Temperature-sensitive regulation of viral vector production

Details Temperature-sensitive regulation of viral vector production Temperature-sensitive regulation of viral vector production

2000-09-29

2003-09-30

Housel, James (Department: 1648)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C424S233100, C435S320100, C435S235100, C435S325000

Reexamination Certificate

active

06627617

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to reagents and methods for producing virus vectors, in particular, reagents and methods for producing adeno-associated virus vectors.
BACKGROUND OF THE INVENTION
Adeno-associated virus (AAV) type 2 is a nonpathogenic human parvovirus that generally depends on coinfection with a helper virus (adenovirus or herpesvirus) for efficient replication (reviewed in Berns (1996). Parvoviridae: The viruses and their replication, p. 2173-2197, in B. N. Fields (ed.), Fields Virology, 3
rd
ed., vol. 2, Raven, Philadelphia). The linear, single-stranded DNA genome of AAV encodes two open reading frames (rep and cap) flanked by 145 bp inverted terminal repeats (ITR) (Srivastava et al., (1983)
J. Virol.
45:555). Replication of the AAV genome requires two viral components, the ITR that serves as the origin of replication (Hauswirth et al., (1977)
Virology
78:488; Straus et al., (1976)
Proc. Natl. Acad. Sci. USA
73:742; Samulski et al., (1983)
Cell
33:135; Senepathy et al., (1984)
J. Mol. Biol.
179:1) and the rep gene products (Senepathy et al., (1984)
J. Mol. Biol.
179:1, Hermonat et al., (1984)
J. Virology
51:329; Tratschin et al., (1984)
J. Virology
51:611). The rep gene encodes four multifunctional proteins (Hermonat et al., (1984)
J. Virology
51:329; Tratschin et al., (1984)
J. Virology
51:611; Mendelson et al., (1986)
J. Virology
60:823; Trempe et al., (1987)
Virology
161:18) that are expressed from two promoters at map units 5 (p5) and 19 (p19). The larger Rep proteins transcribed from the p5 promoter (Rep78 and Rep68), are essentially identical except for unique carboxy termini generated from unspliced (Rep78) and spliced (Rep68) transcripts, respectively (Srivastava et al, (9183)
J. Virol.
45:555). Two smaller rep proteins (Rep52, Rep40), transcribed from the p19 promoter are amino terminal truncations of Rep78 and Rep68, respectively.
Several biochemical activities of Rep78 and Rep68 have been characterized as necessary for AAV replication. These include specific binding to the AAV ITR (Ashktorab et al., (1989)
J. Virology
63:3034; Im et al., 1989)
J. Virology
63:3095; Snyder et al., (1993)
J. Virology
67:6096) and site-specific endonuclease cleavage at the terminal resolution site (trs) (Im et al., (1990)
J. Virology
63:447; Im et al., (1992)
J. Virology
66:1119; Snyder et al., (1990)
Cell
60:105; Snyder et al., (1990)
J. Virology
64:6204). Rep78/68 also possess ATP dependent DNA-DNA helicase ((Im et al., (1990)
J. Virology
63:447; Im et al., (1992)
J. Virology
66:1119) and DNA-RNA helicase as well as ATPase activities (Wonderling et al., (1995)
J. Virology
69:3542). In addition to these activities required for replication, Rep78/68 also regulate transcription from the viral promoters (Beaton et al., (1989)
J. Virology
63:4450; Labow et al., (1986)
J. Virology
60:251; Tratschin et al., (1986)
Mol. Cellular Biol.
6:2884; Kyostio et al., (1994)
J. Virology
68:2947; Pereira et al., (1997)
J. Virology
71:1079), and have been shown to mediate viral targeted integration (Xiao, W., (1996), “Characterization of cis and trans elements essential for the targeted integration of recombinant adeno-associated virus plasmid vectors”, Ph.D. Dissertation, University of North Carolina-Chapel Hill; Balague et al., (1997)
J. Virology
71:3299; LaMartina et al., (1998)
J. Virology
72:7653; Pieroni et al., (1998)
Virology
249:249).
Mutant studies of the Rep proteins have indicated that the activities of Rep can be divided into partially distinct functional domains (
FIG. 1A
) that are spread throughout the protein (Chejanovsky et al., (1989)
Virology
173:120; McCarty et al., (1992)
J. Virology
66:4050; Yang et al., (1992)
J. Virology
66;6058; Owens et al., (1993)
J. Virology
67:997; Weitzman et al., (1996)
J. Virology
70:2440; Walker et al., (1997)
J. Virology
71:2722; Walker et al., (1997)
J. Virology
71:6996; Davis et al., (1999)
J. Virology
73:2084; Urabe et al., (1999)
J. Virology
73:2682). These include regions required for binding to the ITR; a putative NTP-binding/ATPase domain, nuclear localization and residues putatively required for nicking and helicase functions. Several mutations within the NTP-binding/ATPase domain that lacked trs endonuclease and viral replication were also defective for trans-activation functions suggesting a need for further mutant analysis (McCarty et al., (1992)
J. Virology
66:4050). Since most mutants disrupt multiple Rep mediated functions for the AAV life cycle, detailed characterization of distinct functions has been difficult (McCarty et al., (1992)
J. Virology
66:4050; Yang et al., (1992)
J. Virology
66:6058; Owens et al., (1993)
J. Virology
67:997; Weitzman et al., (1996)
J. Virology
70:2440; Walker et al., (1997)
J. Virology
71:2722; Walker et al., (1997)
J. Virology
71:6996; Davis et al., (1999)
J. Virology
73:2084; Urabe et al., (1999)
J. Virology
73:2682).
One of the considerations in designing methods for production or delivery of AAV is the toxicity of the AAV Rep proteins to helper viruses (e.g., adenovirus). Thus the AAV rep/cap genes are typically provided on a separate vector from the helper virus or silenced in the cell chromosome. In addition, the rep gene products are frequently cytotoxic to the cells used to package rAAV vectors.
The use of temperature sensitive (ts) mutations has proven to be an effective method for elucidating the essential functions of viral proteins (Murphy et al., (1988)
Virus Research
11:1; Crowe et al., (1996)
Virus Genes
13:269; Rhode (1978)
J. Virology
25:215; Burns et al., (1992)
Virology
189:568). One approach for generating ts mutants has been to utilize the charged-to-alanine mutagenesis strategy (Cunningham et al., (1989)
Science
244:1081; Bennett et al., (1991)
J. Biological Chemistry
266:5191; Bass et al., (1991)
Proc. Nat. Acad. Sci. USA
88:4498; Wertman et al., (1992)
Genetics
132:337; Diamond et al., (1994)
J. Virology
68:863; Parkin et al., (1996)
Virus Research
46:31). The rationale of this approach is that since most charged residues are found on the protein surface they are expected to exert little effect on protein folding or stability (Cunningham et al., (1989)
Science
244:1081; Wertman et al., (1992)
Genetics
132:337; Dao-Pin et al., (1991)
Biochemistry
30:11521), but could feasibly make a protein more thermosensitive by disrupting electrostatic and H-bonding interactions (Diamond et al., (1994)
J. Virology
68:863). This technique does not always yield ts proteins, but is a popular approach when the crystal structure of the protein in question is lacking.
Accordingly, ts AAV Rep mutants would be advantageous to provide a functional Rep protein that may be controlled or inactivated at non-permissive temperatures so that the toxicity normally associated with AAV Rep proteins may be diminished or avoided.
SUMMARY OF THE INVENTION
The present invention addresses a need in the art for improved strategies for producing AAV vectors. In addition, the present invention is directed to a need in the art for improved reagents and methods for gene delivery.
Current techniques for packaging AAV vectors are not readily amenable to large-scale production. These difficulties arise, in part, from toxicity of the AAV Rep proteins to helper viruses and host cells, thus requiring that the AAV rep/cap genes be provided on a separate vector from the helper virus or silenced in the cell chromosome. In addition, the rep gene products are frequently cytotoxic to the cells used to package rAAV vectors. The present invention provides temperature-sensitive (ts) AAV Rep proteins, and nucleotide sequences encoding the same, that may be used according to the methods disclosed herein to reduce or mitigate the problems posed by Rep protein toxicity. The activity of the inventive ts Rep proteins may be readily controlled by shifting the cells packaging the rAAV vectors to permissive or non-permissive temperatures as desired.
As one aspect, the present invention prov

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