8</td>

<td>transgenic</td><td>Galactose</td><td>na</td><td> -</td><td> -</td>

<td></td><td>Glucose</td><td> 23,290</td><td> 0,155</td><td> 48,8</td>

<td></td><td>Manose</td><td> 5, 642</td><td> 0,038</td><td> 11, 8</td>

<td></td><td>Mannose-6-phosphate</td><td>ndm</td><td> -</td><td> -</td>

<td></td><td>N-acetyl neuraminic acid</td><td>ndm</td><td> -</td><td> -</td>

nd = not detected; ndm = not determined.

Structural Characteristics of LAL

LAL has 6 potential sites in its Asn-linked glycosylation amino acid sequence³⁶, Asn⁷², Asn¹⁰¹ Asn¹⁶¹, Asn²⁷³ and Asn³²¹. It was found that five of these, Asn³⁶, Asn¹⁰¹, Asn¹⁶¹, Asn²⁷³ and Asn are glycosylated, while Asn is unglycosylated or substantially unglycosylated (substantially unglycosylated means that in a mixture of LAL molecules, they are said to be minus Asn from Asn gualguer, Asn Asn, Asn²⁷³ and Asn³²¹). Therefore, one aspect of the disclosure is LAL (eg, human LAL) which is unglycosylated and / or substantially unglycosylated on Asn.⁷², and production and use of such LAL. However, LAL having an Asn⁷² glycosylate is within the scope of the disclosure. N-glycan structures consist mainly of a mixture of bi-, tri- and tetra-antenary structures with N-acetylglucosamine, mannose and mannose-6-phosphate (M6P) as the major sugars. Each site appears to have a favorite set of structures (Table 2 and FIG. 17), as is one aspect of the disclosure. For example, M6P-modified N -glycans reside at least in Asn¹⁰¹Asn¹⁶¹ and Asn²⁷³ . Non-phosphorylated structures are typical of N-glycans found in endogenous egg white proteins. No 0-linked glycans were detected as determined by the absence of N-acetylgalactosamine (GalNac). No sialic acid was detected, which is consistent with the previously determined N-glycan structures and exogenous proteins produced according to the disclosure. The disclosure includes glycosylated LAL with one or more of the disclosed acute oligosaccharide structures.

Table 2. Site residence of LAL glycan structures as determined by glycopeptide LC / MS.

<td>Site</td><td>Glycan Structure</td>

<td>Asn³⁶</td><td>GlcNAc4Man3GlcNAc2 HexlGlcNAc4Man3GlcNAc2</td>

<td>Asn⁷²</td><td>None detected</td>

<td>Asn¹⁰¹</td><td>Phos2Man7GlcNAc2</td>

<td>Asn¹⁶¹</td><td>PhoslMan6GlcNAc2 GlcNAclPhoslMan6GlcNAc2 Man3GlcNAc2 GlcNAc2Man3GlcNAc2 GlcNAc2Man3GlcNAc2 GlcNAc3Man3GlcNAc2 GlcNAc4Man3GlcNAc2 HexlGlcNAc4Man3GlcNAc2</td>

<td>Asn²⁷³</td><td>Man7GlcNAc2 Man8GlcNAc2 Man9GlcNAc2 PhoslMan8GlcNAc2 PhoslMan9GlcNAc2</td>

<td>Asn³²¹</td><td>GlcNAc2Man3GlcNAc2 GlcNAc3Man3GlcNAc2 GlcNAc4Man3GlcNAc2 HexlGlcNAc4Man3GlcNAc2 GlcNAc5Man3GlcNAc2 HexlGlcNAc5Man3GlcNAc2 GlcNAc6Man3GlcNAc2 HexlGlcNAc6Man3GlcNAc2</td>

<td>Hex, galactose;</td><td>Phos, phosphate; Man, mannose; GlcNAc2, N-acetylglucosamine</td>

Methods

The composition of monosaccharides, including neutrals, amino and M6P, was determined qualitatively and quantitatively by high pH anion-exchange pulsed amperometric detection chromatography (HPAEC-PAD).

The structures of the predominant glycans were determined with data from various mass spectrometry methods (MALDI-TOF, NSI-MS / MS and glycopeptide LC-MS).

MALDI-TOF was useful for the determination of neutral N-glycans and was able to detect phosphorylated N-glycans (FIG. 18). NSI-MS / MS was employed to determine the nature of minor peaks in MALDI-TOF spectra, some of which were attributed to phosphorylated N-glycans (FIG. 19). Efforts to improve MALDI-TOF's ability to detect N-glycans have not been fruitful.

Glycopeptide LC / MS was able to detect neutral and phosphorylated structures and was able to determine the position of specific structures in the LAL amino acid sequences (data summarized in FIG. 17 and Table 2).

To determine which peaks in the HPAEC-PAD chromatogram are due to phosphorylated N-glycans, LAL was treated with phosphatase and analyzed (Figure 3). Peaks in groups C and D decreased in area under the curve (AUC) while a peak in group A became more prominent. The peaks in group B did not change in proportion to the other peaks. Based on the knowledge that retention time is proportional to the degree of charge (due to phosphorylation or sialylation), it is contemplated that group C is composed of N -glycans with a phosphate (mono M6P) and group D is composed of N phosphate glycans (bis-M6P).

Retention time was also affected by the composition and relative structural position of neutral and amino monosaccharides. Such examples include the presence of galactose, the presence of a bisecting GlcNac and the degree of GlcNac substitution. Such factors contribute to the multiplicity of peaks in the HPAEC-PAD chromatogram.

Example 12

In vitro Enzyme Activity Analysis of hLAL Transgenic Bird Derivative in Egg White

The lysosomal acid lipase activity in egg white was determined using the fluorogenic substrate assay.

4-methylumbelliferyl oleate essentially as described in Yan et al. (2006), American Journal of Pathology_f Vol. 169, N² 3, p 916-92 6.

A stock solution of 4-methylumbelliferyl oleate (4-MUO) was prepared consisting of 2.5 mM 4-MUO in 4% Triton X-100. The assay was performed in a microtiter plate, each well containing 62.5 pL 0.2 M sodium citrate (pH 5.5) in 0.01% Tween80, 12.5 pL egg white sample and 25 4-MUO pL 2.5 mM. The change in fluorescence was monitored for 30 minutes at 37 ° C.²C, using a Bio-Tek Synergy HT fluorometric plate reader (360 nm excitation and 460 nm emission). Prior to the assay, hLAL-containing egg white was diluted to an enzymatic concentration which resulted in the reaction continuing linearly for at least minutes. The reaction was stopped with 50 pL of 0.75 M Tris-HCl, pH 8.0 and the endpoint fluorescence signal was measured on the same plate reader as above (360 nm excitation and 460 nm emission).

Activity units were determined using

4-methylumbelliferyl as a standard. One unit (U) is defined as the amount of enzyme that results in the formation of 1 æmol of 4-methylumbelliferyl / min under the assay conditions described above. Non-hLAL-containing egg white was used as a negative control.

The hLAL positive egg white samples contained between 1 U and 100 U activity per mL egg white. Egg white from 21 G1 chickens was analyzed. Egg white from 10 of the chickens tested positive for hLAL activity.

Example 13

In Vitro Analysis of LAL Transgenic Bird Derivative

The ability of LAL produced in transgenic bird oviduct cells (referred to herein as SBC-102, LAL or hLAL-derived bird) to bind to cells and to be internalized in the lysosomal compartment was examined in vitro using macrophage and fibroblast cells. . When incubated with macrophage cells, fluorescently labeled SBC-102s were found to localize to the lysosome. This effect could be attenuated using a polysaccharide mannose competitor, implying the N-acetylglucosamine / mannose (GlcNAc / mannose) receptor as a mechanism for recognition and absorption by these cells. SBC-102 increased cell-associated LAL activity in LAL-deficient human fibroblasts and normal murine fibroblasts following in vitro incubation, indicating that exposure to SBC-102 may result in substantial substitution of deficient enzyme activity.

Mannose-6-phosphate (M6P) is present in the oligosaccharide structures of SBC-102, which have been shown to be involved in the distribution of lysosomal enzymes to a wide variety of cell types via the ubiquitous M6P receptor.

LAL was purified from transgenic chicken egg white. Oregon Green NHS was obtained from Invitrogen ™ (N² 0-10241). The rat alveolar macrophage line, NR8383 and the mouse fibroblast line, NIH-3T3, were obtained from ATCC. LAL-deficient Wolman fibroblasts were obtained from the Coriell Institute for Medical Research and LysoTracker® Red was obtained from Invitrogen ™.

Enzyme Labeling: 4 mg of transgenic bird-derived LAL in PBS was labeled with Oregon Green according to the manufacturer's recommendations and the reaction was subsequently dialyzed against PBS, then concentrated.

Macrophage Absorption: Fluorescently labeled transgenic bird derived LAL (5 pg / ml) and LysoTracker® Red were incubated with NR8383 cells for 2 hours. Cells were examined by confocal fluorescence microscopy using a sequential scanning method at 488nm and then 514 nm.

Competitive inhibition with mannan: Fluorescently labeled SBC-102 (5 µg / ml) and mannan were incubated with NR8383 cells for 2 hours. Cells were trypsinized and LAL uptake measured by fluorescence activated cell sorting using median fluorescence intensity as the end point.

The ability of transgenic bird derived LAL to be absorbed and subsequently incorporated into target cell lysosomes was examined using the macrophage cell line, NR8383. Fluorescently labeled transgenic bird-derived LAL and lysosomal marker, LysoTracker® Red (Invitrogen ™), were incubated with cells for 2 hours. The co-localization of transgenic bird-derived LAL and lysosomal marker in the lysosomes of these cells was subsequently examined by confocal fluorescence microscopy using a sequential scanning mode (FIG. 20). LAL has demonstrated localization to lysosomes, which is consistent with similar in vitro studies using rhLAL from a variety of sources.

The specificity of binding of transgenic bird derived LAL to the GlcNAc / mannose receptor was assessed by competitive binding assays using the macrophage cell line, NR8383 (FIG. 21). Fluorescently labeled transgenic bird derived LAL (Oregon Green) at 5 pg / ml and various concentrations of the mannose-containing mannose oligosaccharide were incubated together with cells for 2 hours. The relative inhibition of mannan-derived transgenic bird LAL uptake compared to the mannan-free control was quantified by fluorescence activated cell screening analysis using median fluorescence intensity as the end point. Dose-dependent inhibition of mannose was observed in the binding / absorption of transgenic bird derived LAL, which is consistent with the transgenic bird derived LAL: GlcNAcR interaction.

In addition, mannose-6-phosphate-mediated absorption in fibroblast cells was demonstrated by competition experiments with mannose-6-phosphate (results not shown).

The ability of exposure of transgenic bird derived LAL to increase LAL activity in cells was examined using normal and LAL deficient cells in vitro. Fibroblasts isolated from a Wolman patient and normal murine fibroblasts (NIH-3T3) were incubated in the presence of transgenic bird derived LAL at concentrations of 0, 0.16 or 0.5 pg / mL for hours. Cells were then washed to remove non-specific signal and Cellular Used were assayed for LAL activity using 4-MUO substrate. Endogenous cell-associated LAL activity was less in Wolman fibroblasts compared to NIH-3T3 and dose-dependent increases in activity were observed in both cell types following incubation with transgenic bird-derived LAL (FIG. 22).

Example 14

In Vivo Analysis of Transgenic Bird Derived LAL

LAL deficient Yoshida rats (ie, Homozygous) (see Kuriyama et al., (1990), Journal of Lipid Research, vol. 31, p 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research, vol. 36, pp. 2212-2218, and Yoshida and Kuriyama (1990) Laboratory Animal Science, vol. 40, p. 486-489) were treated with SBC-102 (5 mg / kg, IV) or placebo once a week. , for four weeks, beginning at four weeks of age. For each administration, SBC-102 was injected into the caudal vein of rats at two equal doses (2.5 mg / kg) 30 minutes apart. Rats and wild-type controls of the same age were examined one week after the final dose. Analyzes were performed in triplicate.

General pathological examination of animals treated with SBC-102 showed normalization in liver color and reduction in organ size. SBC-102 treated rats showed essentially normal liver histology, in marked contrast to the substantial accumulation of foamy macrophages in vehicle treated animals (data not shown). Serum alanine and aspartate transferase levels, which are elevated in LAL_y mice, were also reduced in SBC-102 treated mice (not shown).

Internal organ mass and tissue were determined for each rat and data are shown in Fig. 23. Organ size is represented as percent body weight, determined at 8 weeks of age, in LAL rats.^_/_ and LAL mice^+/+after weekly administration of vehicle or SBC-102 at 5 mg / kg for 4 weeks.

Body weights of vehicle treated Yoshida or SBC-102 mice were compared to wild type mice as shown in FIG. 24. SBC-102 (5 mg / kg) or vehicle were administered by IV injection either as a single dose or as separate doses (given within 4 hours) to LAL4 mice. The LAL Rats^+/+ were controls of the same litter at the same age.

Example 15

Triglyceride Analysis

Triglyceride analysis was performed in liver and spleen tissue from homozygous SBC-102 treated homozygous, homozygous placebo wild-type animals. Triglyceride analyzes were performed using standard methodologies (ie, MBL International's Triglyceride Quantification Kit, Catalog N² JM-K622-100) and were performed in triplicate.

Table 3: Liver and Spleen triglyceride levels in LAL-deficient wild-type rats

<td colspan="4">Triglycerides (µg / mg wet tissue)</td>

<td></td><td>Wild Type (n</td><td>Placebo</td><td>SBC-102</td>

<td></td><td> = 3)</td><td>(n = 3)</td><td>(n = 3)</td>

<td>Liver</td><td> 48</td><td> 84</td><td> 57</td>

<td>Spleen</td><td> 3</td><td> 22</td><td> 4</td>

Liver Substrate Levels

Fig. 25 shows liver cholesterol, cholesteryl ester and triglyceride levels determined at 8 weeks of age in WT and LAL deficient rats following weekly administration of vehicle or SBC-102 at 5 mg-kg.^-1 for 4 weeks.

Example 16

Dose Study Response

Based on the studies performed above, the pharmacodynamic effects (PD) of a range of doses and dosing regimens (qw and qow) of LAL (SBC-102) in LAL rats were examined.^_/_. In these studies, SBC-102 was administered by IV injections at dosages of 0.2, 1, 3 and 5 mg / kg qow, or 0.35, 1.0 and 5.0 mg / kg qw for 1 month, beginning at 4 weeks of age. The results demonstrate improvements in body weight gain (BW) (Fig. 26), organomegaly (Fig. 27) and tissue substrate levels (Fig. 28). Serum transaminase levels were also reduced as the dose of SBC-102 increased, with levels essentially reaching wild-type levels at the highest doses.

Example 17

Administration of Recombinant LAL in a Mouse Model

The repeat effects of recombinant human lysosomal acid lipase (LAL) dosing on weight, tissue triglycerides and cholesterol, hepatomegaly, splenomegaly, lymphadenopathy, intestinal weight and other parameters were evaluated in LAL-deficient Donryu rats described in Yoshida and Kuriyama ( 1990) Laboratory Animal Science, vol 40, p. 486-489 (See also Kuriyama et al., (1990) Journal of Lipid Research, vol. 31, pp. 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research, vol. 36, p. 2212-2218).

At 4 weeks of age, homozygous Donalu mice for the LAL deletion (LAL_Y) were divided into groups for administration with recombinant human LAL produced in a transgenic chicken oviduct system or in a saline placebo. at the same age were used as controls.^_/_ were administered once a week for four weeks (four doses total) or once every two weeks for four weeks (two doses total) by injection into the caudal vein as a single dose or in two equal doses given 30 minutes interval. LAL doses were 1 mg / kg or mg / kg. The dosing schedule shown in Table 4

(5 mg / kg) for rats were pretreated with diphenhydramine to counteract potential anaphylactic reactions, a procedure that builds on previous experiences in animal models of enzyme replacement therapy for the treatment of lysosomal storage disease (Shull et al. the National Academy of Science, vol.

et al. (1999) The

P. 36335; Vogler et al, (1994) Proceedings of

91, p. 12937; Bielicki Journal of Biological Chemistry, 274,., (1999) Pediatric Research, 45, p. 838).

Figure 29 shows the daily progress of weight gain in rats given 1 mg / kg LAL per week or 5 mg / kg LAL per week or 5 mg / kg LAL at two weeks. It can be seen from the figure that there is little or no difference in therapeutic effect between the two dose sizes and frequencies.

Table 4: Weighing and Dosing Schedule

<td>Day from from birth</td><td colspan="2">Evaluations / Injections Performed</td>

<td>Day 13</td><td rowspan="9">P AND s THE D 0 s</td><td></td>

<td>Day 14</td><td></td>

<td>20th day</td><td></td>

<td>Day 21</td><td>Weaned puppies</td>

<td>Day 24</td><td></td>

<td>Day 25</td><td></td>

<td>Day 27</td><td></td>

<td>Day 28</td><td>First injection for administration once a week and once every two weeks</td>

<td>31st</td><td></td>

<td>Day 32</td><td rowspan="12"></td><td></td>

<td>Day 34</td><td></td>

<td>Day 35</td><td>Second injection for once weekly administration</td>

<td>Day 38</td><td></td>

<td>Day 39</td><td></td>

<td>Day 41</td><td></td>

<td>Day 42</td><td>Third Injection for administration once per week; Second administration for once every two weeks</td>

<td>Day 45</td><td></td>

<td>Day 48</td><td></td>

<td>Day 49</td><td>Fourth injection for once weekly administration</td>

<td>Day 55</td><td></td>

<td>Day 56</td><td>Necropsy</td>

Pathological Examination of LAL Rats ¹ Treated with Recombinant LAL

At the end of the study described in Example 1, the study animals were humanly euthanized and necropsied to examine general pathology, histopathology and clinical chemistry. General necropsy included examination of the outer surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Internal organ and tissue mass was determined for the rats and organs and tissues were harvested and fixed in 10% buffered neutral formalin. After fixation, tissues were processed and histological slides of hematoxylin and eosin stained sections were prepared and evaluated.

General pathological examination of the treated animals analyzed showed substantial normalization in liver size and color, as can be seen in the dissection shown in Figure 30. Organ to body weight ratios were determined and a reduction in relative organ size to liver, spleen, mesenteric tissue, duodenum, jejunum, and ileum in successfully treated animals that were dissected compared to placebo-treated mice (FIG. 23). The liver tissue histopathology of the analyzed recombinant LAL treated rats shows essentially normal liver histology, in marked contrast to the substantial accumulation of foamy macrophages, in the placebo treated animals (Figure 30).

Example 18

Treatment of Wolman's Disease (WD) by Recombinant LAL Administration

At 7 weeks of age a female patient is admitted to the hospital because of the difficulty in gaining weight and little progress since birth. On initial physical examination, the patient weighs 3.6 kg (birth weight 3.7 kg) and is thin with loose skin folds. The abdomen is distended, with firm hepatomegaly of 6 cm and firm splenomegaly of about 4 cm. Increased lymph nodes are noted in the groin and muscle activity is poor.

Initial hemoglobin level is 9.2 gm, platelets 506000 and white blood cells 11550. Urinalysis is normal and bone marrow smears reveal vacuolated lymphocytes and numerous foam cells. Serum chemical measurements: total lipids 834 mg / 100 mL, phospholipids 176 mg / 100 mL, triglycerides 141 mg / 100 mL, cholesterol 129 mg / 100 mL, bilirubin 0.3 mg / 100 mL, alkaline phosphatase 9.0% BU , 90 SGOT units, 50 TGP units, 20 cholinesterase units, urea nitrogen

8.3 mg, fasting sugar 45 mg / 100 mL. Computed tomography of the abdomen shows bilateral hepatosplenomegaly and symmetrically enlarged adrenal glands with calcification.

The patient is surgically implanted with a venous vascular access port for dosing. After connecting the door to an ambulatory infusion machine, the patient is pretreated with 1 mg / kg diphenhydramine 20 minutes before LAL infusion to counteract potential anaphylactic infusion reactions. The patient is then administered with 1 mg / kg LAL over 5 hours by intravenous infusion. This therapy is repeated once every 7 days indefinitely.

Within two weeks after administration of the first dose of LAL, the patient is evaluated for weight gain and size of the major abdominal organs as determined by ultrasound. Laboratory results demonstrate that LAL infusion restores lysosomal acid lipase activity in the patient and leads to correction of related abnormalities.

Example 19

Treatment of Cholesteryl Ester Storage Disease (CESD) by Recombinant LAL Administration

A 3-year-old boy with a pruritic abdominal rash is examined by his pediatrician. After abdominal examination, hepatomegaly is noted by the doctor and confirmed by ultrasound. At this point, no diagnosis is made and the patient is monitored periodically.

At age 8, he is admitted to the hospital with gastroenteritis. Light microscopy of a liver biopsy shows increased intracytoplasmic glycogen and poor lipid droplets in the hepatocytes. Electron microscopy shows membrane-bound lipid droplets with small electron dense granules. A working diagnosis of type III glycogen storage disease (DeBrancher disease) is made, but Debrancher activity of skin fibroblasts is normal.

At 10 years, hepatomegaly persists and a second liver biopsy is performed. Light microscopy shows alteration of the lobular hepatic parenchyma with distended hepatocytes containing cytoplasmic granules and vacuoles with mild periportal fibrosis. Fibroblast acid lipase activity is found to be 7% of normal, confirming the diagnosis of CESD. Plasma concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C) are each above the 95th percentile for age and sex at 7.51, 3.24 and 5.58 mmol / l. L, respectively, while plasma high-density lipoprotein (HDL-C) is below the 5th percentile to 0.47 mmol / L; it combined hyperlipidemia (hypercholesterolemia, hypertriglyceridemia, hypoalphalipoproteinemia and hyperbetalipoproteinemia).

The patient is surgically implanted with a venous vascular access port for dosing. After connecting the door to an ambulatory infusion machine, the patient is pretreated with 5 mg / kg diphenhydramine 20 minutes prior to LAL infusion to counteract potential anaphylactic infusion reactions. The patient is then administered LAL at 5 mg / kg over 5 hours by intravenous infusion. This therapy is repeated once every 14 days indefinitely.

Within two weeks after administration of the first dose of LAL, the patient is evaluated for weight gain and size of the major abdominal organs as determined by ultrasound. Laboratory results demonstrate that LAL infusion restores lysosomal acid lipase activity in the patient and leads to correction of related abnormalities.

Example 20

Drug Description and Composition

The pharmacological substance of LAL described herein (SBC-102) is a recombinant human lysosomal acid lipase (rhLAL) purified from egg white produced from transgenic Gallus. The excipients used in SBC-102 are

100 similar to those used for other lysosomal storage disorder (LSD) products currently on the market and were selected to maintain the stability of the pharmacological product.

SBC-102 is a clear, colorless, sterile liquid provided in a clear Type I borosilicate glass vial with an unnatural latex (Butyl) FluroTec® coated rubber and aluminum crimp sealant. SBC-102 is provided as an aqueous solution composed of SBC-102 (2 mg / mL), Trisodium Citrate Dihydrate (13.7 mg / mL, USP), Citric Acid Monohydrate (1.57 mg / mL) , USP), Human Serum Albumin (10 mg / mL, USP) and Water for Injection (to final volume, USP). The pH of SBC-102 is 5.9 ± 0.2. SBC-102 contains no preservatives and the vials are for single use only.

Table 5

Excipients in SBC-102 (LAL)

<td>Excipient</td><td>CAS number</td><td>Degree</td><td>Occupation</td>

<td>Trisodium Citrate Dihydrate</td><td> 6132-04-03</td><td>USP</td><td>Plug</td>

<td>Citric Acid Monohydrate</td><td> 5949-29-1</td><td>USP</td><td>Plug</td>

<td>Human Serum Albumin</td><td> 70024-90-7</td><td>USP</td><td>Stabilizer</td>

101

Drug Components

Table 6

Formulation of SBC-102

<td>Component</td><td>Concentration</td>

<td>SBC-102 (rhLAL)</td><td>2 mg / mL *</td>

<td>Trisodium Citrate Dihydrate</td><td>13.7 mg / mL</td>

<td>Citric Acid Monohydrate</td><td>1.57 mg / mL</td>

<td>Human Serum Albumin</td><td>10 mg / mL</td>

<td>Water for Injection, QS for</td><td>1.0 mL</td>

Lisbon, May 8, 2017

102