Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1997-02-25
1998-11-10
Degen, Nancy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
424 932, 435 6, 4351723, C12N 1587, C12N 500, C12N 1586, C12Q 170
Patent
active
058341820
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates generally to adeno-associated virus (AAV) vectors for gene transfer and, more specifically, to methods for increasing transduction of cells with recombinant adeno-associated virus (AAV) vectors.
BACKGROUND OF THE INVENTION
The efficient delivery of therapeutic genes to non-dividing cells with resultant long-term stable expression remains a major goal in the development of gene therapy. None of the currently available vector systems has been shown to be capable of both efficient transduction of non-dividing cells and long-term expression through stable integration of the vector genome into host cell DNA. Retroviral vectors based on Moloney murine leukemia virus, while capable of integration and stable long-term expression, require cell division for efficient transduction. Miller D. G. et al., Mol. Cell. Biol 10, 4239-4242 (1990). In contrast, vectors based on adenovirus and herpes simplex virus are capable of transducing non-dividing cells but do not integrate into host cell DNA with any appreciable frequency. See Grunhaus A. et al., Seminars in Virol 3, 237-252 (1992); Geller A. I. et al., Proc. Natl Acad Sci U.S.A. 87, 1149-1153 (1990). The less well characterized vectors based on the dependent parvovirus, adeno-associated virus (AAV), have been shown to integrate, but their potential for transducing non-dividing cells has yet to be fully investigated. See Hermonat P. L. et al., Proc. Natl. Acad Sci. U.S.A. 81, 6466-6470 (1984); Lebrowski J. S. et al., Mol. Cell. Biol. 8, 3988-3996 (1988); McLaughlin S. K. et al., J Virol. 62, 1963-1973 (1988); and Samulski R. J. et al., J Virol. 63, 3822-3828 (1989).
Adeno-associated virus (AAV) vectors are among a small number of recombinant virus vector systems which have been shown to have utility as both in vitro and in vivo gene transfer vectors (reviewed in Carter, 1992, Curr. Opinion Biotech. 3, 533-539 (1992); Muzcyzka, Curr. Top. Microbiol. Immunol. 158, 97-129) (1992) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of stable DNA integration and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (Flotte et al., Am. J Respir. Cell Mol Biol. 7, 349-356 (1992a)); Egan et al., Nature, 358, 581-584 (1992); Flotte et al., J Biol. Chem. 268, 3781-3790 (1993a); human bone marrow-derived erythroleukemia cells (Walsh et al., Proc. Natl. Acad Sci. US.A. 89, 7257-7261 (1992)), and several others. See also Flotte et al., Proc. Natl. Acad Sci. U.S.A., 90, 10613-10617 (1993).
We have recently demonstrated that AAV vectors preferentially transduce cells in S phase of the cell cycle. However, transduction events do occur independent of S phase at low frequency. The factors within a S phase cell that facilitate transduction by AAV vectors remain undefined, but are likely to directly involve or be closely linked to DNA synthesis. Possible factors include host cell polymerases required for the conversion of the single-stranded input genomes to double-stranded molecules and/or cellular factors facilitating vector integration.
SUMMARY OF THE INVENTION
Of the viral vector systems currently available for gene transfer applications, none has been demonstrated to be capable of both efficient transduction of non-dividing cells and long-term expression through stable integration into host cell DNA. We have discovered that the transduction efficiency on non-dividing cells of the integrating vector system based on adeno-associated virus (AAV) can be greatly increased by treatment with agents which affect DNA metabolism. Increased transduction, particularly of non-dividing cells, can facilitate gene transfer and is useful in many applications, including the manufacture of gene products and therapeutic applications. In addition, we have discovered that the transduction efficiency of both dividing and non-dividing cells with recombinant AAV is increased by treatment with agents that alter certain aspects of DNA metabolism, more specifically, that al
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Alexander Ian E.
Miller A. Dusty
Russell David W.
Degen Nancy
Fred Hutchinson Cancer Research Center
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