Production of rAAV in vero cells using particular adenovirus helpers
The present invention relates to methods and materials for recombinant adeno-associated virus production. More particularly, in some embodiments the invention contemplates the use of an adenovirus known as Simian Adenovirus 13 (SAdV-13) and Vero cells for production of recombinant adeno-associated virus (rAAV).
1. A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
a) stably transforming a Vero cell with a rAAV genome and AAV rep/cap genes to generate a Vero producer cell,
b) infecting the Vero producer cell with simian adenovirus 13 (SAdV-13) helper virus and
c) culturing the Vero producer cell of step b) to produce rAAV.
2. A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
a) stably transforming the Vero cell with AAV rep/cap genes to generate a Vero packaging cell,
b) introducing a rAAV genome into the Vero packaging cell,
c) infecting the Vero packaging cell of step b) with simian adenovirus 13 (SAdV-13) helper virus and
d) culturing the Vero packaging cell of step c) to produce rAAV.
3. The method of claim 2 , wherein the rAAV genome is introduced into the Vero packaging cell by infection with a rAd/AAV hybrid.
4. A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
a) introducing a rAAV genome and AAV rep/cap genes into a Vero cell,
b) infecting the Vero cell of step a) with simian adenovirus 13 (SAdV-13) helper virus and
c) culturing the Vero cell of step b) to produce rAAV.
5. The method of claim 4 wherein steps a) and b) occur concurrently.
6. The method of claim 4 wherein the Vero cell is a packaging cell.
7. The method of any of claims 1 - 6 further comprising the step of isolating the rAAV produced by the cell.
8. The method of claim 3 , wherein the Vero cell is infected with the rAd/AAV 16-24 hours after helper virus infection.
9. A method of producing infectious rAAV comprising culturing a Vero producer cell under conditions permissive for rAAV production, wherein the Vero producer cell comprises simian adenovirus 13 (SAdV-13) helper virus.
10. The method of any of claims 4 - 6 , 3 , wherein the SAdV-13 helper virus is SAdV-13 PME-12.
11. The method of 7 wherein the SAdV-13 helper virus is SAdV-13 PME-12.