Method for direct rescue and amplification of integrated viruses from cellular DNA of tissues
AssigneeThe Trustees of the University of Pennsylvania
InventorsGuangping Gao, James M. Wilson, Mauricio R. Alvira
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Abstract
A method for isolating AAV viruses from cellular DNA of non-human primate (NHP) tissues by transfecting the DNA of NHP into 293 cells, rescuing the virus and amplifying it through serial passages in the presence of adenovirus helper functions is provided. Also provided are kits useful for performing this method.
Claims31
A method of direct rescue of adeno-associated viruses (AAV) from cellular DNA from tissues:
- (a) digesting DNA in a sample of genomic DNA from a mammalian cellular source with a restriction enzyme which does not cleave in a native AAV genome;
- (b) transfecting the digested DNA into cells;
- (c) culturing the transfected cell under conditions in which at least the minimal adenoviral functions necessary for packaging and replication of the AAV are expressed in the cells;
- (d) passaging lysate obtained from the culturing step of (c) in cells expressing helper functions;
- and
- (e) optionally subjecting the lysate from the passaging step to further passaging.
A method of direct rescue and detection of adeno-associated viruses (AAV) from cellular DNA from tissues:
- (a) digesting DNA in a sample of genomic DNA from a mammalian cellular source with a restriction enzyme which does not cleave in a native AAV genome;
- (b) transfecting the digested DNA into cells;
- (c) incubating the transfected cells;
- (d) superinfecting the transfected cells with adenoviral helper functions so that the cells contain at least the minimal adenoviral functions necessary for packaging and replication of the AAV;
- (e) culturing the superinfected cell under conditions in which the helper functions necessary for replication of AAV are expressed;
- (d) harvesting crude lysate from the superinfected culture;
- and
- (e) passaging the crude lysate.
A method of direct rescue of adeno-associated viruses (AAV) from cellular DNA from tissues:
- (a) digesting DNA in a sample of genomic DNA from a mammalian cellular source with a restriction enzyme which does not cleave in a native AAV genome;
- (b) co-transfecting the digested DNA and a non-infectious adenoviral helper plasmid comprising adenovirus sequences consisting of the minimal adenoviral functions necessary for packaging and replication of the AAV which are not supplied by the host cell;
- (c) culturing the transfected cell under conditions in which only the minimal adenoviral functions necessary for packaging and replication of the AAV are expressed in the cells;
- (d) treating the cell culture to obtain crude lysate;
- (e) subjecting the crude lysate to a first passage by transfecting the crude lysate into cells and superinfecting the cells with adenoviral helper functions so that the cells contain at least the minimal adenoviral functions necessary for packaging and replication of the AAV;
- and
- (f) optionally passaging two or more additional times.
A method of direct rescue of adeno-associated viruses (AAV) from cellular DNA from tissues:
- (a) digesting DNA in a sample of genomic DNA from a mammalian cellular source with a restriction enzyme which does not cleave in a native AAV genome;
- (b) co-transfecting the digested DNA and an infectious adenoviral helper plasmid comprising the minimal adenoviral functions necessary for packaging and replication of the AAV which are not supplied supplied by the host cell;
- (c) culturing the transfected cell under conditions in which the minimal adenoviral functions necessary for packaging and replication of the AAV are expressed in the cells;
- (d) treating the cell culture to obtain crude lysate;
- and
- (e) optionally subjecting the crude lysate to two or more passages by transfecting the crude lysate into cells.
Dependencies are inferred from the claim text.