Compositions and methods for generating adeno-associated viral vectors with undetectable capsid gene contamination

1. A method of reducing capsid gene contamination in a population of AAV vector particles, comprising:

(a) introducing an AAV vector into a suitable host cell, wherein the host cell comprises an intron-modified capsid expression cassette, wherein said expression cassette comprises: (i) a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a promoter and a polyadenylation signal, wherein the encoded AAV capsid protein packages AAV vector in the presence of rep and adenovirus helper functions, and (ii) at least one heterologous excisable intron sequence operably linked to the nucleotide sequence encoding the AAV capsid protein; wherein the heterologous excisable intron sequence comprises a splice donor site and splice acceptor site permitting proper splicing of the nucleotide sequence encoding an AAV capsid protein; wherein the nucleic acid molecule has a length of less than 5 kilobases; wherein the total size of the at least one heterologous excisable intron is at least 2 kilobases; and wherein the nucleic acid molecule and the at least one heterologous excisable intron together have a length of more than 5 kilobases;

(b) expressing rep and adenovirus helper functions in the host cell; and

(c) culturing the host cell to produce a population of AAV vector particles.

2. The method of claim 1 , further comprising purifying at least a portion of the population of AAV vector particles produced in accordance with step (c) over a heparin column.

3. The method of claim 1 , wherein the AAV vector comprises a nucleic acid sequence encoding a therapeutic molecule under the control of sequences which direct expression thereof in a cell.

4. The method of claim 3 , wherein the nucleic acid sequence encoding a therapeutic molecule is flanked by AAV terminal repeat sequences (ITRs).

5. A method of reducing capsid gene contamination in a population of AAV vector particles, comprising:

(a) introducing an AAV vector into a suitable host cell, wherein the host cell comprises an intron-modified capsid expression cassette, wherein said expression cassette comprises: (i) a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a promoter and a polyadenylation signal, wherein the encoded AAV capsid protein packages AAV vector in the presence of rep and adenovirus helper functions, and (ii) at least one heterologous excisable intron sequence operably linked to the nucleotide sequence encoding the AAV capsid protein; wherein the heterologous excisable intron sequence is contiguous with the nucleotide sequence encoding an AAV capsid protein; wherein the nucleic acid molecule has a length of less than 5 kilobases; wherein the total size of the at least one heterologous excisable intron is at least 2 kilobases; and wherein the nucleic acid molecule and the at least one heterologous excisable intron together have a length of more than 5 kilobases;

(b) expressing rep and adenovirus helper functions in the host cell; and

(c) culturing the host cell to produce a population of AAV vector particles.

6. The method of claim 5 , wherein the heterologous intron sequence is positioned within the nucleotide sequence encoding an AAV capsid protein, between the promoter and the nucleotide sequence encoding an AAV capsid protein, or in 3′ untranslated region of the nucleotide sequence encoding an AAV capsid protein.

7. The method of claim 5 , further comprising purifying at least a portion of the population of AAV vector particles produced in accordance with step (c) over a heparin column.

8. The method of claim 5 , wherein the AAV vector comprises a nucleic acid sequence encoding a therapeutic molecule under the control of sequences which direct expression thereof in a cell.

9. The method of claim 8 , wherein the nucleic acid sequence encoding a therapeutic molecule is flanked by AAV terminal repeat sequences (ITRs).

10. A method of reducing capsid gene contamination in a population of AAV vector particles, comprising:

(a) introducing an AAV vector into a suitable host cell, wherein the host cell comprises an intron-modified capsid expression cassette, wherein said intron-modified capsid expression cassette comprises: (i) a nucleic acid molecule comprising a nucleotide sequence encoding an AAV capsid protein operably linked to a first promoter and a polyadenylation signal, wherein the encoded AAV capsid protein packages AAV vector in the presence of rep and adenovirus helper functions, and (ii) at least one heterologous excisable intron sequence operably linked to the nucleotide sequence encoding the AAV capsid protein; wherein the intron-modified capsid expression cassette lacks a sequence encoding a functional AAV replication protein; wherein the nucleic add molecule has a length of less than 5 kilobases; wherein the total size of the at least one heterologous excisable intron is at least 2 kilobases; and wherein the nucleic acid molecule and the at least one heterologous excisable intron together have a length of more than 5 kilobases;

(b) expressing rep and adenovirus helper functions in the host cell; and

(c) culturing the host cell to produce a population of AAV vector particles.

11. The method of claim 10 , wherein the rep function is expressed from a rep expression cassette comprising a nucleic acid molecule comprising a nucleotide sequence encoding an AAV replication protein operably linked to a second promoter.

12. The method of claim 10 , further comprising purifying at least a portion of the population of AAV vector particles produced in accordance with step (c) over a heparin column.

13. The method of claim 10 , wherein the AAV vector comprises a nucleic acid sequence encoding a therapeutic molecule under the control of sequences which direct expression thereof in a cell.

14. The method of claim 10 , wherein the nucleic acid sequence encoding a therapeutic molecule is flanked by AAV terminal repeat sequences (ITRs).