Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-10-20
2002-02-12
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100, C435S325000, C435S235100, C435S440000, C435S455000, C435S456000, C435S457000
Reexamination Certificate
active
06346415
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of recombinant adeno-associated virus (AAV) vectors and preparations thereof that can be used for gene transfer.
BACKGROUND
AAV vectors are among a small number of recombinant virus vector systems which have been shown to be useful as in vivo gene transfer agents (reviewed in Carter, 1992,
Curr. Opin. Biotech.,
3:533-539; Muzyczka, 1992,
Curr. Top. Microbiol. Immunol.
158:97-129) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of high-frequency stable DNA integration and expression in a variety of cells, including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992,
Am. J. Respir. Cell Mol. Biol.
7:349-356; Egan et al., 1992,
Nature,
358:581-584; Flotte et al., 1993a,
J. Biol. Chem.
268:3781-3790; and Flotte et al., 1993b,
Proc. Natl. Acad. Sci. USA,
93:10163-10167); human bone marrow-derived erythroleukemia cells (see, e.g., Walsh et al., 1992,
Proc. Natl. Acad. Sci. USA,
89:7257-7261); and several others. Unlike retroviruses, AAV does not appear to require ongoing cell division for stable integration; a clear advantage for gene therapy in tissue such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
AAV is a defective parvovirus that generally replicates only in cells in which certain functions are provided by a co-infecting helper virus. General reviews of AAV may be found in Carter, 1989,
Handbook of Parvoviruses,
Vol. I, pp. 169-228; and Berns, 1990,
Virology,
pp. 1743-1764, Raven Press, New York. Examples of co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. The nature of the helper function is not entirely known but it appears that an indirect effect of the helper virus is to render the cell permissive for AAV replication. This belief is supported by the observation that in certain cases AAV replication may occur at a low level of efficiency in the absence of helper virus co-infection if the cells are treated with agents that are genotoxic or that disrupt the cell cycle.
Generally, in the absence of helper virus, AAV infection results in high-frequency, stable integration of the AAV genome into the host cell genome. The integrated AAV genome can be rescued and replicated to yield a burst of infectious progeny AAV particles if cells containing an integrated AAV provirus are superinfected with a helper virus such as adenovirus. Since the integration of AAV appears to be an efficient event, AAV can be a useful vector for introducing genes into cells for stable expression for uses such as human gene therapy.
AAV has a very broad host range without any obvious species or tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin, provided that an appropriate helper is present. AAV appears to be ubiquitous as it has been isolated from a wide variety of animal species, including most mammalian and several avian species.
AAV has not been associated with the cause of any disease and AAV is not a transforming or oncogenic virus. AAV integration into chromosomes of human cell lines does not cause any significant alteration in the growth properties or morphological characteristics of the cells. These properties of AAV further recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application (such as retroviruses, adenoviruses, herpesviruses, or poxviruses) are disease-causing viruses.
AAV particles are comprised of a capsid having three proteins, VP1, VP2, and VP3, and enclosing a DNA genome. The AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5×10
6
daltons or approximately 4680 nucleotides long. Strands of either sense (“plus” or “minus”) are packaged into individual particles but each particle has only one DNA molecule. Equal numbers of AAV particles contain either a plus or minus strand. Virus particles containing either strand are equally infectious and replication occurs by conversion of the parental infecting single stranded DNA to a duplex form and subsequent amplification of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids. Duplex or single-strand copies of AAV genomes inserted into bacterial plasmids or phagemids can result in infectious particles when transfected into adenovirus-infected cells, and this has allowed the study of AAV genetics and the development of AAV vectors.
In the case of subtype AAV2, the genome has two copies of a 145-nucleotide-long ITR (inverted terminal repeat), one on each end of the genome, and a unique sequence region of about 4470 nucleotides long (Srivastava et al., 1983,
J. Virol.,
45:555-564) that contains two main open reading frames for the rep and cap genes (Hermonat et al.,
J. Virol.
51:329-339; Tratschin et al., 1984a,
J. Virol.,
51:611-619). The unique region contains 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.
ITR sequences are involved in a variety of activities in the AAV life cycle. The ITR sequences, each of which can form a hairpin structure, provide a functional origin of replication (ori) and are required in cis for AAV DNA replication and for rescue and excision from prokaryotic plasmids (Samulski et al., 1983,
Cell
33: 135-143; Samulski et al., 1987,
J. Virol.
61: 3096-3101; Senapathy et al., 1984,
J. Mol. Biol.
179: 1-20; Gottlieb and Muzyczka, 1988,
Mol. Cell. Biol.
6: 2513-2522). In addition, the ITRs appear to be the minimum sequences required for AAV proviral integration and for packaging of AAV DNA into virions (McLaughlin et al., 1988,
J. Virol.
62: 1963-1973; Samulski et al., 1989,
J. Virol.
63: 3822-3828; Balague et al., 1997,
J. Virol.
71: 3299-3306). In the case of DNA replication, it is clear that most of the terminal 125 nucleotide palindrome is required for DNA replication and terminal resolution (Bohenzky et al., 1988,
Virology
166: 316-327; LeFebvre et al., 1984,
Mol. Cell. Biol.
4:1416-1419; Im and Muzyczka, 1989,
J. Virol.
63:3095-3104; Ashktorab and Srivastava, 1989,
J. Virol.
63: 3034-3039).
Several reports indicated that ITRs generally do not behave as transcriptional regulatory sequences (Muzyczka, 1992; and Walsh et al., 1992) and the deletion of the ITR does not have a major effect on AAV p5 promoter activity (Flotte et al., 1992). Since ITRs were not thought to provide transcriptional activity, AAV vectors have been constructed using AAV promoters to express heterologous genes. See, for example, Carter et al., U.S. Pat. No. 4,797,368, issued Jan. 10, 1989. Subsequent reports by Carter and collaborators have shown ITRs to have a low amount of transcriptional activity in transient and stable expression assays. See, e.g., Carter et al. U.S. Pat. No. 5,587,308, issued Dec. 24, 1996, and Flotte et al., 1993a.
In addition to the requirement that ITR sequences be present in cis, the AAV rep and cap genes are required, in cis or in trans, to provide functions for the replication and encapsidation of the viral genome, respectively. As described below, recombinant AAV (rAAV) vectors for use in gene therapy preferably do not contain the AAV cap or rep genes, but rather these genes can be provided by a host cell used for packaging (typically referred to as an “AAV producer cell”).
In the intact AAV genome, the rep gene is expressed from two promoters, p5 and p19, as noted above. Transcription from p5 yields an unspliced 4.2 kb mRNA which encodes a nonstructural protein, Rep78, and a spliced 3.9 kb mRNA which encodes a second nonstructural protein, Rep68. Transcription from p19 yields an unspliced mRNA which encodes Rep52 and a spliced 3.3 kb mRNA which encodes Rep40. Thus, the four Rep proteins all comprise a common internal region
Morrison & Foerster / LLP
Mosher Mary E.
Targeted Genetics Corporation
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