High-efficiency AAV helper functions
The present invention provides methods and compositions for producing high titer preparations of recombinant AAV (“rAAV”) virions. The compositions of the present invention include AAV helper function systems and host cells. The present invention also includes methods of using AAV helper function vectors that effect the production of only small amounts of the long forms of Rep protein, and rAAV virions produced by such methods.
1 . A method of producing recombinant AAV virions, comprising the steps of
(a) introducing an AAV vector into a host cell;
(b) introducing an AAV helper function vector comprising an AAV rep coding region into the host cell, wherein said rep coding region comprises a nucleotide sequence coding for long forms of Rep protein and short forms of Rep protein, such that the host cell produces greater quantities of the short forms of Rep protein than of the long forms of Rep protein;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce recombinant AAV virions.
2 . A recombinant AAV virion produced according to the method of claim 1 .
3 . The method of claim 1 , wherein the AAV helper function vector further comprises an AAV cap coding region.
4 . The method of claim 1 , wherein the host cell produces at least ten-fold greater quantities of the short forms of Rep protein than of the long forms of Rep protein.
5 . A host cell comprising an AAV helper function vector, said AAV helper function vector comprising an AAV rep coding region, wherein the rep coding region comprises a nucleotide sequence that codes for long forms of Rep protein and short forms of Rep protein, such that the host cell produces greater quantities of the short forms of Rep protein than of the long forms of Rep protein.
6 . The host cell of claim 5 , wherein the AAV helper function vector further comprises an AAV cap coding region.
7 . A method of producing recombinant AAV virions, comprising the steps of:
(a) introducing an AAV vector into a host cell;
(b) introducing an AAV helper function vector comprising an AAV rep coding region into the host cell, wherein said rep coding region comprises a nucleotide sequence coding for long forms of Rep protein and short forms of Rep protein, and said AAV helper function vector causes the host cell to produce an amount of the long forms of Rep protein that is substantially less than an amount produced from an AAV helper function vector expressing the long forms of Rep protein under control of an AAV p5 promoter;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce recombinant AAV virions.
8 . A recombinant AAV virion produced according to the method of claim 7 .
9 . The method of claim 7 , wherein the AAV helper function vector further comprises an AAV cap coding region.
10 . A host cell comprising an AAV helper function vector, said AAV helper function vector comprising an AAV rep coding region, wherein the rep coding region comprises a nucleotide sequence that codes for long forms of Rep protein and short forms of Rep protein, and said AAV helper function vector causes the host cell to produce an amount of the long forms of Rep protein that is substantially less than an amount produced from an AAV helper function vector expressing the long forms of Rep protein under control of an AAV p5 promoter.
11 . The host cell of claim 10 , wherein the AAV helper function vector further comprises an AAV cap coding region.
12 . A method of producing recombinant AAV virions, comprising the steps of:
(a) introducing an AAV vector into a host cell;
(b) introducing an AAV helper function vector comprising AAV rep and cap coding regions into the host cell to express Rep and Cap gene products, wherein the rep coding region is under the control of an inducible promoter that expresses an amount of rep RNA that is substantially less than an amount expressed from an AAV p5 promoter;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce recombinant AAV virions.
13 . A recombinant AAV virion produced according to the method of claim 12 .
14 . A method of producing recombinant AAV virions, comprising the steps of:
(a) introducing an AAV vector into a host cell;
(b) introducing an AAV helper construct comprising AAV rep and cap coding regions into the host cell to express Rep and Cap gene products, said rep coding region comprising a nucleotide sequence coding for long forms of Rep protein and short forms of Rep protein, wherein the rep coding region is regulated by an inducible promoter such that the host cell produces greater quantities of the short forms of Rep protein than of the long forms of Rep protein;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce recombinant AAV virions.
15 . A recombinant AAV virion produced according to the method of claim 14 .
16 . The method of claim 14 , wherein the host cell produces at least ten-fold greater quantities of the short forms of Rep protein than of the long forms of Rep protein.