Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-04-14
2003-04-15
Ketter, James (Department: 1643)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S369000
Reexamination Certificate
active
06548286
ABSTRACT:
1. INTRODUCTION
The present invention relates to methods and compositions for increasing the production of high titre stocks of recombinant AAV (rAAV) through regulation of expression of the AAV REP proteins. The methods and compositions of the invention are based on the observation that low level expression of the AAV REP protein increases the efficiency of rAAV DNA replication and the production of AAV viral capsid protein resulting in production of higher titre recombinant viral stocks. The invention encompasses methods and compositions for controlling the level of REP expression at the transcriptional or translational level. Additionally, the invention provides methods for regulating the biological activity and/or stability of the REP proteins at the post-translational level. The methods and compositions of the invention can be used to produce high titre stocks of rAAV which can be used in gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human diseases including inherited and aquired disorders.
2. BACKGROUND OF THE INVENTION
2.1. GENE THERAPY
Gene therapy is generally understood to refer to techniques designed to deliver functionally active therapeutic genes into targeted cells. Such therapeutic genes may encode proteins that complement genetic deficiencies, cytokines, cell surface membrane proteins or any protein that functions to regulate cell growth and/or differentiation. Such proteins may function intracellularly, for example, by regulating a signalling pathway or transcriptional pathway. Alternatively, the proteins may be secreted by the cell and exert their effect extracellularly.
Initial efforts toward somatic gene therapy have relied on indirect means of introducing genes into tissues, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and reimplanted into the body. These types of techniques are generally referred to as in vitro treatment protocols.
In addition, recombinant replication-defective viral vectors have been used to infect cells both in vitro and in vivo. Perhaps the most widely studied viral vectors for use in gene therapy have been the retroviral vectors. The major disadvantages-associated with the use of retroviral vectors include the inability of many viral vectors to infect non-dividing cells, problems associated with insertional mutagenesis and potential helper virus production. Recently, attention has turned to the use of other types of recombinant viral vectors such as adenovirus and adeno-associated virus based vectors, that may be used to deliver genes of interest to cells.
In particular, recombinant adeno-associated virus has many features of interest in the field of gene therapy. The vectors are based on a defective, nonpathogenic human parvovirus that can infect both dividing and non-dividing cells without a marked tropism. In addition, the viral genome can stably integrate within the host genome, facilitating long term gene transfer.
2.2. AAV VIRAL VECTORS
The AAV genome is composed of a linear single stranded DNA molecule of 4680 nucleotides which contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two 145 nucleotide inverted terminal (ITR) repeat sequences that contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. In addition, the ITR sequences are needed for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions (Muzyczka, N., 1992, Current Topics in Microbiology & Immunology. 158, 97-129).
AAV can assume two pathways upon infection into the host cell depending on whether helper virus is present. In the presence of helper virus, AAV will enter the lytic cycle whereby the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome will integrate as a provirus into a specific region of the host cell genome through recombination between the AAV termini and host cell sequences (Cheung, A. et al., 1980, J. Virol. 33:739-748; Berns, K. I. et al., 1982, in Virus Persistence, eds. Mahey, B. W. J., et al. (Cambridge Univ. Press, Cambridge), pp. 249-265).
The use of AAV as a vehicle for the transfer of genetic information has been facilitated by the discovery that when a plasmid containing an intact AAV genome is transfected into a host cell the recombinant AAV vector will integrate into the host cell genome and remain as a provirus until the host cell subsequently becomes infected with a helper virus. Upon infection of the host cell with helper virus, the AAV is rescued out from the plasmid vector and enters the lytic cycle leading to production of mature virions.
The production of rAAV particles, utilizes a vector containing a transgene flanked by the inverted terminal repeats (ITR), which are the sole AAV cis sequences required for DNA replication, packaging and integration. To produce rAAV particles, the AAV (Rep) and capsid (Cap) gene products are normally provided in trans from a different template, usually a helper plasmid.
The three viral coat proteins, VP1, VP2, and VP3 which are required for virion expression are derived from mRNA initiated at the p40 promoter, while the four overlapping non-structural Rep proteins are essential for AAV DNA replication. Rep78 and 68 are expressed from unspliced and spliced transcripts initiating at the p5 promoter, while Rep52 and Rep40 are similarly produced from transcripts initiating at the p19 promoter. Although Rep52/40 have been implicated in AAV single stranded DNA formation (Chejanovsky et al., 1989, Virology 173:120-128) and gene regulation, Rep appear to display all enzyme functions essential for AAV DNA replication, (ITR binding, DNA helicase, and DNA site-specific nicking activity), (Muzyczka, N., 1991, Seminars in Virology 2:281-290). In addition to these functions, Rep both positively and negatively regulate AAV promoters (Labow et al., 1986, Journal of Virology 60:215-258; Pereira et al., 1997, J. Virol, In Press; Tratschin et al., 1986, Mol. Cell Biol. 6:2884-2894) and repress numerous heterologous promoters (Antoni et al., 1991, Journal of Virology 65:396-404; Heilbronn et al., 1990, Journal of Virology 64:3012-3018; Hermonat, P. L., 1994, Cancer Letters 81:129-36; Horer, et al., 1995, Journal of Virology 69:5485-5496; Labow et al., 1987, Molecular & Cellular Biology 7:1320-1325).
Rep gene expression appears to be critical for all steps of the AAV life cycle, including a latent state which occurs in the absence of a helper virus (Berns, K. I., 1990, Virology, 2ed, vol. 2; Berns, K. I., 1996, B. N. Fields et al. ed.; Samulski et al., 1989, Journal of Virology 63:3822-3828). Recently, Rep have also been associated with AAV site-specific integration (Giraud et al., 1994, Proceedings of the National Academy of Sciences of the United States of America; Kotin et al., 1990, Proceedings of the National Academy of Sciences of the United States of America 87:221-2215; Samulski et al., 1991, EMBO Journal 10:3941-3950; Weitzmann et al., 1994, Proceedings of the National Academy of Sciences of the United States of America 91:5808-5812). Repression of viral gene expression by rep and host YY1 protein appears to be required for establishment and maintenance of the latent state (Labow et al., 1986, Journal of Virology 60:251-258; Laughlin et al., 1982, Journal of Virology 41:868-876; Periera et al., 1997, J. Virol In Press; Shi et al., 1991, Cell 67:377-388). Such repression may be necessary to avoid the demonstrated cytostatic effect on the host cell by Rep gene products (Yang et al., 1994, Journal of Virology 68:4847-4856). During a lytic infection, the AAV promoters, particularly p5, are transactivated by the adenovirus ElA proteins and YY1 (Lewis, et al., 1995, J. Virol. 69:1628-1636; Shi et al., 1991, Cell. 67:377-388). The p5 products then positively regulat
Samulski Richard Jude
Snyder Richard
Xiao Xiao
Cell Genesys Inc.
Cell Genesys, Inc.
Judge Linda R.
Ketter James
Pennie & Edmonds LLP
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