In vitro packaging of adeno-associated virus DNA
AssigneeThe Research Foundation of State University of New York
InventorsXiaohuai Zhou, Nicholas Muzyczka, Sergei Zolotukhin, Tiehua Ni
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Abstract
A method for in vitro packaging of adeno-associated viral particles is described. The procedure involves the preparation of cell-free extracts containing all the essential components for packaging. Homogeneous purified substrate DNA for packaging may be prepared separately. The in vitro packaged AAV particles are useful in transduction of mammalian cells and for gene therapy in animals. In one described method, the DNA packaged into AAV particles is not limited by the size constraints characteristic of in vivo packaged AAV particles.
Claims19
A method for in vitro packaging of a DNA substrate into AAV particles that transduce a recipient mammalian cell comprising the steps of:
- (a) transfecting a mammalian host cell culture permissive for AAV replication with a dAAV vector containing AAV capsid and Rep gene coding sequences;
- (b) infecting said host cell culture with a helper virus;
- (c) after said transfecting and said infecting, preparing an extract free of DNA from the transfected cell culture;
- (d) combining said extract with said DNA substrate;
- and,
- (e) incubating said extract under conditions to promote packaging of said DNA substrate.
Chloroform sensitive AAV particles that transduce a recipient mammalian cell prepared by the method comprising the steps of:
- (a) transfecting a mammalian host cell culture permissive for AAV replication with a dAAV vector containing AAV capsid and Rep gene coding sequences;
- (b) infecting said host cell culture with a helper virus;
- (c) after said transfecting and said infecting, preparing an extract free of DNA from the transfected cell culture;
- (d) combining said extract with a DNA substrate;
- and,
- (e) incubating said extract under conditions to promote packaging of said substrate DNA.
Chloroform sensitive AAV particles that transduce a recipient mammalian cell prepared by the method comprising the steps of:
- (a) transfecting a mammalian host cell culture permissive for AAV replication with a dAAV vector containing AAV capsid and Rep gene coding sequences;
- (b) infecting said host cell culture with a helper virus;
- (c) after said transfecting and said infecting, preparing an extract free of DNA from the transfected cell culture;
- (d) combining said extract with a DNA substrate;
- and,
- (e) incubating said extract under conditions to promote packaging of said substrate DNA;
- and
- (f) heating said extract at sufficient temperature for sufficient time to inactivate the helper virus.
A method for preparing a composition that packages in vitro a DNA substrate into AAV particles that transduce a recipient mammalian cell, said method comprising:
- (a) transfecting a mammalian host cell culture permissive for AAV replication with a dAAV vector containing AAV capsid and Rep gene coding sequences;
- (b) infecting said host cell culture with a helper virus;
- (c) after said transfecting and said infecting, preparing an extract free of DNA from the transfected cell culture.
Dependencies are inferred from the claim text.