Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-01-09
1998-11-17
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352351, 4353201, 435369, C12N 1563
Patent
active
058374841
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to gene therapy and more specifically to stable cell lines that are useful for packaging recombinant adeno-associated virus type 2 (AAV) vectors without producing wild-type AAV.
BACKGROUND
AAV vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents (reviewed in Carter, 1992, Current Opinion in Biotechnology, 3:533-539; Muzcyzka, 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 (Flotte et al., 1992a, 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; Flotte et al., 1993b, Proc. Natl. Acad. Sci. USA, (1993) 90:10613-10617), human bone marrow-derived erythroleukemia cells (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261), and several others. AAV may not require active cell division for stable expression which would be a clear advantage over retroviruses, especially in tissue such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
AAV is a defective parvovirus that grows 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. 1, pp. 169-226, Carter, 1989, Handbook of Parvoviruses, Vol II, pp. 247-281, Berns, 1990, Virology, pp. 1743-1764, Raven Press, N.Y.). 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 known but appears to be some indirect effect of the helper virus which renders the cell permissive for AAV replication. This concept 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 either genotoxic or that disrupt the cell cycle.
AAV has a very broad host range with neither obvious species or tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin provided an appropriate helper is present. AAV is ubiquitous and has been isolated from a wide variety of animal species including most mammalian and several avian species.
AAV has not been identified as the cause of any disease. 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 also 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.
The AAV genome has one copy of the 145-nucleotide-long ITR (inverted terminal repeat) on each end 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 p.sub.5, P.sub.19, and p.sub.40 (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA, 76:5567-5571) that are used to express the rep and cap genes. The ITR sequences are required in cis and are sufficient to provide a functional origin of replication (ori) and also are sufficient to provide signals required for integration into the cell genome as well as for efficient excision and rescue from host cell chromosomes or from recombinant plasmids. In addition, it has been shown that the ITR can function directly
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Trempe James P.
Yang Qicheng
Ketter James
Medical College of Ohio
Yucel Irem
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