AAV production in insect cells, methods and compositions therefor
Compositions and methods are disclosed for producing adeno-associated virus (AAV) in insect cells in vitro. Recombinant baculovirus vectors include an AAV Capsid gene expression cassette (Cap), an AAV Rep gene expression cassette (Rep), and a baculovirus homologous region (hr) located up to about 4 kb from a start codon in an AAV expression cassette. Production levels of baculovirus and AAV in insect cells harboring recombinant baculovirus comprising a Cap, a Rep, and an hr are higher compared to controls comprising a Cap and a Rep but no hr. Furthermore, levels of baculovirus and AAV production in insect cells infected with recombinant baculovirus comprising a Cap, a Rep, and an hr are comparatively stable over serial passages of cells, whereas levels of baculovirus and AAV production decline over serial passages of insect cells comprising recombinant baculovirus comprising a Cap and a Rep, but no hr.
1. A baculovirus vector for adeno-associated virus (AAV) production, comprising:
an AAV Cap expression cassette;
an AAV Rep expression cassette; and
a baculovirus homologous region 2 (hr2) located up to about 4 kb from a start codon of an AAV expression cassette, wherein the vector has no Rep binding element (RBE) sequence, no inverted terminal region (ITR) containing an RBE sequence, and no promoter containing an RBE sequence.
2. The vector in accordance with claim 1 , wherein the hr2 region is between the Rep expression cassette and the Cap expression cassette, and wherein the Rep expression cassette and the Cap expression cassette are in a head to head (5′ to 5′) orientation.
3. An insect cell line comprising cells comprising a vector in accordance claim 1 .
4. The insect cell line in accordance with claim 3 , wherein the cells further comprise a second vector, said second vector comprising a transgene flanked by AAV ITRs.
5. A method of growing baculovirus in vitro, comprising: providing a culture of insect cells in accordance with claim 3 ; and incubating the cells.
6. The method in accordance with claim 5 , wherein the incubating the cells comprises passaging the cells, and wherein AAV production yield at passage 7 is at least 2-fold greater compared to a control insect cell line comprising a baculovirus vector comprising an AAV Cap expression cassette and an AAV Rep expression cassette but no baculovirus hr.
7. The method in accordance with claim 5 , wherein the titer at passage 7 of baculovirus comprising the AAV Cap expression cassette is greater than 21.5% of total baculovirus titer.
8. A method of growing AAV in vitro, comprising:
providing a culture of insect cells;
infecting or transfecting the insect cells with a baculovirus vector in accordance with claim 1 ; and
incubating the cells.
9. The method in accordance with claim 8 , wherein the yield at P7 of AAV from the insect cells is at least 50% greater than the yield at P7 of AAV from insect cells comprising a baculovirus vector without the hr.
10. The method in accordance with claim 8 , wherein the yield at P7 of AAV from cells comprising the baculovirus hr is at least 20% greater than the yield of AAV from cells comprising a baculovirus vector without the hr.
11. The method in accordance with claim 8 , wherein the baculovirus vector is exclusive of a Rep binding element (RBE).
12. A method of producing AAV in vitro, comprising growing an insect cell culture comprising a vector in accordance with claim 1 , and a vector comprising a transgene flanked by AAV ITRs.