High-efficiency wild-type-free AAV helper functions
The present invention provides methods and compositions for producing high titer, wild-type-free preparations of recombinant AAV (“rAAV”) virions. The compositions of the present invention include novel nucleic acids encoding AAV helper functions and AAV helper function vectors. The present invention also includes host cells transfected by the claimed nucleic acids, methods of using the claimed vectors, and rAAV virions produced by such methods.
1 . A nucleic acid molecule which encodes one or more AAV helper functions, said nucleic acid molecule comprising:
an AAV rep coding region;
an AAV cap coding region; and
a nucleotide sequence comprising a modified AAV p5 promoter region, such that the modified AAV p5 promoter region no longer functions in transcription initiation.
2 . An AAV helper function vector comprising the nucleic acid molecule of claim 1 .
3 . The AAV helper function vector of claim 2 , wherein the vector is a plasmid.
4 . The AAV helper function vector of claim 2 , further comprising one or more accessory function genes.
5 . A method of producing recombinant AAV (rAAV) virions comprising:
(a) introducing an AAV vector into a suitable host cell;
(b) introducing the AAV helper function vector of claim 2 into the host cell;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce rAAV virions.
6 . A recombinant AAV (rAAV) virion produced by the method of claim 5 .
7 . A host cell comprising the nucleic acid molecule of claim 1 .
8 . The host cell of claim 7 further comprising accessory function genes.
9 . A nucleic acid molecule which encodes one or more AAV helper functions, said nucleic acid molecule comprising:
an AAV rep coding region;
an AAV cap coding region; and
a nucleotide sequence comprising a modified AAV p5 promoter region, wherein the modified p5 promoter region lacks an intact TATA box.
10 . An AAV helper function vector comprising the nucleic acid molecule of claim 9 .
11 . The AAV helper function vector of claim 10 , wherein the vector is a plasmid.
12 . The AAV helper function vector of claim 10 , further comprising one or more accessory function genes.
13 . A method of producing recombinant AAV (rAAV) virions comprising:
(a) introducing an AAV vector into a suitable host cell;
(b) introducing the AAV helper function vector of claim 10 into the host cell;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce rAAV virions.
14 . A recombinant AAV (rAAV) virion produced by the method of claim 13 .
15 . A host cell comprising the nucleic acid molecule of claim 9 .
16 . The host cell of claim 15 further comprising accessory function genes.
17 . The nucleic acid molecule of claim 9 , wherein the nucleotide sequence is arranged in the nucleic acid molecule such that the modified AAV p5 promoter region is situated 3′ relative to the rep coding region.
18 . An AAV helper function vector comprising the nucleic acid molecule of claim 17 .
19 . The AAV helper function vector of claim 18 , wherein the vector is a plasmid.
20 . The AAV helper function vector of claim 18 , further comprising one or more accessory function genes.
21 . A method of producing recombinant AAV (rAAV) virions comprising:
(a) introducing an AAV vector into a suitable host cell;
(b) introducing the AAV helper function vector of claim 18 into the host cell;
(c) expressing accessory functions in the host cell; and
(d) culturing the host cell to produce rAAV virions.
22 . A recombinant AAV (rAAV) virion produced by the method of claim 21 .
23 . A host cell comprising the nucleic acid molecule of claim 17 .
24 . The host cell of claim 23 further comprising accessory function genes.
25 . The nucleic acid molecule of claim 9 , wherein the nucleotide sequence is arranged in the nucleic acid molecule such that the modified AAV p5 promoter region is situated 3′ relative to the cap coding region.