Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-02-19
2002-12-03
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S455000, C435S456000, C435S235100, C435S320100
Reexamination Certificate
active
06489162
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to a method for producing substantially pure stocks of recombinant adeno-associated virus (AAV), free of the adeno-associated helper virus found associated with previously available recombinant AAV. According to the invention, the substantially pure stocks of recombinant AAV may be used to introduce exogenous genetic sequences into cells, cell lines, or organisms; in the absence of the adeno-associated helper virus, the recombinant AAV will remain stably integrated into cellular DNA. In another embodiment of the invention cells containing integrated recombinant AAV may be exposed to helper viruses, resulting in excision, replication, and amplification of integrated sequences, thereby providing a means for achieving increased expression of gene product. The present invention also provides for novel recombinant AAV vectors and adeno-associated helper viruses.
2. BACKGROUND OF THE INVENTION
2.1. VIRAL VECTORS
Viral vectors permit the expression of exogenous genes in eukaryotic cells, and thereby enable the production of proteins which require postranslational modifications unique to animal cells. Viral expression vectors (reviewed in Rigby, 1983, J. Gen. Virol. 64:255-266) have been developed using DNA viruses, such as papovaviruses (i.e. SV40), adenoviruses, herpes viruses, and poxviruses (i.e. vaccinia virus, Mackett et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419; Panicoli et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4927-4931) and RNA viruses, such as retroviruses.
In disclosing the construction and applications of a murine retrovirus shuttle vector, Cepko et al. (1984, Cell 37:1053-1062) cites several properties which may be desirable in a mammalian gene transfer system, including, the ability of the vector to be introduced into a wide range of hosts and the recoverability of transferred sequences as molecular clones (i.e. a vector which can “shuttle” between animal and bacterial cells; see DiMaio et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4030-4034). As efficient shuttle vectors, retroviruses have become a popular vehicle for transferring genes into eukaryotic cells. Retrovirus packaging cell lines (Mann et al., 1983, Cell 33:153-159; Watanabe and Temin, 1983, Mol. Cell. Biol. 3:2241-2249; Cohn and Mulligan, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:63496353; Sorge et al., 1984, Mol. Cell. Biol. 4:1730-1737) allow production of replication-defective retrovirus vectors in the absence of helper virus; the defective retroviral vectors are able to infect and integrate into cells but cannot replicate. The ability to produce helper-free stocks of defective retroviruses using packaging cell lines protects against spread of the recombinant virus, and avoids possible dissemination of recombinant virus-induced disease. However, some retrovirus packaging lines have been shown to produce only low titers of retroviral vectors, or produce high levels of helper virus; furthermore, some retroviruses exhibit limited host ranges (Miller and Baltimore, 1986, Mol. Cell. Biol. 6:2895-2902). The recognition of human retroviruses over the past decade as the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS) and some cases of T-cell and hairy cell leukemia, and the numerous examples of oncogenic animal retroviruses, have created an awareness of health risks potentially associated with the use of retrovirus vectors, particularly relevant to future prospects in human gene therapy. Many of the alternative viral vectors currently available either do not integrate into host cells at high frequency, are not easily rescuable from the integrated state, are limited in their host range, or include other viral genes, thereby creating a need for the development of a safe and efficient viral vector system.
2.2. ADENO-ASSOCIATED VIRUS
Adeno-associated virus (AAV) is a defective member of the parvovirus family. The AAV genome is encapsidated as a single-stranded DNA molecule of plus or minus polarity (Berns and Rose, 1970, J. Virol. 5:693-699; Blacklow et al., 1967, J. Exp. Med. 115:755-763). Strands of both polarities are packaged, but in separate virus particles (Berns and Adler, 1972, Virology 9:394-396) and both strands are infectious (Samulski et al., 1987, J. Virol. 61:3096-3101).
The single-stranded DNA genome of the human adeno-associated virus type 2 (AAV2) is 4681 base pairs in length and is flanked by inverted terminal repeated sequences of 145 base pairs each (Lusby et al., 1982, J. Virol. 41:518-526). The first 125 nucleotides form a palindromic sequence that can fold back on itself to form a “T”-shaped hairpin structure and can exist in either of two orientations (flip or flop), leading to the suggestion (Berns and Hauswirth, 1979, Adv. Virus Res. 25:407-449) that AAV may replicate according to a model first proposed by Cavalier-Smith for linear-chromosomal DNA (1974, Nature 250:467-470) in which the terminal hairpin of AAV is used as a primer for the initiation of DNA replication. The AAV sequences that are required in cis for packaging, integration/rescue, and replication of viral DNA appear to be located within a 284 base pair (bp) sequence that includes the terminal repeated sequence (McLaughlin et al., 1988, J. Virol. 62:1963-1973).
At least three regions which, when mutated, give rise to phenotypically distinct viruses have been identified in the AAV genome (Hermonat et al., 1984, J. Virol. 51:329339). The rep region codes for at least four proteins (Mendelson et al., 1986, J. Virol. 60:823-832) that are required for DNA replication and for rescue from the recombinant plasmid. The cap and lip regions appear to encode for AAV capsid proteins; mutants containing lesions within these regions are capable of DNA replication (Hermonat et al., 1984, J. Virol. 51:329-339). AAV contains three transcriptional promoters (Carter et al., 1983, in “The Parvoviruses”, K. Berns ed., Plenum Publishing Corp., NY pp. 153-207; Green and Roeder, 1980, Cell 22:231-242; Laughlin et al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5567-5571; Lusby and Berns, 1982, J. Virol. 41:518-526; Marcus et al., 1981, Eur. J. Biochem. 121:147-154). The viral DNA sequence displays two major open reading frames, one in the left half and the other in the right half of the conventional AAV map (Srivastava et al., 1985, J. Virol. 45:555-564).
AAV-2 can be propagated as a lytic virus or maintained as a provirus, integrated into host cell DNA (Cukor et al., 1984, in “The Parvoviruses,” Berns, ed., Plenum Publishing Corp., NY pp. 33-66). Although under certain conditions AAV can replicate in the absence of helper virus. (Yakobson et al., 1987, J. Virol. 61:972-981), efficient replication requires coinfection with either adenovirus (Atchinson et al., 1965, Science 194:754-756; Hoggan, 1965, Fed. Proc. Am. Soc. Exp. Biol. 24:248; Parks et al., 1967, J. Virol. 1:171-180); herpes simplex virus (Buller et al., 1981, J. Virol. 40:241-247) or cytomegalovirus, Epstein-Barr virus, or vaccinia virus. Hence the classification of AAV as a “defective” virus.
When no helper virus is available, AAV can persist in the host cell genomic DNA as an integrated provirus (Berns et al., 1975, Virology 68:556-560; Cheung et al., 1980, J. Virol. 33:739-748). Virus integration appears to have no apparent effect on cell growth or morphology (Handa et al., 1977, Virology 82:84-92; Hoggan et al., 1972, in “Proceedings of the Fourth Lepetit Colloquium, North Holland Publishing Co., Amsterdam pp. 243-249). Studies of the physical structure of integrated AAV genomes (Cheung et al., 1980, supra; Berns et al., 1982, in “Virus Persistence”, Mahy et al., eds., Cambridge University Press, NY pp. 249-265) suggest that viral insertion occurs at random positions in the host chromosome but at a unique position with respect to AAV DNA, occurring within the terminal repeated sequence. Integrated AAV genomes have been found to be essentially stable, persisting in tissue culture for greater than 100 passages (Cheung et al., 1980 supra).
Although AAV is believed to be a human virus, its host range for lytic
Chang Long-Sheng
Samulski Richard Jude
Shenk Thomas E.
Judge, Esq. Linda
Mosher Mary E.
Pennie & Edmonds LLP
The Trustees of Princeton University
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