ES2399232T3

Methods for generating high titer helper-free preparations of recombinant AAV vectors

Abstract

A method of generating a population of adeno-associated recombinant virus particles (AAVr) that comprises the steps of: (a) incubating the AAV producing cell under conditions that are permissive for AAV replication and that comprise inducing sublethal stress in the producing cell of AAV to enhance the level of AAV production, in which the AAV producing cell is a mammalian cell, and wherein said cell comprises: (i) one or more AAV packaging genes, in which each of said AAV packaging genes encodes an AAV replication or encapsidation protein; (ii) a recombinant AAV (AAVr) provider comprising a heterologous non-AAV polynucleotide flanked by at least one inverted terminal repeat (RTI); and (iii) an AAV helper virus, (b) lysing the producing cell after the incubation of step a) to produce an AAV producing cell lysate; and (c) purifying the AAV-producing cell lysate to generate a population of adeno-associated recombinant virus (AAVr) particles, in which the sublethal stress is selected from: (I) a nutritional stress imposed by one or more of the following: (i) culturing the producing cell in a medium that is deficient in one or more amino acids, optionally aspartic acid or glutamic acid; (ii) culturing the producing cell in a medium containing less than 10 μmol / l of aspartic acid; (iii) culturing the producing cell in a medium containing less than 2 μmol / l glutamic acid; or (iv) culturing the producing cell in a medium that is deficient in serum; or (II) a thermal stress imposed by culturing the producing cell for 3-6 days at: (i) a temperature lower than the optimum growth temperature of the producing cell; or (ii) a temperature greater than the optimum growth temperature of the producing cell; or (III) an osmotic stress imposed: (i) by culturing the producing cell in a hypoosmotic medium; or (ii) by culturing the producer cell in a hyperosmotic medium; or (IV) a pH stress, in which the pH stress comprises subjecting the producing cell to a pH of approximately pH 7.2 continuously during culture; or (V) a toxic stress, in which toxic stress comprises exposing the producing cell to a genotoxic agent selected from a chemical carcinogen, radiation, UV, a metabolic inhibitor of DNA synthesis and a drug that affects topoisomerases.

Term

Term ended

Projected expiry passed 4 September 2018, 8.1 years ago.

36 claims: 18 independent, 18 dependent

1
REIVINDICACIONES 1. Un procedimiento de generación de una población de partículas de virus adenoasociados recombinantes (AAVr) que comprende las etapas de:(a) incubar la célula productora de AAV en condiciones que sean permisivas para la replicación de AAV y que comprenden inducir un estrés subletal en la célula productora de AAV para potenciar el nivel de producción de AAV, en el que la célula productora de AAV es una célula de mamífero, y en el que dicha célula comprende: (i) uno o más genes de empaquetamiento de AAV, en el que cada uno de dichos genes de empaquetamiento de AAV codifica una proteína de replicación o encapsidación de AAV;(ii) un provector de AAV recombinante (AAVr) que comprende un polinucleótido no de AAV heterólogo flanqueado por al menos una repetición terminal invertida (RTI);y (iii) un virus auxiliar de AAV, (b) lisar la célula productora después de la incubación de la etapa a) para producir un lisado de célula productora de AAV;y (c) purificar el lisado de célula productora de AAV para generar una población de partículas de virus adenoasociados recombinantes (AAVr), en el que el estrés subletal se selecciona de: (I) un estrés nutricional impuesto por uno o más de los siguientes: (i) cultivar la célula productora en un medio que sea deficiente en uno o más aminoácidos, opcionalmente ácido aspártico o ácido glutámico;(ii) cultivar la célula productora en un medio que contenga menos de 10 μmol/l de ácido aspártico;(iii) cultivar la célula productora en un medio que contenga menos de 2 μmol/l de ácido glutámico;o (iv) cultivar la célula productora en un medio que sea deficiente en suero;o (II) un estrés térmico impuesto al cultivar la célula productora durante 3-6 días a: (i) una temperatura menor que la temperatura de crecimiento óptima de la célula productora;o (ii) una temperatura mayor que la temperatura de crecimiento óptima de la célula productora;o (III) un estrés osmótico impuesto: (i) al cultivar la célula productora en un medio hipoosmótico;o (ii) al cultivar la célula productora en un medio hiperosmótico;o (IV) un estrés de pH, en el que el estrés de pH comprende someter la célula productora a un pH de aproximadamente pH 7,2 continuamente durante el cultivo;o (V) un estrés tóxico, en el que el estrés tóxico comprende exponer la célula productora a un agente genotóxico seleccionado de un carcinógeno químico, radiación, UV, un inhibidor metabólico de la síntesis de ADN y un fármaco que afecte a las topoisomerasas.
2
El procedimiento de la reivindicación 1, en el que el estrés subletal es un estrés tóxico y el agente genotóxico es un inhibido metabólico de la síntesis de ADN seleccionado de hidroxiurea, metotrexato y afidocolina.
3
El procedimiento de la reivindicación 1, en el que el estrés subletal es un estrés tóxico y el agente genotóxico es un fármaco que afecta a topoisomerasas seleccionado de amsacrina, camptotecina, etopósido y novobiocina.
4
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha etapa de purificación c) comprende someter a cromatografía el lisado de célula productora de AAV de la etapa b) mediante una pluralidad de cromatografías de intercambio iónico que comprenden al menos una cromatografía de intercambio aniónico cargada positivamente y al menos una cromatografía de intercambio catiónico cargada negativamente, o someter a cromatografía el lisado de célula productora de AAV de la etapa b) mediante una cromatografía de intercambio aniónico seguida de filtración por filtro tangencial para generar una población purificada de partículas de vector de AAVr.
5
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicho lisado de célula productora de AAV se purifica también por afinidad en una resina que tiene un ligando que es específico de una o más moléculas de superficie presentes en AAV, y en el que la purificación por afinidad se realiza opcionalmente después de una cromatografía de intercambio aniónico y/o catiónico y dicho ligando es opcionalmente un anticuerpo que es específico de una molécula de superficie presente en AAV.
6
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que la célula productora de AAV de la etapa a) se concentra antes de la lisis, opcionalmente mediante centrifugación o mediante filtración por flujo tangencial.
7
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha etapa de lisis de la célula productora de AAV se realiza sometiendo la célula a una técnica lítica seleccionada de microfluidificación, sonicación y liofilización.
8
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que el lisado de célula productora de AAV de la etapa b):(i) se trata con una nucleasa, opcionalmente benzonasa, antes de la cromatografía;y/o (ii) se clarifica antes de la cromatografía, opcionalmente mediante filtración o centrifugación.
9
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que la célula productora de AAV se concentra antes de la lisis, se resuspende en un tampón que comprende disolución salina a una fuerza iónica de al menos una disolución de NaCl 50 mM, se lisa y se clarifica entonces mediante filtración antes de la cromatografía.
10
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en el que las fracciones cromatográficas que contienen partículas de AAVr se concentran mediante filtración o centrifugación después de elución de una cromatografía de intercambio iónico.
11
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en el que las fracciones cromatográficas que contienen partículas de AAVr se concentran mediante filtración por flujo tangencial después de elución de una cromatografía de intercambio iónico.
12
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en el que dicha cromatografía de intercambio aniónico se efectúa en una resina amino o imino N-cargada, opcionalmente seleccionada de una resina POROS 50 PI, una resina de dietilaminoetilo (DEAE), una resina de trimetilaminoetilo (TMAE), una resina de amina cuaternaria y una resina de polietilenimina (PEI).
13
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en el que dicha cromatografía de intercambio catiónico se efectúa en una resina catiónica basada en azufre, fósforo o carboxilo, opcionalmente seleccionada de una resina de sulfato de heparina (HS), una resina de sulfopropilo (SP) y una resina de carboximetilo (CM).
14
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha etapa a) de incubación de la célula productora se realiza en un recipiente seleccionado de un matraz de cultivo de tejido, un frasco giratorio, un matraz de agitación, un reactor en tanque, un fermentador tal como un fermentador de agitación de aire, un biorreactor tal como un biorreactor de fibra hueca, de lecho empaquetado o lecho fluidificado o un biorreactor multilecho en el que en el biorreactor multilecho se aíslan al menos 109 unidades replicativas de AAVr por litro de volumen de biorreactor.
15
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha etapa a) de incubación de la célula productora se realiza usando un microportador.
16
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha etapa a) de incubación de la célula productora se realiza durante al menos 5 días.
17
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en el que dicha población purificada de partículas de vector de AAVr contiene:- menos de 1 AAV competente de replicación por 104 partículas de AAV;- no más de 103 partículas infecciosas de virus auxiliar por 108 unidades replicativas de partículas de AAVr;- menos de un 5% de contaminación por virus auxiliar basada en proteína (p/p), detectada por análisis densitométricos de geles de SDS o mediante inmunoensayo de proteína específica de virus auxiliares, y - menos de un % de contaminación por virus auxiliar o proteína celular (p/p), detectada por análisis densitométricos de geles de SDS o mediante inmunoensayo de virus auxiliares o proteínas específicas celulares.
18
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 4 o cualquier reivindicación dependiente de la misma, en que la elución de la cromatografía de intercambio aniónico y/o intercambio catiónico se realiza aumentando la concentración salina y los eluyentes cromatográficos que comprenden las partículas de AAVr se tratan posteriormente para reducir la concentración salina eficaz mediante dilución, diálisis, diafiltración o concentración.
19
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, que comprende adicionalmente la etapa de someter una fracción que comprende partículas de AAV a cromatografía de sulfato de heparina.
20
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicho provector de AAVr comprende un polinucleótido no de AAV heterólogo flanqueado por dos repeticiones terminales invertidas (RTI) de AAV.
21
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha célula productora de AAV comprende al menos un gen de empaquetamiento de AAV que se integra establemente en el genoma de dicha célula productora de AAV, preferiblemente un gen rep de AAV y un gen cap de AAV.
22
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicha célula productora se proporciona mediante un proceso que comprende:(i) introducir el virus auxiliar en la célula productora en que ya se ha introducido el gen o genes de empaquetamiento de AAV y el provector de AAVr;(ii) introducir el provector de AAVr y el virus auxiliar simultánea o secuencialmente en la célula productora en que ya se ha introducido el gen o genes de empaquetamiento de AAV;o (iii) introducir el gen o genes de empaquetamiento de AAV y el provector de AAVr simultánea o secuencialmente en la célula hospedadora en que ya se ha introducido el virus auxiliar.
23
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que la célula productora se proporciona mediante un proceso que comprende introducir en la célula productora al menos un gen de división-empaquetamiento de AAV.
24
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicho virus auxiliar es un adenovirus, un adenovirus termosensible o adenovirus Ad-ts149.
25
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que dicho virus auxiliar es un virus auxiliar termosensible y dicha etapa de incubación de la célula productora se realiza a una temperatura que es permisiva de replicación de AAV pero no permisiva de replicación del virus auxiliar termosensible.
26
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones precedentes, en el que la célula productora es una línea celular de mamífero dependiente de unión, una línea celular de mamífero adaptada a suspensión, una célula N3S de 293 o una célula S3 de HeLa.
27
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 1, en el que dicha etapa de purificación c) comprende una combinación de una cromatografía de intercambio aniónico cargada positivamente y una cromatografía de intercambio catiónico cargada negativamente para generar una población purificada de partículas de AAVr.
28
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 27, en el que dicha cromatografía de intercambio aniónico cargada positivamente se efectúa antes de dicha cromatografía de intercambio catiónico cargada negativamente.
29
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 27, en el que dicha cromatografía de intercambio catiónico cargada negativamente se efectúa antes de dicha cromatografía de intercambio aniónico cargada positivamente.
30
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones 27 a 29, que comprende adicionalmente la etapa de someter a cromatografía el lisado que contiene partículas de AAVr en sulfato de heparina, efectuándose dicha etapa después de dicha cromatografía de intercambio aniónico cargada positivamente y de dicha cromatografía de intercambio catiónico cargada negativamente.
31
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones 27 a 30, que comprende adicionalmente la etapa de someter la célula productora a filtración por flujo tangencial.
32
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 31, en el que la filtración por flujo tangencial se efectúa antes de la cromatografía.
33
Un procedimiento de generación de una población de partículas de AAVr según la reivindicación 31, en el que dicha filtración por flujo tangencial se efectúa después de la cromatografía.
34
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones 27 a 33, en el que dicha cromatografía de intercambio aniónico se efectúa en una resina de amino o imino N-cargada, opcionalmente seleccionada de una resina POROS 50 PI, una resina de dietilaminoetilo (DEAE), una resina de trimetilaminoetilo (TMAE), una resina de amina cuaternaria y una resina de polietilenimina (PEI).
35
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones 27 a 33, en el que dicha cromatografía de intercambio catiónico se efectúa en una resina catiónica basada en azufre, fósforo o carboxilo, opcionalmente seleccionada de una resina de sulfato de heparina (HS), una resina de sulfopropilo (SP) y una resina de carboximetilo (CM).
36
Un procedimiento de generación de una población de partículas de AAVr según una cualquiera de las reivindicaciones 27 a 35, en el que la célula productora se cultiva en condiciones de suspensión.

Independent claims36

518 paragraphs in 4 sections, as filed

Procedures for the generation of high-titre aav recombinant vector preparations free of auxiliaries

Field of the Invention

[0001] The present disclosure relates generally to the field of recombinant adeno-associated virus (AAV) vectors and preparations thereof that can be used for gene transfer. More specifically, it refers to methods for generating high titre preparations of recombinant AAV vectors that are substantially free of helper viruses (eg adenovirus) as well as cellular proteins.

Background

[0002] Adeno-associated viruses (AAV) have unique traits that make them attractive as vectors for gene therapy. Adeno-associated viruses infect a wide range of cell types. However, they are not transformants and are not involved in the etiology of any human disease. The introduction of DNA into recipient host cells generally leads to long-term persistence and DNA expression without altering the normal metabolism of the cell.

[0003] There are at least three desirable features of a recombinant AAV vector preparation for use in gene transfer, especially in human gene therapy. First, it is preferred that the vector be generated at titers high enough to transduce an effective proportion of cells in the target tissue. In vivo gene therapy typically requires a high number of vector particles. For example, some treatments may require more than 108 particles, and the treatment of cystic fibrosis by direct delivery to the airways may require more than 1010 particles. Secondly, it is preferred that the vector preparations are essentially free of replication competent AAVs (namely, phenotypically wild type AAVs that can be replicated in the presence of auxiliary viruses or auxiliary virus functions). Thirdly, it is preferred that the preparation of AAVr vector as a whole is essentially free of other viruses (such as an auxiliary virus used in the production of AAV) as well as auxiliary viruses, cellular proteins and other components such as lipids and carbohydrates. , to minimize or eliminate any risk of generating an immune response in the context of gene therapy. This last point is especially significant in the context of AAVs, because AAV is an "auxiliary dependent" virus that requires co-infection with an auxiliary virus (typically adenovirus) or other provision of auxiliary virus functions to replicate and package effectively during AAV production process and, in addition, it has been observed that adenoviruses generate an immune response in the host in the context of gene therapy applications (see, for example, Byrnes et al., Neuroscience

66: 1015, 1995; McCoy et al., Human Gene Therapy 6: 1553, 1995 and Barr et al., Gene Therapy 2: 151, 1995). The methods of the present disclosure are directed to these and other desirable features of AAVr vector preparations as described and illustrated in detail below.

[0004] General reviews of the virology and genetics of AAVs are available elsewhere. The reader can refer, among others, to Carter, "Handbook of Parvoviruses", vol. I, p. 169-228 (1989) and Berns, "Virology", p. 1743-1764, Raven Press, (1990). The following is a brief synopsis for the convenience of the reader. AAV is a replication defective virus, which means it relies on an auxiliary virus to complete its replication and packaging cycle in a host cell. The AAV genome generally comprises the rep and cap packaging genes, other necessary functions being provided in trans by the helper virus and the host cell.

[0005] The AAV particles comprise a protein capsid having three capsid proteins, VP1, VP2 and VP3, which encompass a linear single stranded DNA genome of ~ 4.6 kb. Individual particles package only one strand of DNA molecule, but this can be the positive or negative strand. The particles containing any strand are infectious, and replication occurs by converting the individual infectious strand progenitor into a duplex form and subsequent amplification, from which the individual strands of progeny move to and package into capsids. Duplex or single stranded copies of AAV genomes (sometimes referred to as "proviric DNA" or "provirus") can be inserted into bacterial plasmids or phagemids, and transfected into adenovirus infected cells.

[0006] By way of illustration, the linear genome of serotype AAV2 is terminated at each end by an inverted terminal repeat sequence (RTI). Among the RTIs there are three transcription promoters p5, p19 and p40 that are used to express the rep and cap genes (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA, 76: 5567-5571). RTI sequences are necessary in cis and are sufficient to provide a source of functional replication, integration into the cell genome and efficient cleavage and recovery of host cell chromosomes or recombinant plasmids. Rep and cap gene products provide functions for viral genome replication and encapsidation, respectively, and it is sufficient that they are present in trans.

[0007] The rep gene is expressed by two promoters, p5 and p19, and produces four proteins designated Rep78, Rep68, Rep52 and Rep40. Only Rep78 and Rep68 are required for AAV duplex DNA replication, but Rep52 and Rep40 appear to be necessary for the accumulation of single-stranded progeny DNA (Chejanovsky et al., Virology 173: 120, 1989). Rep68 and Rep78 specifically bind to the AAV RTI hairpin conformation and possess several enzymatic activities necessary to resolve AAV end replication. Rep78 and Rep68 also exhibit pleyotropic regulatory activities that include positive and negative regulation of AAV genes and expression by some heterologous promoters, as well as inhibitory effects on cell growth. The cap gene encodes capsid proteins VP1, VP2 and VP3. These proteins share a common overlapping sequence, but VP1 and VP2 contain additional amino terminal sequences transcribed by the p40 promoter through the use of alternative initiation codons. All three proteins are required for effective capsid production.

[0008] AAV genomes have been introduced into bacterial plasmids by methods such as prolongation of GC (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79: 2077-2081), addition of synthetic linkers that contain restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 59: 4661-4666 ). Transfection of said recombinant AAV plasmids into mammalian cells with an appropriate auxiliary virus results in recovery and cleavage of the AAV genome free of any plasmid sequence, replication of the recovered genome and generation of progeny-infected AAV particles.

[0009] Recombinant AAV vectors comprising a heterologous polynucleotide of therapeutic interest can be constructed by replacing portions of the AAV coding sequence in bacterial plasmids with the heterologous polynucleotide. The general principles of construction of an AAVr vector are also reviewed elsewhere. See, for example, Carter, 1992, Current Opinions in Biotechnology, 3: 533-539 and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158: 97-129). AAV RTIs are generally retained, since vector packaging requires that they be present in cis. However, other elements of the AAV genome may be omitted, in particular, one or more of the packaging genes. The vector plasmid can be packaged in an AAV particle by supplying the omitted packaging genes in trans through an alternative source.

[0010] In one approach, the AAV RTI-flanked sequence (the AAVr vector sequence) and AAV packaging genes to be provided trans in the host cell in separate bacterial plasmids are introduced. Examples of this approach are described in Ratschin et al., Mol. Cell Biol. 4: 2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984); Tratschin et al., Mol. Cell Biol. 5: 3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988) and Lebkowski et al., 1988 Mol. Cell Biol., 7: 349 (1988). Samulski et al. (1989, J. Virol., 63: 3822-3828) have described a packaging plasmid called pAAV / Ad, which consists of the Rep and Cap coding regions surrounded by adenovirus RTI. Human airway epithelial cells of a cystic fibrosis patient have been transduced with an AAV vector prepared using the pAAV / Ad packaging plasmid and a plasmid comprising the neo selective marker gene expressed through the AAV p5 promoter (Flotte and col., Am. J. Respir. Cell. Mol. Biol. 7: 349,1992).

[0011] It is a second approach to provide the vector sequence or AAV packaging genes in the form of an episomic plasmid in a mammalian cell used for AAV replication. For example, US Pat. 5,173,414 describes a cell line in which the vector sequence is present as an episomic plasmid of high copy number. Cell lines can be transduced with complementary AAV functions in trans rep and cap, generating AAV vector preparations. This approach is not ideal, because the number of copies per cell cannot be rigorously controlled and episomic DNA is much more likely to undergo redisposition, leading to the production of vector by-products.

[0012] It is a third approach to provide the vector sequence or AAV packaging genes, or both, stably integrated into the genome of the mammalian cell used for replication.

[0013] An exemplary technique is set forth in international patent application WO 95/13365 (Targeted Genetics Corporation and Johns Hopkins University) and the corresponding US Pat. No. 5,658,776 (de Flotte et al.). This example uses a mammalian cell with at least one intact copy of a stably integrated AAVr vector, in which the vector comprises an AAV RTI and a transcription promoter operably linked with a target polynucleotide, but in which the expression of Rep is limiting. In a preferred case, an AAV packaging plasmid comprising the rep gene operatively linked with a heterologous AAV is introduced into the cell, and the cell is then incubated under conditions that allow replication and packaging of the AAV vector sequence in particles

[0014] A second exemplary technique is set forth in patent application WO 95/13392 (Trempe et al.). This example uses a stable mammalian cell line with an AAV rep gene operably linked with a heterologous promoter in order to express functional Rep protein. In various preferred cases, the AAV cap gene can also be provided stably or may be introduced transiently (for example, in a plasmid). A stable or transient recombinant AAV vector can also be introduced.

[0015] Another exemplary technique is set forth in patent application WO 96/17947 (from Targeted Genetics Corporation. J. Allen). This example uses a mammalian cell comprising a stably integrated AAV cap gene and an integrated AAV rep gene stably operably linked with a heterologous promoter and inducible by helper viruses. In various preferred cases, a plasmid comprising the vector sequence (stable or transiently) is also introduced into the cells. The recovery of AAV vector particles is then initiated by the introduction of the helper virus.

[0016] These various examples address the problem of providing AAV at a sufficiently high titer, minimizing recombination between vector and packaging components and reducing or avoiding the potential difficulties associated with the expression of the AAV rep gene in the mammalian cell line. (Since Rep proteins can not only limit their own expression but can also affect cell metabolism). However, the packaging of an AAV vector into viral particles continues to rely on the presence of an auxiliary virus suitable for AAV or the provision of auxiliary virus functions. Auxiliary viruses capable of supporting AAV replication are exemplified by adenovirus, but include other viruses such as herpesviruses and poxviruses. The presence of significant amounts of infectious auxiliary virus in a preparation of AAV vectors is problematic because the preparation is intended for use in human administration. Even the presence of non-replicative auxiliary virus components can cause an unacceptable immune reaction in the treated subject.

[0017] The potential problems triggered by the helper virus antigen have been illustrated in several recent studies. Bymes et al. (Neuroscience 66: 1015, 1995) injected a non-replicative human type 5 adenovirus with deletion of the E1 region in the brains of inbred rats. An inflammation response was observed that was attributed to the administered particles rather than to the expression of new viral proteins due to viral infection of the cells. The presence of the virus was associated with an increase in MHC class I expression and a large infiltration of macrophages and lymphocytes T. McCoy et al. (Human Gene Therapy 6: 1553, 1995) instilled the lungs of mice with intact adenovirus, adenovirus with incomplete genomes or adenovirus inactivated with ultraviolet light. All induced pulmonary inflammation, and the number of inflammatory cells in lung tissue was quantitatively similar for all three forms of the virus. Comparative experiments using adenovirus constructs in normal and immunodeficient mice performed by Barr et al. (Gene Therapy

2: 151, 1995) indicate that the antiadenovirus immune response is mediated primarily by T lymphocytes and results in a memory response that affects subsequent doses.

[0018] Therefore, in the development of recombinant AAV vectors such as those for use in gene therapy, there is a need for strategies that minimize the amount of auxiliary virus, as well as auxiliary virus proteins and cellular proteins, present in the final preparation, while still achieving a high AAV titre so that the procedures can be used effectively at a scale that is suitable for the practical application of gene therapy techniques.

[0019] Since high titers of AAVr vector preparations are particularly useful, but the production of high titres of AAVr, particularly in large-scale procedures, can lead to the generation of significant amounts of auxiliary viruses (eg, adenovirus or "Ad"), helper virus proteins (eg, Ad proteins) and / or contaminating cell proteins, It has become especially important to design scalable procedures for the production of AAVr that can be used for the generation of high titre preparations that are substantially free of virus and / or contaminating viral or cellular proteins. The present disclosure provides procedures to achieve these concurrent objectives and demonstrates that such techniques can be used for large-scale production of recombinant AAV vector preparations.

Summary of the Invention

[0020] This disclosure provides methods and materials for generating high titre preparations of adeno-associated viruses (AAV) that are substantially free of helper viruses, helper virus proteins and cellular proteins and other components.

[0021] The disclosure includes the following cases, but without limitation:

A method for generating a population of recombinant adeno-associated virus (AAVr) particles comprising the steps of: a) providing an AAV producing cell comprising: (a) one or more AAV packaging genes, in which each of said AAV packaging genes encodes an AAV replication or encapsidation protein; (ii) a recombinant AAV (AAVr) provider comprising a heterologous non-AAV polynucleotide flanked by at least one reverse terminal repeat (RTI) of AAV; and (iii) an AAV auxiliary virus; b) incubating the producer cell provided in step a) under conditions that are permissive for AAV replication; c) lyse the producing cell after the incubation of step b), producing an AAV producing cell lysate and d) chromatography the AAV producing cell lysate of step c) in a plurality of ion exchange resins that they comprise at least one positively charged anion exchange resin and at least one negatively charged cation exchange resin, generating a purified population of AAVr vector particles, or chromatography the AAV producing cell lysate of step c) on an anion exchange resin followed by tangential flow.

[0022] A method of generating a population of AAVr particles according to the above procedure, wherein said auxiliary virus is an adenovirus or a thermosensitive auxiliary virus, and said step of incubating the producing cell is performed at a temperature that is permissive for AAV replication but not permissive for thermosensitive auxiliary virus replication.

[0023] A method of generating a population of AAVr particles, in which the incubation of the producing cell is performed in a container selected from the group consisting of a tissue culture flask, a rotating flask, a shake flask, a tank-type reactor, a fermenter and a bioreactor, optionally using a microcarrier, and preferably using a mammalian cell line adapted to the suspension.

[0024] A method of generating a population of recombinant adeno-associated virus (AAVr) particles comprising the steps of: a) providing an AAV producing cell comprising:

(i) one or more AAV packaging genes, in which each of said AAV packaging genes encodes an AAV replication or encapsidation protein; (ii) a recombinant AAV (AAVr) provider comprising a heterologous non-AAV polynucleotide flanked by at least one reverse terminal repeat (RTI) and

(iii) an AAV helper virus or a polynucleotide sequence of said helper virus encoding at least one helper virus function; b) subjecting the producer cell provided in stage a) to sublethal stress and c) incubating the stressed producer cell of stage b) under conditions that are permissive for AAV replication. Possible forms of sublethal stress can be selected, but not limited to, from those in the group consisting of nutritional stress, osmotic stress, pH stress, temperature stress, aerobic stress, mechanical stress, radiation stress and toxic stress. It is a non-limiting example of how nutritional stress is imposed to grow the producer cells in a medium that is deficient in one or more amino acids. Additional illustrations are provided below.

[0025] A method of generating a population of AAVr particles in which said purified population of AAVr vector particles is substantially free of competent AAV replication and helper viruses and cellular proteins.

[0026] A method of generating a population of recombinant adeno-associated virus (AAVr) particles comprising the steps of: a) providing an AAV producing cell comprising:

(i) one or more AAV packaging genes, wherein said AAV packaging gene encodes an AAV replication or encapsidation protein; (ii) a recombinant AAV (AAVr) provider comprising a heterologous non-AAV polynucleotide flanked by at least one inverted terminal repeat (RTI) and (iii) an AAV auxiliary virus; b) incubating the producer cell provided in step a) under conditions that are permissive for AAV replication and that comprise inducing sublethal stress in the AAV producing cell; c) lyse the producing cell after the incubation of step b), producing an AAV producing cell lysate and d) purifying the AAV producing cell lysate, generating a population of recombinant adeno-associated virus (AAVr) particles. Suitable purification procedures include those described elsewhere in this disclosure. An exemplary purification process comprises chromatography the AAV producing cell lysate of step c) on at least one chromatographic resin selected from the group consisting of a positively charged anion exchange resin and a negatively charged cation exchange resin, generating a purified population of AAVr vector particles (preferred procedures include anion exchange followed by cation exchange or tangential flow filtration (FFT)). Illustrative chromatographic procedures, including ion exchange chromatography and chromatographic purification on heparin sulfate, are given below by way of example.

[0027] A host cell for producing recombinant adeno-associated virus (AAVr) particles with high efficiency comprising: a) one or more AAV packaging genes, in which each of said AAV packaging genes encodes a replication protein or AAV encapsidation; b) a heterologous polynucleotide introduced into said host cell using an AAVr provider, wherein the AAVr provider comprises the heterologous polynucleotide flanked by at least one inverted terminal repeat repeat (RTI) of AAV and is deficient in said packaging gene or genes AAV; c) an auxiliary virus such as an AAV thermosensitive auxiliary virus (VAts), wherein said VAts is heat sensitive for self-replication.

[0028] A population of AAVr particles, produced according to any of the production processes of this disclosure. Preferably, the population of particles contains no more than about one infectious adenoviral particle per 1000 infectious AAVr particles, preferably less than one per 106 AAVr, even more preferably less than about one in 109.

[0029] High performance assay techniques are also provided which can be used, for example, in the titration of viral preparations as well as in the screening of agents that affect viral replication.

[0030] Based now on the disclosure contained herein, the present invention provides a method of generating a population of recombinant adeno-associated virus (AAVr) particles comprising the steps of:

<dl><dt>(to) </dt><dd>incubating the AAV producing cell under conditions that are permissive for AAV replication and comprising inducing sublethal stress in the AAV producing cell to enhance the level of AAV production, in which the AAV producing cell is a cell of AAV mammal, and wherein said cell comprises: </dd></dl>

<dl><dt>(i) </dt><dd>one or more AAV packaging genes, in which each of said AAV packaging genes encodes an AAV replication or encapsidation protein; </dd></dl>

<dl><dt>(ii)</dt><dd> a recombinant AAV (AAVr) provider comprising a heterologous non-AAV polynucleotide flanked by at least one inverted terminal repeat (RTI); and</dd></dl>

(iii) an AAV auxiliary virus,

<dl><dt>(b)</dt><dd> lyse the producing cell after the incubation of step a), producing an AAV producing cell lysate, and </dd></dl>

<dl><dt>(c)</dt><dd> purify the AAV-producing cell lysate, generating a population of recombinant adeno-associated virus (AAVr) particles, </dd></dl>

in which the sublethal stress is selected from:

<dl><dt>(I)</dt><dd> a nutritional stress imposed by one or more of the following: </dd></dl>

<dl><dt>(i)</dt><dd> culturing the producing cell in a medium that is deficient in one or more amino acids, optionally aspartic acid or glutamic acid; </dd></dl>

<dl><dt>(ii)</dt><dd> culturing the producing cell in a medium containing less than 10 μmol / l of aspartic acid; </dd></dl>

(iii) culturing the producing cell in a medium containing less than 2 μmol / l glutamic acid; or

<dl><dt>(iv)</dt><dd> culturing the producing cell in a medium that is deficient in serum; or</dd></dl>

<dl><dt>(II) </dt><dd>a thermal stress imposed by cultivating the producing cell for 3-6 days at: </dd></dl>

<dl><dt>(i)</dt><dd> a temperature lower than the optimum growth temperature of the producing cell; or</dd></dl>

<dl><dt>(ii)</dt><dd> a temperature greater than the optimum growth temperature of the producing cell; or</dd></dl>

(III) an osmotic stress imposed:

<dl><dt>(i)</dt><dd> by culturing the producing cell in hypoosmotic medium; or</dd></dl>

<dl><dt>(ii)</dt><dd> by culturing the producing cell in a hyperosmotic medium; or</dd></dl>

<dl><dt>(IV)</dt><dd> a pH stress, in which the pH stress comprises subjecting the producing cell to a pH of approximately pH 7.2 continuously during culture; or</dd></dl>

<dl><dt>(V)</dt><dd> a toxic stress, in which toxic stress comprises exposing the producing cell to a genotoxic agent selected from a chemical carcinogen, radiation, UV, a metabolic inhibitor of DNA synthesis and a drug that affects topoisomerases. </dd></dl>

[0031] These and other embodiments of the invention are set forth in the following description.

Brief description of the drawings

[0032]

Figure 1 is a semitonic reproduction of a Southern analysis of the production of AAVr vector using a probe for a model CF therapeutic gene contained in the vector. The highlighted band at 1.4 kb indicates the presence of AAVr in the preparation. The auxiliary function was provided by a subtype 5 adenovirus (Ad5) or by the thermosensitive strain ts149 of adenovirus.

Figure 2 is a semitonic reproduction of a slot transfer analysis for the production of an AAVr vector to quantify the level of AAVr present in each preparation. When the auxiliary function is supplied by ts149, the amount of AAVr produced under standard culture conditions is several logarithmic units less than that produced in the presence of Ad5.

Figure 3 is a semitonic reproduction of a Southern analysis for AAVr, which indicates that increasing the level of ts149 does not improve the level of AAVr production.

Figure 4 is a bar graph indicating a drastic increase in the amount of AAVr produced in the presence of ts49 (striped bars) if the cultivation periods extend beyond 5 days. This is in marked contrast to the substantial reduction in AAVr that appears beyond day 5 when non-thermosensitive adenoviruses are used to provide the auxiliary function (black bars).

Figure 5 is a linear graph showing the viable cell density (DCV) of HeLa S3 cells grown in suspension at 37 ° C (circles) or 32 ° C (square).

Figure 6 is a linear graph showing the effect of tangential flow filtration at two different speeds in HeLa S3 cells grown in suspension culture.

Figure 7 is a bar graph showing the production of ts149 detected in infected HeLa S3 cells cultured for 3-7 days in suspension at the permissible temperature of 32 ° C, compared to the level detected on day 7 after microfluidification (MF) .

Figure 8 is a combination graph showing the purification of ts149 by anion exchange chromatography on an eluted PI matrix with a linear gradient of 900-1300 meq of NaCl at pH 8.0.

Figure 9 is a combination graph showing the purification of adenovirus in an anion exchange matrix of eluted PI with a gradient of 800-1300 meq of NaCl at pH 8.0. Bars: viral activity measured in an infectivity test; solid line: A280 (a measure of total protein); dotted line: buffer conductivity (ms).

Figure 10 is a combination graph showing the separation of adenovirus and recombinant AAV. The upper panel shows the separation in anion exchange matrix of eluted PI with a gradient of 0-1000 meq of NaCl at pH 8.0. The lower panel shows the subsequent adenovirus separation of contaminants in HS cation exchange matrix eluted with a gradient of 0-500 meq of NaCl at pH 8.0.

Figure 11 is two bar graphs showing the effect of fetal bovine serum levels (FBS) on the culture medium on the production of AAVr. Serum deficiency in the culture medium is one of a series of stress factors that the producing cells can undergo to enhance the production of viral particles.

Figure 12 is a semitonic reproduction of a polyacrylamide-SDS gel analysis for AAV proteins during the purification steps. The AAV preparation was subjected to tangential flow filtration after chromatography on an anion exchange column (POROS 50 PI). The silver-stained gel shows the highly purified AAV capsid proteins VP1, VP2 and VP3 in the final crude material.

Figure 13 is a chromatogram showing the concentration of AAV in a heparin sulfate column. The narrow absorbance peak at 280 nm (left axis) at approximately 18 minutes of elution time represents the AAV fraction (after anion exchange and tangential flow filtration) eluted from heparin sulfate with a linear gradient of 0 to 1 NaCl M (shown the conductivity in ms on the right axis).

Detailed description

[0033] It is an object of this disclosure to provide methods and materials for generating high titre preparations of adeno-associated viruses (AAVs) that are substantially free of helper viruses, helper virus proteins and cellular proteins and other components.

[0034] Various methods for the generation and processing of AAV particles in mammalian cells are described in detail below, and illustrations of the use of such techniques are provided in the following examples. The methods of the present invention are defined by the appended claims.

[0035] By way of introduction, it is typical to employ a host or "producer" cell for replication and packaging of AAVr vector. Said producing cell (usually a mammalian host cell) generally comprises, or is modified to comprise, several different types of components for the production of AAVr. The first component is a recombinant adeno-associated viral vector genome (AAVr) (or "AAVr provider") that can be replicated and packaged into vector particles by the host packaging cell. The AAVr provider will normally comprise a heterologous polynucleotide (or "transgene") with which it is desired to genetically alter another cell in the context of gene therapy (since the packaging of said transgene into AAVr vector particles can be effectively used to deliver the transgene to a variety of mammalian cells). The transgene is generally flanked by two inverted terminal repeat (RTI) of AAV, comprising sequences that are recognized during cleavage, replication and packaging of the AAV vector, as well as during integration of the vector into a host cell genome. A second component is an auxiliary virus that can provide auxiliary functions for AAV replication. Although adenoviruses are commonly used, other auxiliary viruses may also be used as is known in the art. Alternatively, the necessary auxiliary virus functions can be genetically isolated from an auxiliary virus and the coding genes can be used to provide functions of the auxiliary virus in trans. The AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell simultaneously or sequentially in any order. The final components for the production of AAV to be provided to the producer cell are "AAV packaging genes" such as the AAV rep and cap genes that provide replication and encapsidation proteins, respectively. Several different versions of AAV packaging genes can be provided (including wild-type repcap modules as well as modified rep and / or cap modules in which the rep and / or cap genes can be left under the control of native promoters or operably linked with promoters heterologists) Such AAV packaging genes may be introduced transiently or stably into the host packaging cell, as is known in the art and described in more detail below.

[0036] After culturing the host cells under conditions that allow AAV replication and encapsidation, the cells and subcellular fractions can be processed to generate high titre preparations of adeno-associated viruses (AAVs) that are substantially free of helper viruses, virus proteins auxiliaries and cellular proteins. Detailed descriptions of processing techniques and illustrative protocols employing these techniques are provided below.

Definitions

[0037] A "vector" as used herein designates a macromolecule or association of macromolecules that comprises or is associated with a polynucleotide and that can be used to mediate the delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles.

[0038] "AAV" is an abbreviation for adeno-associated virus, and can be used to designate the virus itself or derivatives thereof. The term covers all subtypes and both forms of natural and recombinant origin, except when otherwise required. The abbreviation "AAVr" designates recombinant adeno-associated virus, also designated as recombinant AAV vector (or "AAVr vector").

[0039] An "AAVr vector" as used herein designates an AAV vector comprising a polynucleotide sequence of origin other than AAV (specifically, a heterologous AAV polynucleotide), typically a sequence of interest for genetic transformation. of a cell. In the preferred vector constructs of this disclosure, the heterologous polynucleotide is flanked by at least one, preferably two inverted terminal repeated sequences (RTI) of AAV. The term AAVr vector comprises both AAVr vector particles and AAVr vector plasmids.

[0040] An "AAV virus" or "AAV viral particle" designates a viral particle composed of at least one AAV capsid protein (preferably all capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (specifically, a polynucleotide other than a wild-type AAV genome such as a transgene to deliver to a mammalian cell), it is typically referred to as "AAVr vector particle" or simply "vector of AAVr. "

[0041] "Packaging" designates a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0042] The AAV "rep" and "cap" genes designate polynucleotide sequences that encode adeno-associated virus replication and encapsidation proteins. They have been found in all the AAV serotypes examined, and are described below and in the art. The AAV rep and cap genes are referred to herein as "packaging genes" of AAV.

[0043] An "auxiliary virus" of AAV designates a virus that allows AAV (eg, wild-type AAV) to replicate and package in a mammalian cell. A variety of such auxiliary viruses, including adenovirus, herpesvirus and poxvirus such as Vaccinia, are known in the art. Adenoviruses encompass a number of different subgroups, although type 5 adenovirus of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human and avian origin are known and are available in deposits such as ATCC. Herpes family viruses include, for example, herpesvirus simplex (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); which are also available in deposits such as the ATCC.

[0044] The term "VAts" designates a thermosensitive auxiliary virus that can provide auxiliary functions for AAV replication and packaging but is thermosensitive with respect to its own replication (specifically, it can replicate at a "permissive" temperature but replicates with lower efficiency, or preferably nothing at all, at a "non-permissive" temperature). The ability of the VAts to provide AAV replication support may also be thermosensitive, but the preferred VAts for use in the practice of the present disclosure that includes the present invention effectively supports AAV replication at temperatures at which the AAV can replicate. but that are not permissive for the replication of the VAts. Examples of such VAts are described below.

[0045] A "infectious" virus or virus particle is one that comprises a polynucleotide component that can be delivered to a cell for which the viral species is trophic. The term does not necessarily imply any capacity for virus replication. Tests for counting infectious viral particles are described elsewhere in this disclosure and in the art.

[0046] A "replication competent" virus (for example, a replication competent AAV, sometimes abbreviated as "ACR") designates a phenotypically wild-type virus that is infectious and can also replicate in an infected cell (specifically, in presence of an auxiliary virus or auxiliary virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. Preferred AAVr vectors as described herein are incompetent of replication in mammalian cells (especially in human cells) due to the lack of one or more of the AAV packaging genes. Preferably, said AAVr vectors lack any AAV packaging gene sequence to minimize the possibility of ACR being generated by recombination between AAV packaging genes and an incoming AAVr vector. Preferred AAVr vector preparations as described herein are those that contain little or no ACR (preferably, less than about 1 ACR per 102 AAVr particles, more preferably less than about 1 ACR per 104 AAVr particles, even more preferably less than about 1 ACR for 108 particles of AAVr, even more preferably less than about 1 ACR for 1012 particles of AAVr, most preferably no ACR).

[0047] The term "polynucleotide" designates a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications of the nucleotide structure can be conferred before or after polymer assembly. The term polynucleotide, as used herein, interchangeably designates bi- and single stranded molecules. Unless otherwise specified or required, each time the disclosure describes herein that there is a polynucleotide, it encompasses both the double stranded form and each of the two complementary single stranded forms known or predicted to constitute the double stranded form.

[0048] A "gene" designates a polynucleotide that contains at least one open reading frame that can encode a particular protein after transcription and translation.

[0049] "Recombinant," as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation stages and other procedures that result in a construct that is distinct from a polynucleotide found in the nature. A recombinant virus is a viral particle that comprises a recombinant polynucleotide. The terms include duplicates of the original polynucleotide construct and the progeny of the original viral construct respectively.

[0050] A "control element" or "control sequence" is a sequence involved in the interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation or degradation of the polynucleotide. . The regulation can affect the frequency, speed or specificity of the process and can be of an enhancer or inhibitory nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA capable, under certain conditions, of binding to RNA polymerase and initiating the transcription of a coding region usually located in the 3 'direction of the promoter.

[0051] "Operationally linked" means a juxtaposition of genetic elements in which the elements are in a relationship that allows them to act as expected. For example, a promoter is operably linked with an encoding sequence if the promoter helps initiate transcription of the coding sequence. There may be intermediate residues between the promoter and the coding region provided that this functional relationship is maintained.

[0052] An "expression vector" is a vector that comprises a region encoding a polypeptide of interest, and is used to effect the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes with which they are operatively linked for expression is sometimes referred to as an "expression module", a large number of which are known and available in the art or can be easily constructed from of components that are available in the field.

[0053] "Heterologous" means derived from a genotypically distinct entity from the rest of the entity with which it is compared. For example, a polynucleotide introduced by genetic engineering techniques in a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter devoid of its native coding sequence and operably linked with a coding sequence with which it is not naturally linked is a heterologous promoter.

[0054] "Genetic alteration" means a process in which a genetic element is introduced into a cell differently from mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. The genetic alteration can be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide by any process known in the art, such as electroporation, precipitation with calcium phosphate or contacting a polynucleotide-liposome complex. The genetic alteration can also be effected, for example, by transduction or infection with a virus or viral vector of DNA or RNA. Preferably, the genetic element is introduced into a chromosome or mini-chromosome of the cell, but any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0055] A cell is said to be altered, transduced or "stably" transformed with a genetic sequence if the sequence is available to perform its function during prolonged cell culture in vitro. In preferred examples, said cell is "inherited" altered because a genetic alteration is introduced that is also inheritable by the progeny of the altered cell.

[0056] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to designate amino acid polymers of any length. The terms also encompass an amino acid polymer that has been modified: for example, by formation of disulfide bond, glycosylation, stoning or conjugation with a marker component.

[0057] Polypeptides such as "CFTR", "p53", "E1A" and the like, when examined in the context of gene therapy and compositions therefor, designate the respective intact polypeptide, or any fragment or derivative engineered genetics that retain the desired biochemical function of the intact protein. Similarly, references to CFTR, p53, E1A and other genes of such genes for use in gene therapy (typically referred to as "transgenes" for delivery to a recipient cell) include polynucleotides encoding the intact polypeptide or any modified fragment or derivative. by genetic engineering that has the desired biochemical function.

[0058] A plasmid, virus or other "isolated" substance designates the preparation of the substance devoid of at least some of the other components that may also be present when the substance or a similar substance appears naturally or is initially prepared. Therefore, for example, an isolated substance can be prepared using a purification technique to enrich it from a source mixture. The enrichment can be measured absolutely, such as by weight per volume of solution, or it can be measured in relation to a second potentially interfering substance present in the source mixture. Increasing enrichments of the cases of this disclosure are more preferred. Thus, for example, a 2-fold enrichment is preferred, a 10-fold enrichment is more preferred, a 100-fold enrichment is more preferred, and a 1000-fold enrichment is even more preferred.

[0059] An AAV preparation is said to be "substantially free of auxiliary virus if the ratio of infectious AAV particles to infectious auxiliary virus particles is at least about 102: 1, preferably at least about 104: 1, more preferably of at least about 106: 1, even more preferably of at least about 108: 1. The preparations are also preferably free of equivalent amounts of auxiliary virus proteins (specifically, proteins as would be present as a result of said level of auxiliary virus if the impurities of auxiliary virus particles observed above were present in disintegrated form). Viral and / or cellular protein contamination can generally be observed by the presence of Coomasie staining bands in SDS gels (for example, the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2 and VP3).

[0060] "Efficiency", when used in the description of viral production, replication or packaging, designates useful properties of the process: in particular, the growth rate and the number of viral particles produced per cell. "High efficiency" production indicates the production of at least 100 viral particles per cell, preferably at least about 10,000 and more preferably at least about

100,000 particles per cell, during the course of the specified culture period.

[0061] An "individual" or "subject" treated in accordance with this disclosure designates vertebrates, particularly members of a mammalian species, and includes, but is not limited to, domestic animals, sports animals and primates, including humans.

[0062] "Treatment" of an individual or cell is any type of intervention in an attempt to alter the natural course of the individual or cell at the time the treatment is initiated. For example, the treatment of an individual can be undertaken to reduce or limit the pathology caused by any pathological condition including (but not limited to) an inherited or induced genetic deficiency, an infection by a viral, bacterial or parasitic organism, a neoplastic or aplastic condition or an immune system dysfunction such as autoimmunity or immunosuppression. Treatment includes (but not limited to) the administration of a composition, such as a pharmaceutical composition, and the administration of compatible cells that have been treated with a composition. The treatment can be carried out prophylactically or therapeutically, that is, before or after the onset of the pathological event or contact with the etiologic agent.

General techniques

[0063] The practice of the present disclosure that includes the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, virology, animal cell culture and biochemistry that are within the skill of a person skilled in the art. . These techniques are explained in detail in the bibliography. See, for example, "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook, Fritsch and Maniatis, 1989); "Animal Cell Culture" (RI Freshney, ed., 1987); "Gene Transfer Vectors for Mammalian Cells" (JM Miller and MP Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel and col., Eds., 1987); "Current Protocols in Protein Science" (John E Coligan, et al. Eds. Wiley and Sons, 1995) and "Protein Purification: Principles and Practice" (Robert K. Scopes, Springer-Verlag, 1994).

Selection and preparation of AAV vectors and AAV packaging genes

[0064] A recombinant AAV vector of this disclosure comprises a heterologous polynucleotide (specifically, not AAV) of interest instead of the AAV rep and / or cap genes that normally constitute the bulk of the AAV genome. As in the wild-type AAV genome, however, the AAVr provider is preferably flanked by two inverted terminal repeat (RTI) AAVs as noted above. Variations in which the AAVr construct is flanked by only one RTI (typically modified) have also been described in the art, and can be employed in relation to the present disclosure that includes the present invention.

[0065] Adeno-associated viruses of any serotype are suitable, since the various serotypes are functionally and structurally related, even at the genetic level (see, for example, Blacklow, p. 165-174 of "Parvoviruses and Human Disease" JR Pattison, ed . (1988) and Rose, Comprehensive Virology 3: 1, 1974). All AAV serotypes apparently show similar replication properties mediated by homologous rep genes; and generally all carry three related capsid proteins such as those expressed in AAV2. The degree of relationship is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes throughout the genome, and the presence of analogous self-associated segments at the ends corresponding to RTI. The similar infectivity pattern also suggests that the replication functions in each serotype are under a similar regulatory control. Among the various AAV serotypes, AAV2 is the most commonly used.

[0066] An AAV vector of this disclosure will typically comprise a polynucleotide that is heterologous to AAV. The polynucleotide is typically of interest because of the ability to provide a function to a target cell in the context of gene therapy, such as the positive or negative regulation of the expression of a certain phenotype. Said heterologous polynucleotide or "transgene" will generally be of sufficient length to provide the desired coding function or sequence. For encapsidation of AAV2 particles, the transgene will preferably be less than about 5 kb, although other serotypes and / or modifications may be used to allow larger sequences to be packaged into the AAV viral particles.

[0067] When transcription of the heterologous polynucleotide in the intended target cell is desired, it can be operably linked with its own promoter or a heterologous one, depending for example on the level and / or specificity of transcription desired in the target cell, as is known in the matter. Various types of promoters and enhancers are suitable for use in this context. Constitutive promoters provide a constant level of gene transcription and are preferred when it is desired to express the therapeutic polynucleotide continuously. Inducible promoters generally exhibit low activity in the absence of the inductor, and are positively regulated in the presence of the inductor. They may be preferred when expression is desired only at certain times or at certain locations, or when it is desirable to titrate the level of expression using an inducing agent. Promoters and enhancers may also be tissue specific, that is, they exhibit their activity only in certain cell types, presumably due to gene regulatory elements found only in those cells.

[0068] Illustrative examples of promoters are the simian virus SV40 late promoter, the Baculovirus polyhedron enhancer / promoter element, herpesvirus thymidine kinase simplex (HSV tk), the immediate early cytomegalovirus (CMV) promoter and various retroviral promoters that include LTR elements. Inducible promoters include promoters inducible by heavy metal ions (such as the mouse mammary tumor virus (MTVm) promoter or various growth hormone promoters) and T7 phage promoters that are active in the presence of T7 RNA polymerase. By way of illustration, examples of tissue specific promoters include various surfactin promoters (for lung expression), myosin promoters (for muscle expression) and albumin promoters (for liver expression). A wide variety of other promoters are known and generally available in the art, and the sequences of many of these promoters are available in sequence databases such as the GenBank database.

[0069] When translation is also desired in the intended target cell, the heterologous polynucleotide will also preferably comprise control elements that facilitate translation (such as a ribosome binding site or "RBS" and a polyadenylation signal). Accordingly, the heterologous polynucleotide will generally comprise at least one coding region operably linked with a suitable promoter and may also comprise, for example, an enhancer, ribosome binding site and operably linked poly-A signal. The heterologous polynucleotide may comprise a coding region or more than one coding region under the control of the same or different promoters. The complete unit, which contains a combination of control elements and coding region, is often referred to as an expression module.

[0070] The heterologous polynucleotide is integrated by recombinant techniques in or preferably instead of the AAV genomic coding region (specifically, instead of the AAV rep and cap genes), but is generally flanked on both sides by repeat regions inverted terminal (RTI). This means that an RTI appears both in the 5 'and 3' direction of the coding sequence, in direct juxtaposition preferably (but not necessarily) without intermediate sequence of AAV origin to reduce the likelihood of recombination that a competent AAV genome could regenerate of replication Recent evidence suggests that a single RTI may be sufficient to carry out the functions normally associated with configurations comprising two RTIs (WO 94/13788) and therefore vector constructs with only one RTI may be used in conjunction with the packaging and Production of the present disclosure including the methods of the present invention.

[0071] The native rep promoters are self-regulating, and may limit the amount of AAV particles produced. The rep gene may also be operably linked to a heterologous promoter, if rep is provided as part of the vector construct, or separately. Any heterologous promoter that is not strongly negatively regulated by the expression of the rep gene is suitable, but inducible promoters are preferred because the constitutive expression of the rep gene can have a negative impact on the host cell. A wide variety of inducible promoters are known in the art which include, by way of illustration, promoters inducible by heavy metal ions (such as metallothionein promoters), steroid hormone inducible promoters (such as MMTV promoter or growth hormone promoters ) and promoters such as those of phage T7, which are active in the presence of T7 RNA polymerase. An especially preferred subclass of inducible promoters are those that are induced by the helper virus that is used to complement the replication and packaging of the AAVr vector. A series of helper virus inducible promoters have also been described which include the promoter of the early adenovirus gene that is inducible by herpesvirus proteins such as VP16 or 1CP4, as well as promoters inducible by Vaccinia or poxvirus.

[0072] Methods for identifying and assaying helper virus inducible promoters have been described in the application being processed and common property hereby published as WO96 / 17947 by Targeted Genetics Corporation (Allen et al.). Therefore, methods are known in the art to determine if the candidate promoters are inducible by auxiliary viruses or not and if they will be useful or not in the generation of high efficiency packaging cells. Briefly, one such procedure involves replacing the p5 promoter of the AAV rep gene with the supposed helper-inducible promoter (known in the art or identified using well-known techniques such as linking with "reporter" genes without promoter). The AAV rep-cap genes (with p5 replaced) preferably linked with a positive selectable marker such as an antibiotic resistance gene, are then stably integrated into a suitable host cell (such as the HeLa or A549 cells exemplified below). Cells that are capable of growing relatively well under selection conditions (for example, in the presence of an antibiotic) are then tested for their ability to express the rep and cap genes after the addition of an auxiliary virus. As an initial assay for the expression of rep and / or cap, cells can be easily screened using immunofluorescence to detect Rep and / or Cap proteins. Confirmation of packaging capacities and efficiencies can then be determined by functional tests of the replication and packaging of incoming AAVr vectors. Using this methodology, an auxiliary virus-inducible promoter derived from the mouse metallothionein gene has been identified as a suitable replacement for the p5 promoter, and has been used to produce high titres of AAVr particles (as described in WO 96 / 17947 from Targeted Genetics Corporation).

[0073] Given the relative encapsidation size limits of the various AAV genomes, the insertion of a large heterologous polynucleotide into the genome requires the removal of a portion of the AAV sequence. The elimination of one or more AAV genes is in any case desirable to reduce the likelihood of generating competent AAV replication ("ACR"). Accordingly, coding sequences or promoters of rep, cap or both are preferably eliminated, since the functions provided by these genes can be provided in trans.

[0074] The resulting vector is designated as "defective" of these functions. To replicate and package the vector, the missing functions are complemented with a packaging gene, or a plurality thereof, which together encode the functions necessary for the various missing rep and / or cap gene products. Gene genes or packaging modules are preferably not flanked by RTI of AAV, and preferably do not share any substantial homology with the AAVr genome. Therefore, to minimize homologous recombination during replication between the vector sequence and the packaging genes provided separately, it is desirable to avoid overlapping the two polynucleotide sequences. The level of homology and the corresponding frequency of recombination increase with increasing length of homologous sequences and with their level of shared identity. The level of homology that will pose a problem in a given system can be determined theoretically and confirmed experimentally, as is known in the art. However, typically the recombination can be substantially reduced or eliminated if the overlapping sequence is less than an approximately 25 nucleotide sequence if it is at least 80% identical in its entire length, or less than an approximately 50 nucleotide sequence if it is at least 70% identical in its entire length. Of course, even lower levels of homology are preferable, since they will further reduce the likelihood of recombination. It seems that, even without overlapping homology, there is some residual frequency of ACR generation. Even greater reductions in the frequency of ACR generation can be obtained (for example, by non-homologous recombination) by "separating" the AAV replication and encapsidation functions, as described by Allen et al. in the US patent application 08 / 769,728, filed on December 18, 1996, published internationally as WO98 / 27204 on June 25, 1998 (Targeted Genetics Corporation).

[0075] The AAVr vector construct, and complementary gene packaging constructs, can be practiced by practicing the present disclosure that includes the invention in a number of different ways. Stably transformed viral particles, plasmids and host cells can be used to introduce said constructs into the packaging cell, transiently or stably.

[0076] In certain cases of this disclosure, for example in certain embodiments of this invention, the AAV vector and the complementary packaging gene or genes, if any, are provided in the form of bacterial plasmids, AAV particles or any combination. thereof. In other cases and embodiments, the AAV vector sequence, the packaging gene or genes or both in the form of genetically altered eukaryotic cells (preferably, hereditary altered) are provided. The development of hereditary altered host cells to express the AAV vector sequence, the AAV packaging genes or both, provides an established source of material that is expressed at a reliable level.

[0077] A variety of different genetically altered cells can therefore be used in the context of this disclosure that includes the invention. By way of illustration, a mammalian host cell with at least one intact copy of a stably integrated AAVr vector can be used. An AAV packaging plasmid comprising at least one AAV rep gene operably linked to a promoter can be used to deliver replication functions (as described in the common property application herein with Flotte et al., Now US Pat. 5,658,776). Alternatively, a stable mammalian cell line with an AAV rep gene operatively linked to a promoter can be used to deliver replication functions (see, for example, Trempe et al. (USSN 08 / 362,608, January 9, 1995 and WO95 / 13392, of May 18, 1995); Burstein et al., (USSN documents 08 / 770.122, filed December 18, 1996 and WO98 / 23018, June 25, 1998) and Johnson et al. ((USSN documents 08 / 254,358, filed June 6, 1994, issued as US 5,656,785 on August 19, 1997)). The AAV cap gene, which provides the encapsidation proteins as described above, can be provided in conjunction with an AAV rep gene or separately (see, for example, the aforementioned applications and patents as well as Allen et al., USSN 08 /769,728, filed on December 18, 1996 and WO98 / 27204 on June 25, 1998 (Targeted Genetics Corporation). Other combinations are possible and are included within the scope of this disclosure that includes the invention.

Introduction of genetic material in cells

[0078] As described in the art, and illustrated herein as well as in the references cited above, the genetic material can be introduced into cells (such as "producer" mammalian cells for the production of AAV) using any of a variety of means to transform or transduce said cells. By way of illustration, such techniques include, for example, transfection with bacterial plasmids, infection with viral vectors, electroporation, precipitation with calcium phosphate and introduction using any of a variety of lipid-based compositions (a process often referred to as "lipofection. "). The procedures and compositions for performing these techniques have been described in the art and are widely available.

[0079] The selection of suitably altered cells can be performed by any technique of the subject. For example, polynucleotide sequences used to alter the cell can be simultaneously introduced to or operatively linked with one or more detectable or selectable markers as is known in the art. By way of illustration, a drug resistance gene can be used as a marker. The drug resistant cells can then be chosen, grown and tested for expression of the desired sequence, specifically a product of the packaging gene or a product of the heterologous polynucleotide, as appropriate. The acquisition, localization and / or maintenance test of an introduced polynucleotide can be performed using DNA-based hybridization techniques (such as Southern blotting and other procedures as are known in the art). The expression assay can easily be performed by Northern analysis of RNA extracted from genetically altered cells, or by indirect immunofluorescence by the corresponding gene product. The test and confirmation of the packaging capacities and efficiencies can be obtained by introducing into the cell the remaining functional components of AAV and an auxiliary virus, to test the production of AAV particles. When a cell with a plurality of polynucleotide constructs is inherited, it is generally more convenient (though not essential) to introduce them into the cell separately, and validate each stage in series. References describing such techniques include those cited herein.

Selection and preparation of auxiliary viruses

[0080] As discussed above, AAV is a parvovirus that is defective of self-replication, and should generally rely on an auxiliary virus to provide certain replicative functions. A number of such auxiliary viruses have been identified, including adenovirus, herpesvirus (including but not limited to HSV1, cytomegalovirus and HHV-6) and poxvirus (particularly Vaccinia). Any such virus may be used when practicing this disclosure that includes the invention.

[0081] Frequently, the helper virus will be an adenovirus of a type and subgroup that can infect the intended host cell. Human adenoviruses of subgroup C are commonly used, particularly serotypes 1, 2, 4, 6 and 7. Serotype 5 is generally preferred.

[0082] Traits and growth patterns of adenoviruses are known in the art. The reader can refer, for example, to Horowitz, "Adenoviridae and their replication," p. 771-816 in "Fundamental Virology", Fields et al., Eds. The packaged adenoviral genome is a linear DNA molecule linked through adenoviral RTIs to the left and right ends through a terminal protein complex, forming a circle. The control and coding regions for early, intermediate and late components overlap in the genome. The genes of the early region are involved in the replication of the adenoviral genome, and are grouped depending on their location in the E1, E2, E3 and E4 regions.

[0083] Although not essential, in principle it is desirable that the auxiliary virus strain be replication defective in the subject who ultimately receives gene therapy. Therefore, any residual auxiliary virus present in an AAVr preparation will be incompetent of replication. Adenoviruses from which the E1A region, or both E1A and E3, have been removed are not infectious to most human cells. They can be replicated in a permissive cell line (for example, the human 293 cell line) that is capable of complementing the missing activity. Adenovirus regions that appear to be associated with the auxiliary function have been identified and described in the art, as well as regions that do not (see, for example, P. Colosi et al., WO97 / 17458 and the references cited therein) .

Use of conditionally sensitive auxiliary virus

[0084] As described herein, a "conditionally sensitive" helper virus can be employed to provide helper virus activity. Said auxiliary virus strain must have at least the property of being able to support AAV replication in a host cell under at least one group of conditions in which it does not undergo effective genomic replication. When auxiliary virus activity is supplied as intact virus particles, it is also generally necessary for the virus to be capable of replication in a host cell under a second set of conditions. The first group of conditions will differ from the second group of conditions in an easily controllable feature, such as the presence or absence of a necessary cofactor (such as a cation), the presence or absence of an inhibitory drug or a displacement of environmental conditions such as temperature Most conveniently, the difference between the two conditions is temperature, and said conditionally sensitive virus is therefore referred to as a thermosensitive auxiliary virus (VAts).

[0085] For the purposes of this disclosure, a "thermosensitive" or "ts" auxiliary virus is one that is capable of replicating its genetic material in a eukaryotic cell in a certain temperature range (the "permissive" temperature range), typically about 15-35 ° C and preferably about 2032 ° C. However, at the "non-permissive" temperature, even when the other conditions remain the same, the rate of replication of the genetic material is substantially lower, at least 10 times lower, usually at least about 100 times lower and preferably at least about 1000 minor times This temperature is typically about 35-50 ° C, generally about 42 ° C. In a typical example of said ts auxiliary virus, the virus is capable of efficient replication at relatively low temperatures, such as temperatures of about 20-32 ° C, but is incapable of efficient replication at relatively high temperatures such as temperatures of about 37-42 ° C. It is understood that the virus-infected cell can nevertheless exhibit some metabolic processes attributable to the virus at the non-permissive temperature, including but not limited to the auxiliary function for AAV production.

[0086] A heat-sensitive auxiliary virus can be produced in large quantities by culturing the infected cells at a permissive temperature. The AAV vector can then be produced by culturing cells comprising vector elements and the thermosensitive helper virus at a non-permissive temperature. The vector preparation will be substantially free of auxiliary virus components.

[0087] A large number of thermosensitive adenovirus variants have been described in the art; see, for example, the variants described by Ensinger et al. (J. Virol. 10: 328. 1972); Williams et al. (J. Gen Virol. 11: 95, 1971); Ishibashi (Proc. Natl. Acad. Sci. USA 65: 304, 1970): Lundholm et al. (Virology 45: 827, 1971) and Shiroki et al., (Virology 61: 474, 1974) among others. Complementation analysis indicates that such variants fall within a plurality of different complementation groups (Ginsberg et al., Cold Spring Harbor Symp. Quant. Biol. 34: 419, 1974). This suggests that a series of stages in the adenovirus replicative cycle may become thermosensitive.

[0088] Since the auxiliary function for AAV replication requires that only part of the adenovirus cycle be intact, testing the auxiliary function of various mutants at the non-permissive temperature provides a means to map the auxiliary function. For example, Ishibashi et al. (Virology 45: 317, 1971) reported that thermosensitive avian adenovirus variants support AAV1 and AAV2 replication. Ito et al. They reported that the thermosensitive ts13 mutant of human adenovirus 7 (Ad7ts13) helps AAV replication at the non-permissive temperature as effectively as the wild strain. Drake et al. (Virology 60: 230, 1974) reviewed the complementation of the synthesis of the AAV4 antigen by 3 groups of thermosensitive mutants of herpesvirus simplex type 1 (HSV1). Handa et al. (J. Gen. Viro. 29: 239, 1975) reported auxiliary activities for the production of the AAV1 virus by human adenovirus mutants Ad5ts36, Ad5ts125, Ad5ts149, Ad12tsA275, Ad12tsB221 and Ad12tsC295. Ostrove et al. (Virology 104: 502, 1980) reported that the heat-sensitive mutants Ad5ts125, Ad5ts135, Ad5ts157, Ad5ts116, and Ad5ts142 and the mutants of the host interval hr6, but not hr3, supported AAV replication. Mayor et al. (J. Gen Virol. 35: 545, 1977) reported that Ad31ts13, but not Ad31ts94, supported the production of AAV1 at the non-permissive temperature.

[0089] Straus et al. (Proc. Natl. Acad. Sci. USA 73: 742, 1976) reported that Ad5ts125 supported AAV2 replication under conditions in which the adenovirus did not replicate on its own. They used this property to study the DNA intermediates formed during AAV replication. Myers et al. (J. Virol. 35: 65, 1980) conducted a quantitative study on auxiliary function and showed that Ad5ts149 supported the production of

20,000 infectious AAV particles per cell at the non-permissive temperature, while Ad5ts107 produced only ~ 100 particles per cell. Since Ad5ts107 has a mutation in the coding region of the 72 kDa DNA binding protein, they concluded that this protein played a role in AAV RNA expression. More recently, Carter et al. (Virology 191: 473; 1992) proposed that a fully functional 72 kDa protein is necessary for quantitative posttranscriptional expression of AAV rep and cap genes.

[0090] As set forth in the background section, the existence of heat sensitive adenoviruses has been known for quite some time. However, there have been no effective teachings or suggestions regarding the actual use of conditional auxiliary viruses in the generation of recombinant AAV vectors, such as those that could be used for gene therapy.

[0091] Part of the explanation may be due to the difficulty in obtaining functional AAV titers when recombinant vectors are used. Among other things, AAV Rep proteins apparently regulate their own expression negatively through the p5 promoter (Tratschin et al., Mol. Cell Biol. 6: 2884, 1986). Furthermore, it has been observed that the expression of the rep gene in packaging cell lines such as those that could be used for the production of recombinant AAV vector tends to inhibit cell growth and / or metabolism (see, for example, Targeted Genetics Corporation WO96 / 17947 of Allen et al.).

[0092] The differences between the generation of wild-type AAV and recombinant AAV vectors tend to be quite drastic when considered in terms of production. In particular, it has been observed that the production of recombinant AAV vectors tends to be substantially less than the production of wild-type AAV particles, and that the presence or generation of even small amounts of contaminating wild-type AAV tend to give as a preferred production of wild-type virus that can eventually overcome recombinant AAV vectors resulted.

[0093] These phenomena are further illustrated by the results described in Examples 1 and 2 of this disclosure and in Figure 1. The ts149 thermosensitive adenovirus mutant is reviewed elsewhere that supports AAV particle replication (Myers et al. J. Virol. 35: 65, 1980). However, Example 2 shows that when this mutant is used to support the production of an AAV vector with a heterologous promoter under standard conditions, the level of production is several orders of magnitude less than that supported by wild-type adenovirus.

[0094] This disclosure shows that heat-sensitive auxiliary viruses can in fact be used to prepare recombinant AAV vectors at functional titers, overcoming apparent production obstacles. The following descriptions illustrate how to select a thermosensitive auxiliary virus and optimize conditions to provide sufficient AAV for gene therapy purposes.

[0095] In particular, it is shown that extending the AAV replication period when tsAd is used as an auxiliary dramatically increases the amount of AAV vector that is produced (Example 3). This is anti-intuitive, because lengthening the replication period when wild-type Ad is used similarly reduces the amount of AAV vector by at least an order of magnitude. A subject professional who seeks to optimize the conditions for the production of AAV would logically go to shorter culture times and higher concentrations of auxiliary virus; It is shown herein that both are ineffective.

[0096] This disclosure further provides improved culture and separation procedures for preparing quantitative amounts of heat sensitive adenoviruses. Although not strictly required for the practice of certain cases of this disclosure, for example for the practice of certain embodiments of this invention, heat sensitive adenovirus preparations obtained by these procedures are particularly suitable for the production of AAV, among other things, with purposes of gene therapy.

[0097] Variants sensitive to the conditions of the selected strain of auxiliary virus can be generated by an appropriate mutegenization and selection strategy. For example, the virus can be mutagenized with nitrosoguanidine, nitrous acid, hydroxylamine or 5-bromo-2-deoxyuridine. Candidates are selected who can multiply in a suitable eukaryotic cell in the desired permissive conditions, but not in the desired non-permissive conditions. As an illustration, thermosensitive adenovirus mutants can be obtained that multiply, for example, at 32 ° C but not at 39.5 ° C. The plating efficiency ratios at 39.5 ° C versus 32 ° C are preferably less than 10-4 and more preferably less than 10-5. An additional illustration of suitable selection processes for thermosensitive adenoviruses can be found, for example, in Ensinger et al., J. Virol. 10: 328, 1972 and Williams et al., J. Gen Virol. 11: 95, 1971. The description of adenovirus variants that are not heat sensitive but sensitive to the type of host can be found in Harrison et al., Virology

77: 319, 1977. Effective thermosensitive mutants can be prepared for use in the practice of this disclosure which includes the invention, for example, from alternative auxiliary viruses such as herpes simplex 1 (HSV1) or herpes simplex 2 (HSV2). See, for example, Schaffer et al., Virology 52: 57, 1973 for HSV1; Esparza et al., Virology 57: 554, 1974 for HSV2. As indicated in the background section, a large number of condition-sensitive auxiliary viruses have been described, and can be obtained from the scientists who developed or described them or from a public deposit.

[0098] Not all variants sensitive to the conditions of the viruses listed above will work with the present invention. In particular, the strain must become sensitive to the condition at a stage of its replicative cycle such that the function that is blocked under non-permissive conditions is not necessary for AAV high efficiency replication. The choice of which auxiliary virus strain to use can be made with reference to the known biology of the auxiliary virus and the AAV replicative requirements.

[0099] It is an exemplary auxiliary virus for use in the practice of the present disclosure that includes the present invention the thermosensitive adenovirus ts149 serotype Ad5 (Ad5ts149). As shown in the examples section, under optimized conditions this strain can be used to produce AAVr at levels that match or exceed those supported by wild-type Ad5. The ts149 has a single transition from CG to AT at position 7563 (Roovers et al., Virus Genes 4: 53, 1990). This results in a change of the amino acid leucine in residue 411 of the DNA polymerase to phenylalanine. DNA polymerase is contained in the E2 transcription unit of the adenovirus. However, other ts mutants that are mapped in this region are less suitable. In particular, the transcription unit E2 also comprises the coding region of the 72 kDa DNA binding protein (DBP). A strain that does not produce undetectable DBP (Add / 802) supports AAV replication, but at a level that is reduced by an order of magnitude (Carter et al., Virology 191: 473, 1992). Adts125, which also comprises a mutation that is mapped in the region encoding DBP, supports AAV replication (Straus et al., J. Virol. 17: 140, 1976), although the levels are generally much lower than with Ad5 of wild type (Myers et al., J. Virol. 35: 65, 1980). Accordingly, thermosensitive adenovirus vectors suitable for use in the practice of the present disclosure that includes the present invention include those for which sensitivity is mapped in the E2A region of the genome, preferably in the region encoding DNA polymerase.

[0100] The expert can easily determine which viral strains are suitable for use as an auxiliary virus when performing an AAVr replication assay using a panel of candidate auxiliary virus strains in a candidate cell under conditions that are not permissive for the self-replication of the auxiliary. . For thermosensitive variants, screening at non-permissive temperature is performed according to the known properties of the strain. Non-permissive temperatures are generally higher than permissive temperatures, typically about 35-50 ° C, preferably 38-45 ° C, more preferably about 39.5 ° C. Variants that support AAV replication at a level that is within an order of magnitude of that supported by the corresponding wild-type virus are preferred. When screening, the expert must incorporate the other teachings of this disclosure. In particular, screening by culture during times that seek maximum AAV replication with wild-type virus is insufficient. A kinetic matrix must be established in which candidate auxiliary viruses are used for longer periods and then compared with the wild-type virus at the time of maximum collection. A more detailed illustration of this analysis is provided in Example 3 of this disclosure.

[0101] Once a suitable auxiliary virus strain has been selected, it can be applied in this invention in a number of different ways. Viral particles, viral plasmids and stably transformed host cells can be used.

[0102] In one embodiment, the auxiliary virus genome (or minimally, regions of the auxiliary virus genome encoding the auxiliary function) is introduced into the host cell to be used for replication of the AAVr vector in the form of a plasmid. of DNA or a plurality of plasmids that provide complementary functions. Methods for experimental manipulation of adenovirus are known in the art. The reader refers to Graham et al., "Manipulation of adenovirus vectors" in: Murray EJ, ed. "Methods in molecular biology: Gene transfer and expression protocols", vol 7. Clifton, NJ: The Human Press, 1991: 109-128, which provides detailed protocols for the propagation, titration and purification of adenovirus, counter-infection and recombination in vivo . Adenoviral plasmids are commercially available from Microbix Biosystems Inc., Toronto, Canada.

[0103] In another embodiment, the host cell is stably transfected with adenoviral genes, or genetically altered to provide the functions necessary for AAVr replication. Alternatively, the host cell can be genetically altered with only a portion of the adenoviral genome, and subsequently infected or transfected with an adenoviral plasmid or particle. Patent applications WO 95/27071 and WO 95/34671 describe hereditary altered host cells to provide an adenoviral function that complements the replicative property of various defective adenoviral constructs.

[0104] In yet another embodiment, the host cell used for AAV replication is infected with an auxiliary virus that is capable of self-replication, but not under non-permissive conditions. Any preparation of the necessary strain that provides sufficient MOI can be used. In order to satisfy GMP and other regulatory requirements, and to facilitate scaling up for commercial purposes, auxiliary virus preparations preferably comprise a high density of infectious particles and are substantially free of cellular debris and other contaminants. Desirable properties include the following:

<dl><dt>• </dt><dd>A density of at least 106, preferably at least about 108, more preferably at least about 1010 IU / ml, determined in a TCID50 assay. </dd></dl>

<dl><dt>• </dt><dd>A ratio of adenoviral DNA to total protein or adenoviral hexon indicating that at least 10%, preferably at least about 50%, more preferably at least about 80% of the viral particles contain adenoviral DNA. </dd></dl>

<dl><dt>•</dt><dd> Less than 20%, preferably less than about 10%, more preferably less than about 1% contamination by non-adenoviral material at the protein or DNA level, detected by protein stained SDS gels or digestion agarose gels with restriction nuclease stained with ethidium bromide. </dd></dl>

<dl><dt>•</dt><dd> A total of at least 109, preferably at least about 1011, more preferably at least about 1013 IU per production lot. </dd></dl>

[0105] Auxiliary virus can be prepared in any cell that is permissible for viral replication. For adenovirus, preferred cells include 293 cells and HeLa cells. Traditionally, when these cells have been used for adenovirus replication, they have been used in plaque cultures. However, as shown in Example 4, these procedures generally support the replication of thermosensitive adenoviruses at levels that are one or two smaller logarithmic units than for wild type adenoviruses.

[0106] Therefore, it is preferable to employ culture techniques that allow an increase in planting density. 293 cells and variants of HeLa cells that have adapted to the suspension culture are available. HeLa is preferable for reasons of cell growth, viability and morphology in suspension. As shown in Example 5, these cells can grow at sufficient density (2 x 106 per ml) to compensate for the lower replication rate of the thermosensitive adenovirus strain. Once established, the cells are infected with the virus and grown at the permissive temperature for a sufficient period, generally 3-7 days and typically approximately 5 days.

[0107] Tangential flow filtration is a technique used in the art to process large volumes of mammalian cells in order to perfuse, concentrate and collect them. See, for example, Dorin et al.,

Biotechnol Prog. 6: 494, 1990; Maiorella et al., Biotechnol. Bioeng 37: 121, 1991. It is recommended to use this technique with suspension cultures for the preparation of auxiliary virus for use in the practice of the present disclosure that includes the present invention. Example 5 demonstrates that HeLa cells withstand shear forces of 750-1500 s-1, allowing cell concentration and diafiltration of depleted media.

[0108] The culture virus is harvested from depleted media or by microfluidification of the cells. The level of auxiliary virus produced in the culture is typically at least 107 IU / ml, and preferably at least about 3 x 107 IU / ml.

[0109] The auxiliary virus prepared according to the above description can be used directly to infect host cells used for AAVr replication. More commonly, the virus is isolated and concentrated before use. Current procedures for purifying and concentrating auxiliary viruses typically involve isopycnic gradients of CsCl. This procedure consumes time and labor, requires numerous open processing stages and is difficult to scale up. Instead, purification by chromatography is recommended. The reader refers in general to Prior et al., Pharmaceut. Technol 19: 30, 1995 and Huyghe et al., Human Gene Therapy 6: 1403, 1995. Particularly preferred for the isolation of thermosensitive strains of adenovirus is anion exchange chromatography, especially in a polyethyleneimine resin using a continuous gradient of NaCl a pH 7.4. A detailed illustration of the separation process with polyethyleneimine is provided in Example 6.

Provision of a host cell comprising auxiliary virus and AAV

[0110] Several criteria influence the selection of cells for use in the production of AAVr particles as described herein. As an initial matter, the cell must be permissive for replication and packaging of the AAVr vector when the selected auxiliary virus is used. However, since most mammalian cells can be productively infected by AAV, and many can also be infected by helper viruses such as adenovirus, it is clear that a wide variety of mammalian cells and cell lines effectively meet these criteria. Among these, the most preferred cells and cell lines are those that can easily grow in culture to facilitate large-scale production of recombinant AAV vector preparations. However, again many of these cells effectively satisfy this criterion. When large-scale production is desired, the choice of the production method will also influence the selection of the host cell. For example, as described in more detail below and in the art, some production techniques and culture vessels or chambers are designed for the growth of adherent or bound cells, while others are designed for the growth of suspended cells. In the latter case, the host cell would therefore preferably be adapted or adaptable to the growth in suspension. However, even in the case of cells and cell lines that are considered adherent or dependent on the anchor, it is possible (as described below) to derive variants adapted to the suspension of a parental line dependent on the anchor by serial cell selection capable of growth in suspension.

[0111] When a thermosensitive auxiliary virus is used, the cell must be able to efficiently replicate the AAVr vector under conditions that are not permissible for replication of the helper virus. By way of illustration, when using ts149 adenovirus as a ts helper virus (as described and illustrated below), the cell should be able to support AAVr replication and packaging at temperatures well above 32 ° C, preferably about 39 , 5 ° C. Human 293 cells are an example of a cell line that satisfies these criteria, but numerous other cells and cell lines are capable of replicating AAVr at this relatively high temperature.

[0112] Ultimately, the helper virus, the AAVr vector sequence and all AAV sequences necessary for replication and packaging must be present in the same cell. When one or more AAV packaging genes are provided separately from the vector, a host cell is provided comprising: (i) one or more AAV packaging genes, in which each of said AAV packaging genes encodes a protein AAV replication or encapsidation; (ii) a heterologous polynucleotide introduced into said host cell using an AAVr vector or provider, wherein said AAVr vector or provider comprises said heterologous polynucleotide flanked by at least one AAV RTI and is deficient in said packaging gene or genes AVV and (iii) an auxiliary virus or sequences encoding the necessary auxiliary virus functions. It should be noted, however, that one or more of these elements can be combined into a single replicon. By way of illustration, an auxiliary virus may also comprise an AAVr provider or an AAV packaging gene.

[0113] The auxiliary virus is preferably introduced into the cell culture at a level sufficient to infect the majority of cells in culture, but can be kept to a minimum to limit the amount of auxiliary virus present in the resulting preparation. A multiplicity of infection or "MOI" of 1-100 may be used, but an MOI of 5-10 is typically suitable.

[0114] Similarly, if the AAV vector and / or packaging genes are introduced transiently into the packaging cell (as opposed to being stably introduced), they are preferably introduced at a level sufficient to genetically alter most of the cells in culture. The quantities required are generally of the order of 10 μg per 106 cells, if supplied as a bacterial plasmid, or 108 particles per 105 cells if supplied as AAV particle. The determination of the optimal amount is a routine titration exercise that is within the skill of the expert.

[0115] These elements can be introduced into the cell simultaneously or sequentially in any order. When the cell is inherited by any of the elements, the cell can be selected and allowed to proliferate before introducing the next element.

[0116] In a preferred case, for example in a preferred embodiment, the auxiliary virus is finally introduced into the cell to recover and package a resident AAVr vector. The cell will generally be already supplemented to the extent necessary with AAV packaging genes. Preferably, the AAVr vector or packaging genes, and more preferably both, are stably integrated into the cell. It is readily appreciated that other combinations are possible. Such combinations are included within the scope of the invention.

[0117] Once the necessary elements are provided to the host cell, the cell is cultured under conditions that are permissible for AAV replication, to allow replication and packaging of the AAVr vector. The cultivation time is preferably adjusted to correspond to the maximum production levels, and is typically 3-6 days. Preferably, at least 100 viral particles are produced per cell; more preferably at least about 1000 per cell, even more preferably at least about

10,000 per cell. Preferably, at least 0.5 x 106, more preferably at least about 1 x 106, even more preferably at least about 2 x 106 UR / ml of AAV vectors per 2 x 105 cells are produced during the culture period. Optionally, large-scale production methods such as suspension culture and tangential flow filtration can be used. The AAV particles are then collected and isolated from the cells used to prepare them.

[0118] The AAVr particle preparations of the present disclosure preferably comprise a high density of infectious AAV particles and are substantially free of helper virus, helper virus proteins and cellular debris and other contaminants. Desirable properties include the following:

<dl><dt>•</dt><dd>A concentration of at least 107, preferably at least about 108, more preferably at least about 1010 UR / ml, determined in a replication assay or comparison of quantitative hybridization with a known standard. </dd></dl>

<dl><dt>•</dt><dd> No more than 103, preferably no more than about 102, more preferably no more than about 10 infectious particles of auxiliary virus per 108 UR of AAVr particles. </dd></dl>

<dl><dt>•</dt><dd> Less than 5%, preferably less than about 1%, more preferably less than about 0.01%, even more preferably less than about 0.001% contamination by protein-based auxiliary virus (w / w), detected by densitometric analysis of SDS gels or by helper virus specific protein immunoassay (such as hexon fiber or adenovirus penton). </dd></dl>

<dl><dt>•</dt><dd> Less than 5%, preferably less than about 1%, more preferably less than about 0.01%, even more preferably less than about 0.001% contamination by helper virus or cellular protein (w / w), detected by densitrometric analysis of SDS gels or by immunoassay for helper viruses or cell-specific proteins. </dd></dl>

<dl><dt>•</dt><dd> Preferably, the preparation is also substantially free of other potential cellular components such as lipids, carbohydrates and / or cellular nucleic acids. </dd></dl>

[0119] The procedures exhibited in this disclosure are suitable for preparing small experimental batches or preparative batches of 10-100 or more. For large-scale batch preparations, the following property is also desirable:

<dl><dt>• </dt><dd>A total of at least 1010, preferably 1012 and more preferably 1014 UR of AAV vector particles in the preparation. </dd></dl>

[0120] Optionally, the AAVr vectors can be further processed to enrich AAVr particles, deplete the auxiliary virus particles or otherwise make them suitable for administration to a subject. Purification techniques may include isopycnic gradient centrifugation and chromatographic techniques. Reduction of infectious auxiliary virus activity may include inactivation by heat treatment or by pH treatment as is known in the art. Other processes may include concentration, filtration, diafiltration or mixing with a suitable pharmaceutical buffer or excipient. The preparations can be divided into unit dose and multiple dose aliquots for distribution, which will retain the essential characteristics of the batch, such as the homogeneity of the antigenic and genetic content, and the relative proportion of contaminating auxiliary virus.

[0121] Exemplary techniques for generating auxiliary virus and AAV preparations that exhibit various desirable properties as described above are provided in the following sections and subsequent examples.

[0122] Various methods for the determination of the infectious titer of a viral preparation are known in the art. However, a high performance titration assay as provided herein is a preferred method for the determination of the title. In an exemplary high yield titration assay, a series of culture wells are established each comprising an aliquot of mammalian cells and an aliquot of viral preparation (as well as control wells comprising, for example, single cells, viruses alone and nothing). The series of culture wells may be, for example, in the form of a microtiter vessel. Typically, aliquots (for example, serially diluted aliquots) of the viral preparation are added to the holder, and then the cells and viruses are incubated under conditions that allow virus replication (typically, growth conditions suitable for the cell mammalian host). After virus replication, viral nucleic acid is generally released by lysis of mammalian cells (using conditions or agents that promote lysis as necessary). In preferred cases, the nucleic acid (including viral nucleic acid) in the multiplicity of lysates is transferred and fixed to a membrane under conditions of nucleic acid binding (washing as appropriate to remove proteins and other contaminants). The membrane is preferably a duplicate or mirror image of the crop series in which the individual wells of the original series are subsequently represented by "sets" of nucleic acid (from the lysate of each culture well) that are joined at corresponding positions in the membrane Hybridization of the membrane can then be used with a virus-specific labeled probe (or viral insert specific) to identify and quantify the relative amount of specific or viral nucleic acid at each point in the series, and correspondingly, in each of the original culture wells. The conditions and materials for transfer, binding, washing and hybridization of nucleic acid can be adapted from routine molecular biology techniques such as "point transfer" hybridization (as described in the art, see for example molecular biological techniques in Sambrook et al., supra and Ausubel et al., supra). Illustrative applications of these techniques are presented below.

[0123] These procedures therefore provide a high performance infectivity assay that can be used in the determination of the infectious titer of a viral preparation. As shown in Example 4, the viral titers determined by this rapid and quantitative procedure correspond closely with the titers determined by more classical techniques. However, in addition, this high-performance procedure allows the simultaneous processing and analysis of many viral replication reactions and therefore has many other uses including, for example, screening of permissive or non-permissive cell lines of viral replication and infectivity, as well as the screening of agents that affect viral replication, as discussed further below.

Production techniques and purification of auxiliary viruses preferred for use in the present invention

[0124] In various preferred aspects of the present disclosure, production and purification procedures are employed for the generation of auxiliary viruses suitable for use in the production of AAVr vectors as described herein. It is an auxiliary virus commonly used for the production of AAV adenovirus, typically Ad5, although other auxiliary viruses can also be employed as discussed herein and in the art.

[0125] For the purpose of illustration, it is convenient to divide the examination of virus production and purification into the "anterior" and "posterior" phases. The "above" process generally designates the production of the virus in suitable host cells and the release or removal of the virus from the cells to produce a "crude" virus preparation such as a lysate. "Subsequent" processing can be used to purify the crude virus preparation (for example, to isolate it from cellular proteins and / or other contaminants).

[0126] A variety of techniques are known for the production and processing of auxiliary viruses, including adenovirus (eg, centrifugation in CsCl, as well as other techniques such as those described in WO 96/27677). The auxiliary virus produced using such techniques can then be employed in the production of AAVr vectors as described herein.

[0127] The following sections describe, for the purpose of illustration, techniques that can be used for the production of adenovirus, although other techniques are known in the art and can be used herein.

(i) Previous auxiliary viruses

[0128] The auxiliary virus, such as Ad5, can be easily produced by infecting mammalian cells (eg, human cells). In the illustrative examples described below, cells grow in media and culture vessels suitable for host cell growth, are concentrated before infection and then infected with helper virus (eg, at an MOI of 1-5 ) with gentle agitation. After infection, the cells can be resuspended in fresh medium and incubated for an additional period of time (typically about 2 days) to allow replication and packaging of the helper virus. After incubation, cells can be collected and lysed to release the helper virus. After lysis, the cell lysate is preferably treated with a nuclease to degrade the free nucleic acid (eg, cell nucleic acid) without degrading the nucleic acid that is encapsidated into viral particles. The lysate can be clarified (for example, by filtration and / or centrifugation) and can also be subjected to additional purification techniques to purify and concentrate the auxiliary virus in the preparation as described and illustrated below.

[0129] As an illustrative example of said process, cells can grow in media at a density of approximately 1 x 10 6 cells / ml in a container such as a shake flask. After incubation, the cells can then be concentrated to approximately 107 cells / ml and infected with Ad5 at 1-2 infectious units / cell with gentle agitation. The cells can then be resuspended in medium at approximately 106 cells / ml and allowed to produce virus during an incubation period of approximately 2 days. The cells can then be collected, resuspended in medium or buffer (for example, at about 5 x 10 6 cells / ml) and then disintegrated, for example by mechanical lysis such as passing through a microfluidizer at 55.16 MPa or equivalent technique ( for example, lyophilization or sonication). The lysate can be treated with a nuclease (for example, benase) for 1 hour at 37 ° C. The lysate can be clarified through a filter, such as a 1.0 µm filter, or by centrifugation. Analogous techniques and modifications thereof are described below.

(ii) Subsequent auxiliary viruses

[0130] Preferred techniques for the subsequent processing of auxiliary viruses, such as adenovirus, employ ion exchange chromatographic procedures for purification of auxiliary virus.

[0131] By way of illustration, the adenovirus filtrate can be loaded as described above in an anion exchange resin, such as an N-charged amino or imino resin (eg, POROS 50 PI, or any DEAE based resin , TMAE, tertiary or quaternary amine or PEI) on a buffer-balanced chromatography column (such as TMEG, also referred to herein as the chromatography buffer

A: 50 mM Tris (pH 8.0), 5 mM MgCl 2, 1 mM EDTA, 5% glycerol).

[0132] The column can then be washed with multiple volumes of TMEG column (for example, 5-6 volumes), followed by multiple volumes of saline wash (for example, 5-6 volumes of TMEG with 800 mM NaCl (chromatography buffer) B: 60% TMEG and 40% TMEG with 2M NaCl) The adenovirus can be eluted with TMEG with 1300 mM NaCl (35% chromatography buffer A and 65% chromatography buffer B).

[0133] The adenovirus peak can be identified in the fractions by an infectivity test or by nucleic acid hybridization or immunoassay, as described in the art. The peak can be sterilized by filtration through a sterile 0.2 μm filter. Optionally, the peak can be concentrated by tangential flow filtration, for example in a Filtron Ultrasette or Millipore Pellicon unit. The peak or concentrate can be diafiltered in this system in a suitable buffer such as PBS + 5% sucrose. Alternatively, the adenovirus can be left in the elution buffer. The final adenoviral product can be sterilized by filtration through a 0.2 μm filter and stored for use. As described and illustrated herein, a thermosensitive auxiliary virus (such as a thermosensitive adenovirus) can also be employed.

[0134] Examples which describe the preparation and use of said auxiliary viruses are given below for further illustration.

AAV production and purification techniques preferred for use in the present invention

[0135] As with the auxiliary viruses, it is convenient for purposes of illustration to divide the examination of the production and purification of AAV into "previous" and "later" process phases, designating the "previous" process generally the production of AAV in cells suitable hosts and the release or removal of the virus from the cells to produce a "crude" AAV preparation. "Subsequent" processing can be used to purify the AAV preparation (for example, to isolate the AAV from cellular proteins and / or other contaminants).

[0136] In preferred aspects of the present disclosure, for example in preferred aspects of the present invention, anterior and subsequent AAV processing is performed in a manner designed to substantially reduce and / or eliminate contaminating cellular proteins, as well as any virus. contaminating auxiliary (for example Ad) or auxiliary virus proteins, any of which could contribute to the triggering of an immune response if they were present at substantial levels in the preparation of final AAVr vector to be used for gene transfer.

[0137] The following sections describe, for purposes of illustration, techniques that can be used for the production of AAV.

(i) Previous AAV processing

[0138] The AAV vector can be produced from a mammalian cell line that contains the necessary AAV packaging genes (eg, a rep and cap AAV gene), a recombinant AAV (AAVr) provider comprising a non-AAV heterologous polynucleotide flanked by at least one inverted terminal repeat (RTI) of AAV and an auxiliary AAV virus (eg, an adenovirus). These components can be introduced into the cell in a variety of configurations, as described above and illustrated below. Since AAV can be replicated and packaged in a variety of mammalian cells, there are a large number of cell lines that can be modified and used for the production of AAV.

[0139] By way of illustration, the AAV vector can be produced from a cell line such as "C12" (as described by KR Clark et al., Gene Therapy, 3: 1124-1132, 1996) or the line "C137.5" (described in a pending and common property application herewith by Targeted Genetics Corporation, J. Allen et al. WO 96/17947), which has been engineered to contain a rep construct and / or cap, as well as a vector construct. Optionally, a cell line such as C12 or c137 containing a rep and / or cap construct can be transfected with a plasmid containing a vector construct such as ptgAAV-CF. Or a cell can be transfected with a plasmid containing rep and cap, such as pRS5, as well as a plasmid containing a vector construct. The cell can be infected with adenovirus or transfected with DNA that contains adenoviral genes.

[0140] A variety of such "AAV" producing cells can be generated, as described in the references cited herein and in the art.

[0141] AAV producing cells can grow under conditions (including media, temperature and the like) that are generally suitable for the growth of mammalian cells, and that are also generally permissible for AAV replication. For example, the DMEM / F12 suspension medium is preferred for cell growth and the DMEM medium is preferred only for the production of AAV vector. As is known in the art, some cell types and cell lines tend to be binding dependent, while others are capable of suspension growth and many binding dependent cells can also "adapt" to suspension growth by cycling the cells in suspension conditions as a means to enrich in and ultimately select variants that are capable of growth in suspension. Accordingly, the growth of cells for the production of AAV can be carried out in any of a variety of containers, depending in part on whether the selected production line is relatively binding dependent or suspension adapted. Such containers for the growth of binding dependent cells include, for example, tissue culture flasks, rotating flasks, microcarriers and bioreactors (such as hollow fiber, packed bed or fluidized bed bioreactors). Containers for suspension adapted mammalian cell lines include, for example, stirring flasks, tank reactors and air stirring fermenters.

[0142] AAV replication takes place over a period of time as well as at a point in the growth cycle where viral production is optimal, preferably in medium to late logarithmic growth (typically one to three days), after whose time the cells can be collected and lysed to release the progeny virus. For example, cells can be resuspended in growth media at approximately 110x106 cells / ml and allowed to produce for 48 hours. The cells can then be collected (for example, by centrifugation) and resuspended in buffer (for example, TMEG (or "chromatography buffer A"): 50 mM Tris, pH 8, 0.5 mM MgCl 2, 1 mM EDTA, 5% of glycerol) at about 1-10 x 106 cells / ml.

[0143] AAV can replicate to a high number of copies (eg, 105-106 genomes / cell) in transduced cells if the AAV Rep proteins and necessary auxiliary virus functions are provided with relative simultaneity. If Cap proteins are also provided, AAV particles are assembled in the nucleus of infected cells, where they tend to assemble in crystalline series. The first stage in product recovery is therefore cell disintegration. Although lyophilization and / or sonication can be used to disintegrate cells (as with adenovirus), such techniques are not very suitable for large-scale preparation. Therefore, mechanical lysis is preferable in this regard, using techniques such as microfluidification. Detergents and other chemical agents can also be used to mediate or facilitate lysis. Treatment of lysates with nucleases (such as benzonease) has been found to be helpful in reducing viscosity and improving filterability. Clarification, for example by microfiltration to separate the vector from at least a portion of the cellular waste, is also helpful in promoting recovery and purification.

[0144] By way of illustration, the cells can be mechanically lysed after the incubation period by sequentially passing through a microfluidifier (typically approximately at 55.16 MPa using two passes). Other commonly employed techniques include lyophilization and sonication cycling, as is known in the art. The lysate can also be treated with a nuclease to degrade nucleic acid (such as cellular or viral nucleic acid) that is not effectively "protected" by being packaged in a viral particle. Gasoline digestion is typically employed for approximately 1 hour at 37 ° C. The lysate can also be clarified. Procedures for clarification include passing through a filter, such as a 1.0 μm filter, and centrifugation.

[0145] Tangential flow filtration (FFT) can be beneficially used to process and collect large volumes of cells. FFT can be used to perfuse, concentrate and collect animal cells. For example, FFT can be used to process cells under laminar flow conditions at average wall shear rates of up to 3000 per second (see, for example, Maiorella, B., et al., Biotechnology and Bioengineering, 37: 121- 126, 1991). Large-scale virus concentration using FFT ultrafiltration has been described by R. Paul et al. Human Gene Therapy, 4: 609-615, 1993.

[0146] Illustrative production routines that employ such techniques are described below.

(ii) Previous processing of AAV

[0147] As described above, it would be particularly advantageous to obtain AAV preparations that were substantially free of auxiliary virus particles (such as Ad particles). In addition, AAV vector preparations will also preferably be free of helper and cellular virus proteins (which can also be immunogenic). However, there is an additional group of limitations that influence the suitability of AAV production techniques. Namely, to be particularly useful for the production of AAV for gene therapy, it is most desirable that the techniques be "scalable", specifically applicable together with large-scale manufacturing devices and procedures. This last group of limitations effectively reduces or eliminates the usefulness of available standard techniques such as separation with cesium chloride (which is not well suited for large-scale preparation procedures).

[0148] A combination of procedures that are both scalable and remarkably effective for the generation of AAV preparations that are substantially free of auxiliary virus particles, as well as auxiliary and cellular virus proteins and other such contaminants, has been discovered. The preferred combination of procedures employs ion exchange chromatographic procedures, which contrasts with the various procedures mentioned in the art for the potential purification of, for example, AAV or Ad. In particular, such procedures as described in the art typically employ a single type of ionic separation, sometimes in combination with other kinds of chromatographic procedures (see, for example, K. Tamayose et al., Human Gene Therapy 7: 507-513 (1996) and WO96 / 27677, September 12, 1996). However, in the case of AAV production, it has been found that a combination of opposite sequential ion exchange chromatography is particularly effective for the generation of AAV preparations that are substantially free of helper virus particles and proteins, as well as of cellular proteins

[0149] In view of these findings, it seems that AAV is not only "adapted" to both anionic and cationic exchange chromatography, but that such combination of both opposite ion exchanges is particularly effective in removing all particulate contaminants and proteins that typically appear in the generation of AAV vector preparations. Any of a variety of cationic and anionic exchange resins may be employed in conjunction with these procedures, whose fundamental properties are the availability of negatively and positively charged groups, respectively, with which AAV can bind at least to some degree (most preferably in a degree that differs substantially from the relative binding affinity of one or more of the aforementioned contaminants, namely Ad particles and proteins, as well as mammalian cell proteins). Without wishing to link to any theory, it is believed that the anion exchange stage is particularly effective in separating AAV from adenovirus, while both stages (but especially the cation exchange stage) are believed to be particularly effective in separating AAV from cellular proteins Anion exchange has also been used followed by tangential flow filtration, as described and illustrated below. As described further below, it has been found that AAV preparations can be largely concentrated by heparin sulfate chromatography.

[0150] By way of illustration, a clarified AAV lysate can be loaded, as described above, on a positively charged anion exchange column such as an N-charged amino or imino resin (eg, POROS 50 PI or any resin based on DEAE, TMAE, tertiary or quaternary amine or PEI) or a negatively charged cation exchange column (such as HS, SP, CM or any cationic resin based on sulfur, phosphorus or carboxyl). The column can be washed with a buffer (such as the A / TMEG chromatography buffer). The column can be eluted with a gradient of increasing NaCl concentration and the fractions can be collected and tested for the presence of AAV and / or contaminants.

[0151] Other methods may be used instead of, or preferably in addition to, the above-described anionic and cationic exchange procedures, based on intermolecular associations mediated by features other than the charge as are known in the art. Such other procedures include intermolecular associations based on ligand-receptor pairs (such as antibody-antigen interactions or lectin carbohydrate), as well as separations based on other attributes of the molecules, such as molecular sieve chromatography based on size and / or shape. To take just one example, a filtered or partially purified AAV preparation can be loaded on a column containing an AAV specific antibody. This column can join AAV. The column can be clarified with buffer to remove the recombinant proteins and then eluted with a gradient or step of increasing NaCl concentration, and the fractions can be collected. Alternatively, said column can be eluted with a different pH buffer than that of the loading buffer.

[0152] The peaks of AAV and adenovirus can be identified in the fractions by infectivity assays

or by nucleic acid hybridization or immunoassays. The peaks can be grouped and the whole can be diluted or dialyzed or diafiltered with a buffer (eg, TMEG or equivalent) to reduce the salt concentration.

[0153] This assembly may be injected into a positively charged anion exchange column and / or a negatively charged cation exchange column (such as those designated above). The column can be washed with a buffer (such as A / TMEG chromatography buffer). The column can be eluted with a gradient of increasing NaCl concentration and the fractions can be collected. The AAV and adenovirus peaks can be identified in the fractions by an infectivity test or by a nucleic acid hybridization or immunoassay. The peaks can be grouped based on the results of any of these tests.

[0154] The set of fractions containing AAV eluted from an anion exchange column as described above can be concentrated and purified by tangential flow filtration (FFT), for example, in a Filtron Ultrasette or Millipore Pellicon unit. A suitable molecular weight cutting membrane (such as a 100,000 or 300,000 cut) is typically composed of a polymer such as a regenerated cellulose or polyethersulfone. The preparation is filtered through the membrane and the product is retained. The retained material can be diafiltered using the membrane with successive washes with a suitable buffer such as Ringer's balanced salt solution + 5% glycerol. The final sample is highly enriched in the product and can be sterilized by filtration through a 0.2 μm filter and stored for use.

[0155] In the purification and concentration of AAV by tangential flow filtration of post-column anion exchange material, the 300,000 molecular weight cutting membrane has resulted in higher yields of replicative units than the molecular weight cutting membrane of 100,000

[0156] An additional step that can be used for adenovirus removal, if desired, involves treating the set of eluents with a thermal inactivation step (as described herein) and then filtration (for example, before submitting FFT preparation). However, it has been found that the "anion exchange to FFT" procedure described above resulted in a preparation of AAV that was free of detectable adenoviruses and resulted in better yields of purified AAV.

[0157] Illustrative production routines that employ such techniques are described below.

Alteration of the growth conditions of AAV producing cells to enhance production

[0158] During the course of production tests with AAV in various media and culture vessels, cultures were typically monitored for various growth and / or metabolic parameters such as cell density, glucose and amino acid availability and production of metabolic by-products such as ammonia and lactic acid. Such components can be easily monitored using standard techniques such as HPLC and enzymatic assays, as described in the art.

[0159] As described in the following examples, it was discovered that certain amino acids, particularly aspartate and glutamate, were rapidly depleted in both batch and infusion cultures. Moreover, in various experiments in batches and perfusion it was observed that substantially 90 to 99% of the available asp and glu is removed after 24 to 48 hours in said cultures. Since the levels of asp and glu appeared to be suboptimal in said media, additional amounts of either or both amino acids were therefore provided. Crop maintenance and optimization techniques such as these have been routinely applied in the context of large-scale bioproduction (see, for example, Glacken, MW. Et al., Biotechnology and Bioengineering, 28: 1376-1389, 1986; Glacken, MW, Bio / Technology 6: 1041-1050, 1988; Bibila, TA, et al., Biotechnol. Prog., 10: 87-96, 1994 and Borys, MC, et al., Biotechnology and Bioengineering, 43: 505-514, 1994).

[0160] Surprisingly, the replacement of these depleted amino acids resulted in a sharp drop in AAV production. For example, in the experiments described below, supplementing the standard medium (DMEM) with additional asp and glu reduced production efficiency by more than an order of magnitude (from approximately 1800 DNase resistant particles (PRA) per cell to approximately 140 PRA per cell), although viability was slightly enhanced.

[0161] Another common component of the means for the growth of mammalian producing cells is the serum component, such as fetal bovine serum (FBS), which is typically included in the media at a level of about 10%. As described below, when the serum level for AAV production was increased (to 20%), AAV vector production fell more than 2 times. In contrast, when cells were subjected to increasingly lower serum levels, the production of AAV vector increased dramatically. For example, when serum levels were reduced to one tenth of normal starting levels (specifically 1%), vector production increased more than 4 times.

[0162] Without wishing to link to any theory, it seems now that stressing the producing cells, metabolically

or by other means as described below, it can dramatically enhance the production of AAV vector.

[0163] Stress can be effectively characterized, and tested, based on the negative effect of the stressful condition or stressful agent on cell growth and / or metabolism. In fact, stress can be achieved by introducing any condition or agent that inhibits cell growth and / or metabolism, or by altering the level of a pre-existing condition or agent such that it becomes suboptimal with respect to cell growth and / or metabolism. . A wide variety of such conditions are known and / or evident including nutritional stress (one or more nutrients present at suboptimal levels for growth and / or metabolism), thermal stress (suboptimal temperature, which may include cell growth at lower temperatures or major, or subject the cells to temporary thermal shocks such as a cold shock or hot shock), osmotic stress (suboptimal osmotic level, which can be hypoosmotic or hyperosmotic), pH stress (suboptimal pH that can be acidic or alkaline), aeration stress (for example, suboptimal oxygen levels or gas exchange), mechanical stress (for example, shear stress as occurs in culture mixing), stress of radiation and toxic stress (presence of one or more chemicals or other agents that inhibit growth and / or metabolism). With most, if not all such agents and conditions, it is possible to subject the cells to stress continuously or temporarily. By way of illustration, in the case of thermal stress, the cells can grow at temperatures that are above or below the optimum (typically, the optimum is approximately the normal body temperature of the animal from which the cells are derived), or the cells they can undergo a temporary thermal shock, such as a cold shock or a hot shock. Currently preferred examples of such stress conditions include: nutritional stresses such as amino acid or serum limitation, alteration of aeration and agitation levels, alteration of osmotic levels (for example, using non-metabolizable carbohydrates such as sorbitol) and inclusion of chemical agents such as saturated aliphatic carboxylic acids (for example , propionic, butyric, isobutyric, valeric and caproic acids and their salts with organic or inorganic bases), N, N'-diacylated diamines (such as pentamethylene bisacetamide, hexamethylene bisacetamide and heptamethylene bisacetamide), organic sulfur compounds (such as dimethylsulfoxide) and glucocorticoids (such as hydrocortisone, dexamethasone, prednisolone, aldoesterone and triazonolone. Other such agents include genotoxic agents such as chemical carcinogens, UV, hot shock, metabolic inhibitors of DNA synthesis (for example, hydroxyurea, methotrexate, afidicoline, drugs that affect topoisomerases (for example, amsacrine, camptecine, etoposide and novobiocin).

[0164] As noted above, producer cells can also undergo sublethal stress by altering the pH. As exemplified below, it was found that induced pH stress by raising the pH of the medium not only increased the AAV, but also caused a drastic shift in the relative proportions of AAV that were released into the culture medium. As further described below, this technique can therefore be used to facilitate the purification of AAV as well as to enhance production.

[0165] Illustrative procedures are provided below to optimize the production of AAVs using various stress conditions, since these are results that demonstrate that the application of a variety of different stress conditions can be used to effectively boost AAV production levels.

[0166] The methods of the present invention that involve incubating an AAV producing cell under conditions that are permissible for AAV replication and that comprise inducing sublethal stress in the AVV of the present invention are set forth in the appended claims.

Use of AAVr for gene therapy

[0167] Vector compositions comprising polynucleotides with a therapeutically relevant genetic sequence are described herein. The AAV viral vectors of this disclosure can be used for administration to an individual for purposes of gene therapy. Suitable diseases for gene therapy include, but are not limited to, those induced by viral, bacterial or parasitic infections, various malignancies and hyperproliferative conditions, autoimmune conditions and congenital deficiencies.

[0168] Gene therapy can be performed to enhance the level of expression of a particular protein within or secreted by the cell. The vectors of this disclosure can be used to genetically alter cells for gene labeling, replacement of a missing or defective gene or insertion of a therapeutic gene. Alternatively, a polynucleotide can be provided to the cell that reduces the level of expression. This can be used for the suppression of an undesirable phenotype, such as the product of an amplified or overexpressed gene during the course of a malignancy, or a gene introduced or overexpressed during the course of a microbial infection. Expression levels can be reduced by providing a therapeutic polynucleotide comprising a sequence capable of forming, for example, a stable hybrid with the target gene or RNA transcript (antisense therapy), capable of acting as a ribozyme to cleave the relevant or capable mRNA. act as a claim for a target gene product.

[0169] Of particular interest is the correction of the genetic defect of cystic fibrosis by providing a regulator of cystic fibrosis transmembrane conductance (CFTR) that is properly functional to the respiratory epithelium. Afione et al. (J. Virol. 70: 3235, 1996) and Conrad et. to the. (Gene Therapy: in press. 1996) have shown a stable in vivo gene transfer of CFTR to primate lung using single dose AAV vectors. There is a variety of CFTR polypeptides that are capable of reconstructing functional deficiencies of CFTR in cells derived from cystic fibrosis patients. Rich et al., Science, 253: 205 (1991) described a CFTR derivative in which amino acid residues 708-835 were missing that was capable of transporting chloride and capable of correcting a CFTR defect of natural origin. Egan et al. Nature, 358: 581 (1992) described another CFTR derivative (comprising approximately 25 amino acids of an unrelated protein followed by the native CFTR sequence from residue 119) that was also capable of restoring the electrophysiological characteristics of normal CFTR. Arispe et al., Proc. Natl. Acad. Sci. USA 89: 1539 (1992) showed that a fragment of CFTR comprising residues 433-586 was sufficient to reconstitute a correct chloride channel in lipid bilayers. Sheppard et al., Cell 76: 1091 (1994) showed that a truncated CFTR polypeptide in residue 836 at approximately half its length was still capable of forming a regulated chloride channel. Therefore, AAV vectors with native CFTR protein coding sequences and mutants and fragments thereof are all preferred cases of this disclosure.

[0170] Also of particular interest is the correction of the p53 tumor suppressor gene, locally defective in certain types of tumor, by supplying a properly functional p53 gene to the tumor site (Huyghe et al., Human Gene Therapy 6: 1403,1995).

[0171] The compositions of this disclosure can be used in vivo as well as ex vivo. In vivo gene therapy comprises administering the vectors of this disclosure directly to a subject. The pharmaceutical compositions can be supplied as liquid solutions or suspensions, as emulsions or as solid forms suitable for dissolution or suspension in liquid before use. For administration to the respiratory tract, it is a preferred mode of aerosol administration, using a composition that provides a solid or liquid aerosol when used with an appropriate aerosolizing device. Another preferred mode of administration to the respiratory tract is using a flexible fiber optic bronchoscope to instill vectors. Typically, viral vectors are in a pharmaceutically acceptable pyrogen-free buffer such as Ringer's balanced salt solution (pH 7.4). Although not necessary, pharmaceutical compositions can optionally be supplied in unit dosage form suitable for administration of a precise amount.

[0172] An effective amount of virus is administered depending on the goals of the treatment. An effective dose can be obtained in single or divided doses. When a low percentage of transduction can cure a genetic deficiency, then the goal of treatment is generally to meet or exceed this level of transduction. In some cases, this level of transduction can be achieved by transducing only about 1 to 5% of the target cells, but it is more typically 20% of the cells of the desired tissue type, usually at least about 50%, preferably at least about 80%, more preferably at least about 95% and even more preferably at least about 99% of the cells of the desired tissue type. As a guide, the number of vector particles present in a single dose attempted by bronchoscope will generally be at least about 1 x 108, and is more typically 5 x 108, 1 x 1010 and sometimes 1 x 1011 particles, including both DNase resistant particles as DNase sensitive. In terms of DNase resistant particles, the dose will generally be between 1 x 106 and 1 x 1014 particles, more generally between about 1 x 108 and 1 x 1012 particles. The treatment may be repeated as often as every two to three weeks, as necessary, although one treatment may be sufficient once every 180 days.

[0173] The effectiveness of the genetic alteration can be monitored by several criteria. Samples removed by biopsy or surgical excision can be analyzed by in situ hybridization, PCR amplification using specific vector probes, RNAse protection, immunohistology or cell count by immunofluorescence. When the vector is administered by bronchoscopy, pulmonary function tests can be performed, and the presence of inflammatory cytokines can be assessed in bronchial lavage. In the treated subject the clinical features can also be monitored, and determine whether the cells express the function that is intended to be transmitted by the therapeutic polynucleotide.

[0174] The decision of whether to use therapy in vivo or ex vivo, and the selection of a particular composition, dose and route of administration will depend on a number of different factors including, but not limited to, the features of the condition and the subject that It is being treated. The assessment of these traits and the design of an appropriate therapeutic regimen are ultimately the responsibility of the prescribing physician.

[0175] The above description provides, among others, methods for generating high-titre preparations of recombinant AAV vectors that are substantially free of helper viruses (eg adenovirus) and cellular proteins.

[0176] The examples presented below are provided as additional guidance for a person skilled in the art and are not intended to be limiting in any way.

EXAMPLES

EXAMPLE 1

Illustrative production of recombinant AAV vector using a wild-type auxiliary virus (Ad5) and a thermosensitive auxiliary virus (Ad TS149)

[0177] This example illustrates the use of a wild-type auxiliary virus (Ad5) and a thermosensitive auxiliary virus (Ad ts149) to provide auxiliary functions for the replication of a recombinant AAV vector particle comprising a model therapeutic gene.

[0178] The ptgAAVCF plasmid consists of the left AAV2 RTI, a complete cystic fibrosis transmembrane regulator cDNA, a synthetic polyadenylation sequence based on the mouse -globin polyadenylation sequence, AAV2 sequences in the 3 'direction of the cap coding sequences and the RTI of right AAV2 in a plasmid skeleton pBR322 (Afione et al., 1996). The packaging plasmid pGEM-RSS was derived from plasmid pHIVrep (Antoni et al., 1991) and consists of the U3 and R regions of LTR of HIV-1, including the rep and cap regions of AAV2 promoters p19 and p40, sequences of plasmids pBR322 and pGEM for bacterial replication and selection and a small region of human Alu repetitive cell DNA in the 5 'direction of the HIV LTR.

[0179] Type 5 adenovirus grew from a stock solution obtained from the American Type Culture Collection (Rockville, Md). Ad5ts149 (Ensinger et al., J. Virol. 10: 328, 1972) was obtained from Harold S. Ginsberg.

[0180] Working stock solutions of Ad5 and Ad5ts149 (ts149) were produced at 37 ° C and 32 ° C, respectively, infecting 293-1 cells at a multiplicity of infection (MOI) of 5 and 1, respectively. After 4 hours, the cultures were fed again with fresh medium and incubated at 37 ° C in a humidified incubator with 10% CO2. After 72 hours, the cells were removed, pelleted at 1000 g at 15 ° C and resuspended in PBS containing 0.1 g / l MgCl2 and 0.1 g / l CaCl2. The cell suspension was then frozen and thawed three times, sheared three times through an 18 gauge needle and clarified by centrifugation at 1000 g at 15 ° C. The clarified lysate was then treated with DNase I at a final concentration of 2 mg / ml for 30 minutes at 37 ° C. The layered treated lysate was spread on a gradient of CsCl discontinuous stage in water comprising 4.0 ml of CsCl (1.25 g / cm3) layered over 2.0 ml of CsCl (1.40 g / cm3 ) in water and centrifuged at 35,000 rpm for 1 to 2 hours in a Beckman SW41 rotor. The adenovirus band was removed from each tube, grouped, diluted in CsCl to 1.35 g / cm 3 in water and centrifuged overnight at 35,000 rpm in a Beckman SW55 rotor. The adenovirus band was grouped, adjusted to 10% glycerol and extensively dialyzed against 10 mM Tris buffer at pH 7.5 supplemented with 1.0 mM MgCl2 and 10% glycerol.

[0181] 293-1 cells (ATCC CRL 1573) were kept in T bottles in a humidified incubator with 10% CO2 in DMEM medium rich in glucose (JRH) supplemented with 10% fetal bovine serum (FBS, Hyclone). For this example, 293-1 cells were inoculated at 4.4 x 104 cells / cm2 in tissue culture flasks with DMEM supplemented with 10% FBS and 2.0 mM L-glutamine and incubated for 24 hours at 37 ° C in a humidified incubator with 10% CO2.

[0182] The cells (approximately 107 cells per flask) were then infected with working stock solutions of Ad5 or ts149 for 1 hour at an MOI of 5, followed by transient transfection of vector and packaging plasmids. Transient plasmid cotransfection of the ptgAAVCF vector and pGEM-RS5 helper plasmid was performed using LIPOFECTAMINE ™ (Gibco). In that process, 37.5 μg of each plasmid was mixed together with 150 μl of LIPOFECTAMINE ™ and diluted in 4.75 ml of serum-free MEM. The adenovirus inoculum was removed and the plasmid-LIPOFECTAMINE ™ mixture was added to the cells and incubated for 4 hours in a 5% CO2 incubator at the appropriate temperature. The plasmid-LIPOFECTAMINE ™ mixture was removed from the culture after 4 hours and replaced by fresh medium.

[0183] Cells infected with wild-type virus were grown at 37 ° C and cells infected with Adts149 were incubated at 39.5 ° C. After 72 hours, the cells were harvested, sedimented and resuspended in 10 mM Tris at pH 7.5. The suspension was then lysed by sonication in a water and ice bath using a Branson spindle cup sonicator using 4 pulses of 15 seconds and the AAVCFr and adenovirus production were tested.

Example 2

Quantification of AAVr and adenovirus titers in vector preparations

[0184] The production of the AAVCFr vector was tested in cell lysates of the preceding example by the C37 replication assay and adenovirus production was analyzed by slot transfer hybridization.

[0185] HeLa C37 was constructed to allow inducible expression of AAV Rep proteins for AAVr vector replication. Briefly, an AAV Rep / Cap expression module consisting of the mouse metallothionein I promoter, the AAV2 rep and cap genes and the AAV transcription termination site was constructed. A neomycin resistance gene under the control of the SV40 early promoter, the small SV40 intron T and the SV40 polyadenylation signal was also included in the plasmid. HeLa cells were transfected with the plasmid and the clones were selected in G418. A panel of clones was screened by an AAVr vector amplification assay. One clone, C37, demonstrated consistent and dose-dependent amplification of AAVr vector after transduction and adenovirus infection.

[0186] Detection of the replicating vector is achieved by DNA isolation followed by hybridization with a CFTR probe. In detail, He37 C37 cells were inoculated at 4.4 x 104 cells / cm2 in tissue culture flasks with DMEM supplemented with 10% FBS and 2.0 mM L-glutamine and incubated for 24 hours at 37 ° C in an incubator humidified with 5% CO2. The cells were then inoculated with adenovirus (MOI = 5) and sample dilutions of AAVCFr for 72 hours. The cells were harvested by scraping and prepared for Southern blot analysis. Total cell DNA was prepared, digested with EcoRI, subjected to 1% agarose gel electrophoresis, transferred to a nylon 66 membrane, followed by hybridization with a 32P-labeled human CFTR cDNA restriction fragment. This probe detects an approximately 1.5 kb fragment of the AAVCF vector (corresponding to the predicted 1,488 kb EcoRI fragment). Vector replication was quantified with respect to an endogenous genomic CFTR band and expressed as units of replication. A replication unit (UR) is defined as a signal strength equivalent to the endogenous genomic CFTR band that is approximately 1.8 kb. In some experiments, linear regression of known vector patterns diluted in series was used to extrapolate and calculate the vector concentration in the samples.

[0187] The adenoviral DNA slot transfer assay was performed as follows. Aliquots of samples were denatured in 0.4 M NaOH, 10 mM EDTA with 1.0 µg / ml salmon sperm DNA at 65 ° C. Adenovirus samples and standards were diluted and filtered through nylon membranes using a slot transfer manifold and washed with 0.4 M NaOH. The filter was hybridized with a 32P labeled probe corresponding to the adenovirus E1A gene sequence. . The complete Ad5 genome is available at Genbank with access number X02996. A 1 kb SspI-XbaI fragment (corresponding to nucleotides 339-1339) was used and the transfers were analyzed in a Phosphorimager (Molecular Dynamics). A genomic equivalent was considered to be equivalent to an adenoviral particle.

[0188] Figure 1 shows the results of the AAVCFr vector replication test in lysates prepared with Ad5 or ts149 at permissive (37 ° C) and non-permissive (39.5 ° C) temperatures. The production of recombinant vector was supported by ts149 at 39.5 ° C, but the productivity was approximately 2 to 3 times lower than with Ad5.

[0189] Figure 2 shows the results of the slot transfer assay to determine the amount of adenovirus. The production of adenoviral genomes was reduced 3-4 logarithmic units by the use of thermosensitive mutant compared to that of wild type.

Example 3

Optimization of the auxiliary function to improve the production of AAVr

[0190] This example illustrates various attempts to improve the level of AAVr obtained when thermosensitive auxiliary viruses are used. Increasing the levels of auxiliary virus infection was not helpful, but adjusting the kinetics was surprisingly effective.

[0191] The effects of increasing the multiplicity of infection on vector production were first evaluated. 293-1 cells were infected with Ad5 at an MOI of 5 or with ts149 at various MOI, followed by transient cotransfection with vector plasmids and packaging. After 72 hours, the cells were lysed and AAVCFr vector production was tested by the C37 vector replication assay and adenovirus production was analyzed by slot transfer hybridization. It was collected at an additional time point at 96 hours for cells infected with ts149 at an MOI of 5.

[0192] Figure 3 shows the results of the AAVCFr replication assay performed on cell lysates prepared with ts149 at various MOIs. Increasing the MOI of ts149 did not restore the productivity of the vector to the levels observed with Ad5 (as shown by the intensity of the 1.4 kb hybridization band). However, a higher level of vector production was observed at the time point of 96 hours. The concentration of ts149 in the lysate detected by the slot transfer analysis increased with increasing MOI, but remained

5 3 to 4 smaller logarithmic units compared to Ad5.

[0193] After the observation of the increased vector productivity with ts149 at 96 hours in the previous experiment, a study of the temporal and kinetic course of production was carried out. 293-1 cells were infected with Ad5 or ts149 at an MOI of 5 followed by transient cotransfection with vector plasmids and

10 packing Cells infected with Ad5 and ts149 were grown at 37 ° C and 39.5 ° C, respectively, for 6 days. Vector production was assayed on lysates on days 3, 4, 5, and 6 by a vector replication assay and adenoviruses were analyzed by slot hybridization.

[0194] Figure 4 illustrates the kinetics of vector production. Black bars represent lysates

fifteen produced using wild-type Ad5 as an auxiliary; The striped bars represent lysates produced using ts149 as an auxiliary. The maximum vector production when Ad5 was used was ~ 2.0 x 106 UR / ml, with day 4 being maximum. At this time point, the vector production obtained using ts149 was less than ~ 0.3 x 106 UR / ml . However, on day 5, there was a drastic alteration of the relative efficacy of the two auxiliary viruses. Ad5-supported vector production fell to less than 0.3 x 106 UR / ml. In contrast, vector production supported by

twenty ts149 rose to more than 2 x 106 UR / ml. The levels of adenoviral genome observed when using ts149 were significantly lower than with Ad5.

Example 4

25 Test of viral titers and high performance assay techniques

[0195] The thermosensitive and wild-type adenovirus stock solutions used in the preceding examples in 293-1 cells in tissue culture flasks were produced. In this example, the levels of adenovirus that are being produced by 293-1 cells were quantified by the titration criteria test by

30 TCID50 or infectivity test.

[0196] The TCID50 assay was performed as follows: 1.0 x 103 293-1 cells were seeded in 96-well microtiter plates, infected with serial dilutions of adenovirus stock solution and allowed to incubate at 37 ° C in a humidified incubator with 5% CO2. 8 duplicates of 100 µl of each dilution were inoculated into the cells. 3 days after infection, the cells were fixed with methanol, washed with PBS and stained with FITC-conjugated anti-hexon antibody (Biodesign) followed by staining with propidium iodide to visualize cell nuclei. After rinsing with PBS, the plate was examined with a fluorescent microscope and the presence of cells containing hexons was scored. The title was calculated in the valuation criteria using a Poisson distribution. A virus dilution that provides 50% samples of positive duplicates of hexons has 0.5

40 IU / 100 μl of inoculum. The infectious titer is the product of the inverse of this dilution, 0.5 IU / 100 µl and 10 (conversion factor in ml), giving the final infectious titer per ml.

[0197] A high performance microtiter infectivity test was performed to measure infectious viruses as follows. Aliquots (10 μl) of cell-free supernatants serially diluted in 45 HeLa cells grown in 96-well microtiter plates were inoculated. After 3 days, the infected cells were treated and lysed with a denaturation solution (addition of one tenth volume of 4.0 M NaOH, 10 μg / ml salmon sperm DNA and 100 mM EDTA). The lysate was transferred to a Silent Monitor BiodyneB plate (Pall) and vacuum filtered on a nylon membrane. The membrane was washed, denatured, hybridized with a 32P-labeled adenovirus E1A cDNA restriction fragment and analyzed in a Phosphorimager

fifty (Molecular Dynamics). Linear regression analysis of serially diluted adenovirus patterns was used to calculate infectious adenovirus titers in the samples, using adenovirus standards titrated by the TCID50 assay.

[0198] Specific viral productivity was calculated by normalizing infectious virus titers in the lysate to 55 cell numbers at the time of infection. The results are shown in Table 1:

<dl><dt>TABLE 1: Adenovirus Production </dt><dd /></dl>

<dl><dt>Adenovirus </dt><dd>Cellphone line Specific productivity (IU / cell) Test </dd></dl>

<dl><dt>293-1 </dt><dd>16 Infectivity microtiter </dd></dl>

<dl><dt>293-1 </dt><dd>fifteen Infectivity microtiter </dd></dl>

<dl><dt>293-1 </dt><dd>10 Infectivity microtiter </dd></dl>

[0199] These results show that the specific production of Adts149 in 293-1 cells was one to two logarithmic units less than that of Ad5.

[0200] An Ad5 virus preparation of known title showed a linear range ranging from 12.5 to 500 IU / well based on the linear regression in the infectivity microtiter assay.

[0201] Combining a viral infectivity assay with a microtiter serial format as described above resulted in a technique that is both rapid and quantitative, and is highly suitable for automation.

[0202] The high performance infectivity test described above can also be applied to the test of other viruses (eg, AAVr and AAVwt). The assay can be carried out essentially as described above using appropriate mammalian cells (for example, He37 C37 cells for AAVr or 293 cells for AAVwt) and under permissible conditions for replication of the virus to be tested (for example, in the presence of auxiliary virus AAVr and AAVwt), and then lysates can be prepared and nucleic acids in said lysates can be transferred to a membrane as described above. Hybridization of the membrane containing the series of bound nucleic acid assemblies (each set of cells being released from the corresponding culture well) is typically carried out with a suitable virus specific probe (for example, a specific rep and / or probe). or AAV cap could be used to detect AAVwt, or a probe specific to an inserted transgene could be used in the case of a recombinant AAV vector).

[0203] The high performance infectivity assay described above exhibited a linear response in the determination of AAVr titers in a relatively wide range of concentrations. For example, when a viral preparation of known title was diluted 1: 2 (determined by a modified infectious center assay), starting from 2400 infectious units or "IU / well", and used as a standard for titre determination of two preparations of purified tgAAVCF of unknown title, each of which was serially diluted 1: 5, The microtiter assay showed a linear range ranging from 75 to 600 IU / well based on linear regression. The determination of the AAVwt titer preferably employed a limiting dilution format (for example, when 8 serial dilutions of a AAVwt preparation of known title were tested, the titer determined by the microtiter test was essentially the same as that determined by the standard TCID50 assay, 3 x 109 IU / ml).

[0204] By limiting dilution or by comparison with a known standard, the infectious virus titer can be determined, which corresponds to the titers determined by more classical techniques (for example, the infectious center assay or the analysis criterion of assessment of % TCID50). In addition to its use in the determination of viral titers, this high-performance infectivity assay has many other uses including, but not limited to, screening of permissive or non-permissive cell lines of viral replication and infectivity (for example, including various cells of mammalian or variants thereof in different wells of a microtiter series), as well as screening agents that affect viral replication (e.g., including various agents in different wells of a microtiter series as described above and determining the effect of the agents on the infectious virus titer). Among other things, the ability to rapidly screen agents or conditions that enhance viral infectivity and / or replication is particularly useful in the context of developing or optimizing the production of viral vectors. Conversely, the ability to rapidly screen agents or conditions that suppress viral infectivity / replication is quite useful in the context of identification of antiviral therapeutic products.

Example 5

Development of suspension cultures to produce auxiliary viruses

[0205] The preceding example shows that the levels of heat sensitive adenoviruses produced by conventional culture techniques are low. This limits the ability to use thermosensitive adenoviruses as auxiliaries in the production of AAV vectors. The present example provides an improved process that allows the production of heat sensitive adenoviruses in much larger amounts. The use of host cells grown in suspension culture is crucial for improvement.

[0206] N3S of 293 and S3 of HeLa are suspended variants of the human embryonic kidney cell lines 293-1 and human HeLa epithelioid carcinoma respectively. The suspension culture was carried out in 500 ml shake flasks (Bellco) with working volumes of 250 to 300 ml. HeLa S3 cells (ATCC 2.2-CCL) were maintained in DMEM / F-12 with 15 mM HEPES supplemented with 7.5% FBS and 2.0 mM L-glutamine. The N3S of 293-1 (Microbix Biosystems Inc.) were subjected to passes with Joklik MEM supplemented with 7.5% FBS and 2.0 mM L-glutamine. The shake flasks were shaken at 50-65 rpm.

[0207] Growth performance was evaluated in the following experiment. They were subjected to N3S series passes of 293 and S3 of HeLa in suspension in shake flasks of duplicate 500 ml and cell growth and viability were monitored. Flasks were inoculated at cell densities of 2 to 5 x 105 cells / ml and then cultured for 2 to 3 days. To control planting density differences, population duplication levels (NDP) were compared for duplicate crops. The mean NDP was 2.0 ± 0.49 (mean of 6 SD) and 1.1 ± 0.62 for S3 of HeLa and N3S of 293, respectively (n = 14). Major cell duplications were observed consistently with HeLa S3 cells. The cell morphology in suspension was drastically different for the two lines. HeLa cells grew as individual cells or small aggregates. In contrast, the N3S cells of 293 formed large aggregates of 50 to 100 cells each. Significant numbers of non-viable cells were observed in the center of large clusters. The stock solutions were subcultured by centrifugation followed by gentle disintegration with a pipette, releasing the non-viable cells from the aggregates. The initial cultivation viability of N3S of 293 was consistently lower compared to He3 S3.

[0208] Based on growth, viability and cell morphology in suspension, the HeLa S3 cell line was selected for further process development. Growth and viability were evaluated at permissive temperatures. S3 HeLa cells were seeded in 500 ml shake flasks at 5 x 105 cells / ml and monitored daily for 7 days.

[0209] Figure 5 shows the density of viable cells (DCV) of HeLa S3 cells, grown at 32 ° C (square) and 37 ° C (circles). The bars around the time points at 32 ° C indicate the range of values observed in shake flasks of 500 ml duplicates. Cells grown at 37 ° C reached a maximum of 2.5 x 106 cells / ml on day 5, while cells grown at 32 ° C reached a maximum of 2 x 106 cells / ml on day 6. The viability (determined by exclusion with trypan blue) was at least about 90% in total.

[0210] Tangential or cross flow filtration is a versatile technique for a wide variety of large-scale biopharmaceutical applications, including cell concentration or removal, macromolecule concentration and media / buffer exchange. Tangential flow processing is necessary to concentrate cells for infection and to collect infected cells on a large scale.

[0211] The effect of laminar shear on cell viability in tangential flow filtration was evaluated by concentrating and diafiltering HeLa S3 cells. HeLa S3 cells were inoculated at a density of 4 x 105 cells / ml in 3 l Applilon bioreactors and grown at 2 x 106 cells / ml in DMEM / F-12 with 15 mM HEPES (JRH) supplemented with 7.5% of FBS, 2.0 mM glutamine, 1 x MEM amino acids, 1 x non-essential MEM amino acids, 0.1% Pluronic F-68 polyol and 2 g / l glucose. The workload of the bioreactor was 2 l. Dissolved oxygen, pH, temperature and stirring at 60% (relative to air saturation), 7.2, 37 ° C and 100 rpm, respectively, were controlled using the FERMCON ™ (Scius Corporation) controller system.

[0212] Tangential flow experiments were performed with hollow mixed cellulose fiber (Microgon) membranes. The pore size and surface area were 0.2 μm and 725 cm2, respectively. A 0.2 μm filter was selected to retain the cells by passing the spent media. The cells (Cole Palmer) were pumped through the internal diameter of the hollow fibers. Recirculation rates were adjusted to provide average wall shear rates of 750 and 1500 s-1. Once the cross flow was established, a permeate flow control of 30 and 90 ml / min, respectively, was achieved with a pump (Cole Palmer) located in the permeate line. During cell concentration, permeate extraction continued until the desired concentration was reached at times. During diafiltration, the feeding of media entering the bioreactor was activated until the desired medium exchange was achieved at times. Viable cells were counted before and after each treatment.

[0213] Figure 6 shows the growth curves of He3 S3 cells before and after tangential flow processing in an exemplary experiment. 2 L of cells were grown in 3 L bioreactors. On day 3 (arrow), the cells were concentrated 7 times from the working volume of 2 l diafiltered against 6 volumes of growth medium and brought to the original work volume. The results show that the cells were not damaged by a wall shear of 750 s-1 (squares) or 1500 s-1 (circles) and continued to grow at high cell densities.

[0214] The suspension cultures of HeLa S3 cells were then tested as host cells for the production of ts149, or their capacity in 300 ml suspension culture was investigated. HeLa S3 cells of 300 ml suspension culture were centrifuged (1 x 10 6 cells / ml), concentrated and infected with ts149 (MOI = 3). After 1 hour, the culture was transferred to a shake flask, resuspended in media and grown for 7 days at 32 ° C. He3 S3 cells continued to grow from about 1 x 10 6 cells / ml at the time of infection to about 2 x 10 6 cells / ml on day 5. Viability was reduced to ~ 60% on day 7.

[0215] Figure 7 shows the production of ts149 by HeLa S3 cell cultures. The culture was sampled daily and lysates were prepared by lyophilization for viral production analysis by the adenovirus infectivity test. Virus production reached ~ 4.5 x 107 IU / ml of culture approximately on day 3-5. On day 7, the cells were collected by centrifugation, resuspended in TMEG buffer and lysed by microfluidification (MF). The infectious titer of the microfluidized lysate was comparable with those of the lyophilized lysate sample, indicating that recovery by microfluidification was comparable with lyophilization procedures.

Example 6

Improved purification procedure for the production of a thermosensitive auxiliary virus (Ad ts149)

[0216] A purification using CsCl gradients is expensive for large-scale production. This example illustrates the purification of ts149 by ion exchange chromatography.

[0217] Chromatography was performed at a Perseptive Biosystem BIOCAD ™ chromatography workstation. The resin used was a weak anionic polyethyleneimine (PI) exchanger (POROS ™ 50 PI). The column was equilibrated with TMEG (50 mM Tris, pH 8.0, 5 mM MgCl2, 1 mM EDTA, 5% glycerol). In-line chromatography was monitored for pH, conductivity and optical density at 280 nm.

[0218] HeLa S3 suspension infected with ts149 was collected at an MOI of 2 and centrifuged. The sediment was resuspended in TMEG and lysed by cavitation at 20.7 MPa using a microfluidifier (Microfluidics). The lysate was clarified by filtration through a 5 µm syringe filter (Millex SV) followed by a 0.45 µm syringe filter (Acrodisc). The clarified lysate was loaded on a 1.6 ml POROS ™ 50 PI anion exchange column processed at 1 ml / min. The column was washed with 10 column volumes of TMEG with 900 mM NaCl and the ts149 was eluted with a linear gradient of 900 to 1300 mM NaCl. The 0.5 ml fractions were collected and the presence of adenovirus was tested by infectivity and slot transfer assay.

[0219] Figure 8 shows the results of the infectivity test performed in consecutive column fractions. The majority of infectious adenovirus was found in fractions 26 to 28, coinciding with the absorbance peak eluting at approximately 100 ms at approximately 25 minutes. The ts149 eluted just before the large peak at higher saline concentration. Infectivity and slot transfer tests performed in parallel confirmed that the infectious particles and viruses were in the same peak fractions.

[0220] The total protein was also tested in lysate and PI peak fractions by the Bradford method. The protein concentration was 1.8 mg / ml in the lysate and less than 30.0 μg / ml in the whole PI. Virions were separated from most of the cellular protein in a single stage and eluted as a single peak. The virions showed a very high affinity for the PI matrix, as evidenced by the relatively high saline concentration necessary to elute them from the column.

[0221] Large-scale procedures for heat-sensitive auxiliary viruses can incorporate all the improvements described in these examples. In an illustration, virus production would comprise the following stages:

<dl><dt>•</dt><dd> Cell culture in a suspended bioreactor </dd></dl>

<dl><dt>•</dt><dd> Media concentration / exchange </dd></dl>

<dl><dt>•</dt><dd> Infection with auxiliary virus </dd></dl>

<dl><dt>•</dt><dd> Virus production </dd></dl>

<dl><dt>•</dt><dd> Collection: concentration / diafiltration </dd></dl>

<dl><dt>•</dt><dd> Lysis by microfluidification </dd></dl>

<dl><dt>•</dt><dd> Ion exchange chromatography in PI </dd></dl>

<dl><dt>•</dt><dd> Concentration / Diafiltration </dd></dl>

<dl><dt>•</dt><dd> Sterilization by filtration </dd></dl>

[0222] This type of approach is inherently scalable and susceptible to current Good Manufacturing Practices.

[0223] Additional exemplary illustrations of such techniques are provided below.

Example 7

Comparison of first and second generation processes for the production of auxiliary viruses

A. Illustrative production and processing of first generation auxiliary virus

[0224] In an exemplary "first generation" process for the production of auxiliary virus, mammalian cells were grown in 40 T225 flasks and then infected with Ad5 at an MOI of approximately 1. After incubation, the cells were collected by centrifugation and were lysed by lyophilization and passed through a needle. The lysate was subjected to DNase I treatment and then processed in a CsCl step gradient and an isopycnic gradient. The purified material was dialyzed and sterilized by filtration.

[0225] Using this first generation process, approximately 1 x 1012 particles (or approx. 1 x 1011 infectious units) were obtained from 4 x 108 cells.

B. Illustrative production and processing of second generation auxiliary virus

[0226] In an exemplary "second generation" process for the production of auxiliary virus, mammalian cells (S3 from HeLa) grew in 10 l bioreactors and were then infected with Ad5 (from ATCC, plaque purified subsequently in 293 cells, serially propagated in HeLa S3 cells and double purified by gradient centrifugation of CsCl) at an MOI of about 1. After incubation, the cells were concentrated, collected by diafiltration and lysed by microfluidification. The lysate was subjected to benzone (nuclease) treatment and then filtered. The filtrate was then processed on an anion exchange (PI) column, concentrated, diafiltered and finally sterilized by filtration.

[0227] Using this second generation process, approximately 1 x 1014 particles (or approx. 5 x 1012 infectious units) were obtained from 1 x 1010 cells.

[0228] Figure 9 illustrates the results of subsequent processing of auxiliary virus using anion exchange chromatography as described above. Bars: viral activity measured in an infectivity test, continuous line: A280, dotted line: buffer conductivity (ms).

[0229] As is evident from the comparison of the fractionation of viral activity against A280 absorbance, these processing procedures resulted in a substantial separation of the auxiliary virus from the bulk of the contaminant materials that would be expected to contain cellular nucleic acids and proteins. .

Example 8

Comparison of first and second generation processes for the production of recombinant AAV vectors

A. Production and processing of first-generation illustrative AAVr

[0230] In an exemplary "first generation" process for the production of AAVr vector, mammalian cells were grown in 40 T225 flasks and then infected with Ad5 at an MOI of approximately 5. After incubation, cells were harvested by centrifugation and lysed by sonication. The lysate was subjected to DNase 1 treatment and then processed in a series of two CsCl gradients. The purified material was dialyzed and sterilized by filtration. Using this first generation process, approximately 5 x 106 UR replicative units were obtained from 4 x 108 cells.

B. Production and processing of second-generation illustrative AAVr

[0231] In an exemplary "second generation" process for the production of AAVr vector, mammalian cells were grown in 10 l bioreactors and then infected with Ad5 at an MOI of approximately 5. After incubation, the cells were concentrated, they were collected by diafiltration and lysed by microfluidification. The lysate was subjected to benzone (nuclease) treatment and then filtered. The filtrate was then processed in an anion exchange column, followed by a cation exchange column. The eluent fractions containing AAV were pooled, concentrated, diafiltered and finally sterilized by filtration. This second generation process is expected to provide more than 1 x 1011 UR replicative units from 1 x 1010 cells.

[0232] Figure 10 shows the results of sequential fractionation in ion exchange columns: first, in an anion exchange matrix (upper panel) and then in a cation exchange matrix (lower panel). Bars: viral activity measured in an infectivity test for adenovirus or AAV, continuous line: A280 (a measure of total protein), dotted line: buffer conductivity (ms). As is evident from the analyzed fractions, it is possible to obtain extremely high levels of separation between AAV and adenovirus, as well as between AAV and A280 absorbent material (mostly proteins) using the techniques of the present invention. In particular, the results revealed that AAV vectors can be retained in both anionic and cationic exchange columns and that differential AAV elution using both anionic and cationic exchange resulted in a dramatically enhanced ability to separate AAV from all major pollutants from interest (including adenovirus as well as cellular proteins).

[0233] In another exemplary second generation process for the production of AAVr vector, the filtrate was prepared as described above and then processed in an anion exchange column, followed by grouping of eluent fractions containing AAV, and the grouped anion exchange eluents were then subjected to tangential flow filtration (FFT). As described below, it was found that this anion exchange procedure to FFT resulted in a highly concentrated and purified AAV preparation.

[0234] A detailed analysis of the AAV obtained using said second generation technology, using techniques as described above and in the art (including infectivity assays, Slot transfer analysis and gel electrophoresis with SDS) provided further confirmation that the material was of high quality and was substantially free of contaminating adenoviral particles (and adenoviral protein and DNA) and also substantially free of contaminating cellular proteins and DNA. SDS gels revealed the presence of bands corresponding to VP1, VP2 and VP3 (specifically, AAV capsid proteins). No other bands were visible after Coomasie staining. These data are consistent with the results of the column fractionation analyzes as shown in Figures 10-11.

[0235] As an illustrative FFT anion exchange procedure, the following is an exemplary purification and concentration process that starts from 1 l of pooled fractions of anion exchange chromatography. If desired (as noted above), this assembly can be subjected to thermal inactivation followed by a filtration step (using, for example, a 0.22 μm filter). For tangential flow filtration (FFT), a sterilized Pellicon XL system equipped with a molecular weight cutting membrane of

300,000 operating at 40/0 for inlet and outlet pressures. 1 L of pooled material was loaded into the system at a volume of 500 ml and then concentrated to 250 ml. Diafiltration was performed with 5 diavolumes (1250 ml) of modified Ringer's solution + 5% glycerol. After diafiltration, the retentate was concentrated to a final volume of 14 ml. The total process time was approximately 3.25 hours (not including sterilization time). Silver-stained SDS gels, slot transfers and infectivity tests confirmed that the AAV preparation (containing approximately 1010 replicative units) was substantially free of contaminating adenoviruses as well as adenoviral and cellular proteins.

[0236] The following are results of said procedure that show infectious and total viral titer such as UR (replicative units) and PRA (DNase resistant particles) respectively:

300 K FFT Total UR volume Total PRA P / I% UR% PRA Input set 1000 ml 8.9 x 1010 3.1 x 1014 3483 100 100 Gross purified product 12.5 ml 7.3 x 1010 2, 3 x 1014 3103 82 74

[0237] The data presented in Figure 12 illustrates the results of an AAV production routine that uses tangential flow filtration after an anion exchange column. Purified material was concentrated on the POROS 50 PI column using a 300,000 molecular weight cutting membrane (Millipore Pellicon XL). The concentrated material was diafiltered with 5 successive volumes of Ringer's balanced salt solution + 5% glycerol. The material was then concentrated on the membrane 10 times. Figure 12, a semitonic reproduction of a polyacrylamide-SDS gel stained with a silver stain, shows highly purified AAV capsid proteins VP1 (85 kDa), VP2 (72 kDa) and VP3 (62 kDa) in a material purified purified final.

[0238] As is evident from the data presented herein, these second generation techniques for the preparation and purification of AAV result in substantially improved procedures compared to those described above.

[0239] The exemplary media for adenovirus helper growth and for preparing AAVr are detailed in the following table:

[0240]

TABLE 2:

<dl><dt>Ad medium </dt><dd>AAVr medium </dd></dl>

<dl><dt>INORGANIC SALTS </dt><dd /></dl>

<dl><dt>Faith (NO3) 3 · 9H2O </dt><dd> 0,05 0,05 </dd></dl>

<dl><dt>NaHCO3</dt><dd> 2200 2200 </dd></dl>

<dl><dt>OTHER COMPONENTS </dt><dd /></dl>

<dl><dt>glucose</dt><dd> 4500 4500 </dd></dl>

<dl><dt>HEPES</dt><dd> 3575 3575 </dd></dl>

<dl><dt>Na hypozantin </dt><dd> 2,39 2,39 </dd></dl>

<dl><dt>linoleic acid </dt><dd> 0,042 0,042 </dd></dl>

<dl><dt>lipoic acid </dt><dd> 0,105 0,105 </dd></dl>

<dl><dt>phenol red sodium salt </dt><dd /></dl>

<dl><dt>OTHER COMPONENTS </dt><dd /></dl>

<dl><dt>putrescine2HCl</dt><dd> 0,081 0,081 </dd></dl>

<dl><dt>sodium pyruvate </dt><dd> 55 55 </dd></dl>

<dl><dt>Pluronic polyol F-68 </dt><dd> 100 100 </dd></dl>

<dl><dt>AMINO ACIDS </dt><dd /></dl>

<dl><dt>L-Alanine</dt><dd> 4,455 4,455 </dd></dl>

<dl><dt>L-arginine · HCl</dt><dd> 273,9 273,9 </dd></dl>

<dl><dt>L-asparagine H2O </dt><dd> 22,5 22,5 </dd></dl>

<dl><dt>L-aspartic acid </dt><dd> 19,95 19,95 </dd></dl>

<dl><dt>L-cysteine · HCl · H2O </dt><dd> 17,56 17,56 </dd></dl>

<dl><dt>L-Cystine-2HCl</dt><dd> 52,29 52,29 </dd></dl>

<dl><dt>L-glutamic acid </dt><dd> 22,05 22,05 </dd></dl>

<dl><dt>L-glutamine</dt><dd> 657 657 </dd></dl>

<dl><dt>glycine</dt><dd> 26,25 26,25 </dd></dl>

<dl><dt>L-histidine · HCl · H2O </dt><dd> 73,48 73,48 </dd></dl>

<dl><dt>L-Isoleucine</dt><dd> 106,97 106,97 </dd></dl>

<dl><dt>L-Leucine</dt><dd> 111,45 111,45 </dd></dl>

<dl><dt>L-Lysine · HCl</dt><dd> 163,75 163,75 </dd></dl>

<dl><dt>L-methionine</dt><dd> 32,34 32,34 </dd></dl>

<dl><dt>L-phenylalanine</dt><dd> 68,48 68,48 </dd></dl>

<dl><dt>L-proline</dt><dd> 17,25 17,25 </dd></dl>

<dl><dt>L-serine</dt><dd> 36,75 36,75 </dd></dl>

<dl><dt>L-threonine</dt><dd> 101,05 101,05 </dd></dl>

<dl><dt>L-tryptophan </dt><dd> 19,22 19,22 </dd></dl>

<dl><dt>L-tyrosine</dt><dd> 91,79 91,79 </dd></dl>

<dl><dt>L-valine</dt><dd> 99,65 99,65 </dd></dl>

<dl><dt>VITAMINS </dt><dd /></dl>

<dl><dt>d-biotin</dt><dd> 0,00365 0,00365 </dd></dl>

<dl><dt>Ca D-pantothenate </dt><dd> 2,24 1,00 </dd></dl>

<dl><dt>choline chloride </dt><dd> 8,98 8,98 </dd></dl>

<dl><dt>folic acid </dt><dd> 2,65 2,65 </dd></dl>

<dl><dt>myoinositol</dt><dd> 12,6 12,6 </dd></dl>

<dl><dt>niacianamide</dt><dd> 2,0185 2,0185 </dd></dl>

<dl><dt>pyridoxal · HCl</dt><dd> 2 2 </dd></dl>

<dl><dt>pyridoxine · HCl</dt><dd> 0,031 0,031 </dd></dl>

<dl><dt>riboflavin</dt><dd> 0,219 0,219 </dd></dl>

<dl><dt>thiamine · HCl</dt><dd> 2,17 2,17 </dd></dl>

<dl><dt>thymidine</dt><dd> 0,365 0,365 </dd></dl>

<dl><dt>b12 vitamin </dt><dd> 0,68 0,68 </dd></dl>

* add an appropriate amount of NaCl for osmolality at 300 mOsm (± 20 mOsm)

C. AAV vector purification using heparin sulfate chromatography

[0241] As discussed above, chromatographic techniques can be used to further purify and concentrate AAV preparations in accordance with the present disclosure that includes the present invention. By way of illustration, a preparation of AAV that is in crude form (e.g. lysate) or that has been eluted from an anion exchange or cation exchange column and / or concentrated by tangential flow filtration can be purified by binding with a column that It comprises heparin sulfate. The AAV can then be eluted from said column using a buffer containing a salt (for example, a linear NaCl gradient).

[0242] As an illustration of the use of heparin sulfate chromatography, AAV obtained from a set of "PI" (as described below in Example 9) was concentrated first four times and diafiltered with TMEG + NaCl 100 mM using a 300K tangential flow filtration membrane. The concentrate was then injected into a 1 ml heparin sulfate column (Pharmacia column "Hi-Trap Heparin") and eluted using a linear NaCl gradient.

[0243] Figure 13 is a chromatogram showing the resulting concentration of AAV in the heparin sulfate column. The narrow absorbance peak at 280 nm (left axis) at approximately 18 minutes of elution time represents the fraction of AAV eluted from heparin sulfate with a linear gradient of 0 to 1 M NaCl (the conductivity is shown in ms on the axis right).

Example 9

Production and assay of recombinant AAV vector

[0244] In another group of production routines, 3-4 x 109 cells grown in a Cell Factory were used, using DMEM + 10% FBS as growth medium. The cells were infected with Ad at an MOI of about 20 and collected 72 hours after infection. The collected cells were suspended in TMEG + NaCl at a concentration of approximately 5 x 10 6 cells / ml. After mechanical lysis (microfluidification, 2 passes at 55.16 MPa), the lysates were treated with benzone (25 units / ml, 37 ° C, 1 hour) and then filtered through a 5 μm filter (Pall Profile II) .

[0245] As an exemplary anion exchange column, the POROS 50 PI column (available from Perseptive Biosystems) was used. Briefly, the filtrate was loaded on the column in approximately 100 ml and then eluted with a gradient of 500 mM NaCl. The fractions that were determined to contain (by infectivity test) the majority of the AAV were collected and pooled (designated as "set of PI").

[0246] The PI pool was then diluted to approximately 1: 7 with TMEG, loaded onto a Toso Haas SP650C column and eluted with a gradient of 500 mM NaCl. The fractions that were determined to contain (by infectivity test) the majority of the AAV were collected and pooled (designated as "SP set"). The whole of SP was concentrated using a Centriprep 10K filter and then sterilized by passing through a 0.2 μm filter.

[0247] The results revealed that recombinant AAV was essentially free of detectable infectious adenoviruses (determined by the detection limit analysis with 293 cell series amplification and the TCID50 assay). The preparation was also essentially free of adenoviral DNA (determined by slot transfer analysis), essentially free of cellular proteins (determined by PAGE-SDS gel analysis), of cellular DNA (determined by PCR analysis) and which was also essentially free of phenotypically wild type AAV (determined by serial amplification and Southern analysis).

Example 10

Enhancement of AAV production due to nutritional stress

[0248] As discussed above, it is believed that the production of AAV can be enhanced using any of a variety of agents and / or conditions that effectively stress (or deoptimize) the growth or metabolism of AAV producing cells. In this example, it is shown that the depletion of certain amino acids that occurs during cultivation is associated with a relative potentiation of AAV production and, conversely, that supplements of means to remove nutritional stress actually result in a drastic Vector performance reduction.

(a) Nutritional stress during batch and perfusion culture [0249] JL14 cells were inoculated at approximately 4x105 cells / ml in 2 l bioreactors and grown in the AAVr medium shown in Table 2 in batch or infusion mode ( using filtration by tangential filter, day 1 to 0.4 volumes / day, days 2-3 to 1-2 vol./day, day 4 to 2 vol./day and day 5 to 4 vol./day). The cell density, glucose, lactate and amino acids were monitored in the cultures using standard techniques.

5 [0250] The analyzes revealed that cell density reached a maximum in the batch culture of 1 x 10 6 cells / ml on day 2 and in the perfusion culture of 8 x 10 6 cells / ml on day 6. Glucose was not limiting in no case (> 1 g / l) and lactate was not inhibitor.

10 [0251] However, amino acid analysis revealed that both glutamate and aspartate were rapidly depleted in both batch and infusion cultures, as shown in the following tables:

TABLE 3: TABLE 4:

<dl><dt>Amino acid analysis of the culture medium by LOTS (time course - μmol / l) </dt><dd /></dl>

<dl><dt>Aspartic acid </dt><dd> 133 96 10 4 9 7 </dd></dl>

<dl><dt>Threonine </dt><dd> 119 687 644 606 552 533 </dd></dl>

<dl><dt>Serina </dt><dd> 105 271 230 157 117 98 </dd></dl>

<dl><dt>Asparagine </dt><dd> 132 130 113 96 68 69 </dd></dl>

<dl><dt>Glutamic acid </dt><dd> 147 90 2 1 1 1 </dd></dl>

<dl><dt>Glutamine </dt><dd> 146 3424 2987 2450 1989 1843 </dd></dl>

<dl><dt>Proline </dt><dd> 115 135 143 162 164 185 </dd></dl>

<dl><dt>Glycine </dt><dd> 75 288 241 194 151 130 </dd></dl>

<dl><dt>Valine </dt><dd> 117 692 631 518 417 342 </dd></dl>

<dl><dt>Cystine </dt><dd> 121 143 133 120 107 99 </dd></dl>

<dl><dt>Methionine </dt><dd> 149 160 132 100 74 58 </dd></dl>

<dl><dt>Isoleucine </dt><dd> 131 617 531 383 264 182 </dd></dl>

<dl><dt>Leucine </dt><dd> 131 645 538 374 248 161 </dd></dl>

<dl><dt>Tyrosine </dt><dd> 181 407 379 355 329 315 </dd></dl>

<dl><dt>Phenylalanine </dt><dd> 165 323 289 259 231 214 </dd></dl>

<dl><dt>Tryptophan </dt><dd> 204 47 41 33 28 26 </dd></dl>

<dl><dt>Ammonia </dt><dd> 17 760 816 941 1021 1033 </dd></dl>

<dl><dt>Ornithine </dt><dd> 71 89 110 128 144 </dd></dl>

<dl><dt>Lysine · HCl </dt><dd> 572 521 463 415 384 </dd></dl>

<dl><dt>Histidine </dt><dd> 155 276 257 243 229 211 </dd></dl>

<dl><dt>Arginine </dt><dd> 174 1020 943 870 791 747 </dd></dl>

<dl><dt>Amino acid analysis of the culture medium by PERFUSION (time course - μmol / l) </dt><dd /></dl>

<dl><dt>Aspartic acid </dt><dd> 133 95 12 5 10 10 10 10 </dd></dl>

<dl><dt>Threonine </dt><dd> 119 709 691 560 596 651 641 657 </dd></dl>

<dl><dt>Serina </dt><dd> 105 281 264 147 156 180 199 185 </dd></dl>

<dl><dt>Asparagine </dt><dd> 132 130 124 75 78 109 119 119 </dd></dl>

<dl><dt>Glutamic acid </dt><dd> 147 88 1 0 1 1 1 0 </dd></dl>

<dl><dt>Glutamine </dt><dd> 146 3525 3299 2517 2640 2906 2986 3082 </dd></dl>

<dl><dt>Proline </dt><dd> 115 145 165 163 174 177 157 171 </dd></dl>

<dl><dt>Glycine </dt><dd> 75 304 267 189 205 217 227 230 </dd></dl>

<dl><dt>Valine </dt><dd> 117 678 635 485 500 532 551 561 </dd></dl>

<dl><dt>Cystine </dt><dd> 121 141 136 112 118 123 119 118 </dd></dl>

<dl><dt>Methionine </dt><dd> 149 157 133 91 99 107 108 107 </dd></dl>

<dl><dt>Isoleucine </dt><dd> 131 616 543 369 401 430 432 442 </dd></dl>

<dl><dt>Leucine </dt><dd> 131 649 554 364 400 430 438 444 </dd></dl>

<dl><dt>Tyrosine </dt><dd> 181 413 398 328 355 379 373 386 </dd></dl>

<dl><dt>Phenylalanine </dt><dd> 165 336 16 244 268 291 287 292 </dd></dl>

<dl><dt>Tryptophan </dt><dd> 204 58 47 36 41 48 44 47 </dd></dl>

<dl><dt>Ammonia </dt><dd> 17 831 1182 956 1202 1219 931 990 </dd></dl>

<dl><dt>Ornithine </dt><dd> 42 97 74 115 112 56 44 </dd></dl>

<dl><dt>Lysine · HCl </dt><dd> 718 651 528 594 643 617 628 </dd></dl>

<dl><dt>Histidine </dt><dd> 155 284 310 223 243 262 266 265 </dd></dl>

<dl><dt>Arginine </dt><dd> 174 1058 948 826 901 978 974 1016 </dd></dl>

(b) Nutritional stress associated with enhanced AAV production

5 [0252] Follow-up studies were conducted to confirm the importance of the relative shortage of glutamate and aspartate in the culture media. JL 14 cells were taken from a shake flask and divided into two groups. Each group was inoculated with 3 x 109 infectious units of 170-37 Ad5. A group of cells was resuspended at 106 cells / ml in AAVr medium (Table 2) containing 10% FBS and 1% L-glutamine (300 ml). He

10 the other was resuspended in AAVr medium containing 10% FBS, 1% L-glutamine, 10 mg / l aspartic acid and 110 mg / l glutamic acid.

[0253] Each group was incubated at 37 ° C for 72 hours in a shake flask. The cells were collected, microfluidified twice at 55.16 MPa, subjected to benzonase, seeded in an infectivity assay, collected and probed.

[0254] The results showed that the control agitation matrix produced 6.2 UR per cell. The stirring flask supplemented with aspartic and glutamic acids produced 0.94 UR per cell.

twenty [0255] This indicates that when the depletion of aspartic and glutamic acids is avoided by providing these amino acids in excess, the production of AAVr is compromised due to the inability to subject the cells to nutritional stress.

[0256] Additional assays were performed using a He6-derived D6 cell line having an integrated AAVr vector 25 (RTI- (CMV promoter) - (-gal reporter gene) -RTI), as well as copies of the rep genes and wild type AAV cap.

[0257] Cells were seeded at 5 x 106 cells per T-225 flask in 30 ml of complete DMEM (10% FBS, 2 mM L-glutamine) and incubated at 37 ° C in 10% CO2 for 2 days, after of which the cells reached

30 a density of 2 x 107 cells per flask. Cells were infected in two duplicated flasks with Ad5 at an MOI of 10. One flask contained complete DMEM, the other contained complete DMEM supplemented with 5 x aspartic acid and glutamic acid. Cells were collected and counted after 72 hours of culture.

[0258] Complete DMEM provided 2.6 x 107 cells with 88% viability. The medium supplemented with

35 aspartate / glutamate provided 3.8 x 107 cells with 91% viability. The cells were resuspended, sonicated, treated with benzone (25 U / ml), clarified and tested by slot transfer analysis.

[0259] The results were as follows: D6 virus was produced in complete DMEM (not supplemented) at 1.8 40 x 1010 PRA / ml (1800 PRA per cell). D6 virus was produced in DMEM supplemented with asparate / glutamate at 1.4 x 109 PRA / ml (140 PRA / cell).

Example 11

Production of recombinant AAV vector under serum stress

5 [0260] As an example of the production of AAVr under stress conditions, reduced serum stress has been used together with techniques as described above. Briefly, JL 14 cells were grown in shaking flasks in modified DMEM + 10% FBS in continuous serial culture mode, and divided every 3-4 days. The cells of the suspension culture were placed in 16 Nunc Cell Factory devices of 10 stacks, at 3

10 x 108 Factory cells / device in a rotation of 3 to 4 days. The medium used for growth had a 10-fold reduction in serum (specifically, DMEM + 1% FBS), thus placing the cells under serum stress.

[0261] At 24 hours after sowing, the medium in the devices was removed and fresh medium containing 3 x 109 units of Ad / ml was added. After 72 hours of culture at 37 ° C, the cells of the 15 Factory devices were dislodged by gentle tapping, the medium containing cells was collected and the cells were sedimented, resuspended in TMEG + 100 mM NaCl and then lysed by pass to through a microfluidifier at 55.16 MPa. The lysate was clarified through a 5 μm filter and the clarified lysate was loaded on a 500 m IP anion exchange column. The column was eluted with an increasing gradient of NaCl (up to 500 mM) in TMEG buffer. Fractions were collected and tested using a clone 37 assay as described by Allen et al. (WO96 / 17947, supra). The fractions containing most of the AAV vector were then pooled and concentrated 10 times using a Centriprep centrifugal concentrate at 1000 xg for 30 minutes. The concentrated material was dialyzed against Ringer's balanced saline solution with 5% glycerol and stored at -70 ° C. AAV was tested by clone 37 assay, as well as by slot transfer and PAGE-SDS. The presence of adenoviruses, adenoviral proteins and DNA can also be tested in the material.

25 cellular as well as other potential contaminants.

[0262] Figure 11 shows the results obtained using GAK-0003 producing cells established in T-225 flasks at 107 cells per flask and inoculated on day 2 with adenovirus DAB-003 at an MOI of 10. Different flasks were cultured for 72 hours at 37 ° C in recent DMEM containing a different percentage of

30 FBS, as shown in the figure. On day 5, each flask was collected, the cells were counted, resuspended, sonicated, benzone and seeded to measure vector production as before.

[0263] Optimal vector production was observed at a percentage of FBS of 1%. Therefore, medium that is deficient in FBS (less than 2.5%, preferably less than 2%, but more than 0%) is preferred as a condition for subjecting the producing cells to serum stress.

Example 12

Production of recombinant AAV vector under pH stress

40 [0264] As a further example of the production of AAVr under stress conditions, pH stress was used in conjunction with techniques as described above. Briefly, AAV producing cells grew in bioreactors as described above. The cells were then infected with Ad5 at an MOI of 10 and inoculated in low serum media (as in Example 11) in suspension in 1.5 l bioreactors. He

Four. Five they maintained the cultures at high pH levels (from 7.2 to 8.0). The cell number, viability, glucose consumption, lactate production, pH, osmolarity and AAV production were then monitored daily in the cultures. As shown below, there was an increase in AAV production when the pH rose to 7.4, coupled with an even more drastic increase in the number of AAV particles released to the supernatant (which increased as the pH was raised ):

<dl><dt>pH </dt><dd>from Associated particles Particles in Particles % from cells % from </dd></dl>

<dl><dt>culture </dt><dd>to cell supernatant totals associated supernatant </dd></dl>

[0265] In sum, as the pH increased, there was a strong increase in the number of AAV particles released to the supernatant and a shift in the percentage of particles in supernatant: cell-associated (of almost all cell-associated at pH 7, 2 a mostly in supernatant (92%) at pH 8.0). The capacity of

55 recovering the AAV particles directly from the supernatant without the need to lyse the producing cells represents a powerful advantage in terms of AAV production and purification. AAV isolated from the supernatant using pH stress can be easily concentrated and purified using techniques as described herein (eg, ion exchange chromatography and / or tangential flow filtration).

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EP1944362B1	European Patent Office (EPO)	B1	2015-10-21
EP2942393A1	European Patent Office (EPO)	A1	2015-11-11
DK1944362T3	Denmark	T3	2016-01-25
PT1944362E	Portugal	E	2016-01-27
ES2557997T3	Spain	T3	2016-02-01
JP2016025852A	Japan	A	2016-02-12
JP5881626B2	Japan	B2	2016-03-09
CY1113731T1	Cyprus	T1	2016-06-22
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CY1117384T1	Cyprus	T1	2017-04-26
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