Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
Sequences of a serotype 8 adeno-associated virus and vectors and host cells containing these sequences are provided. Also described are methods of using such host cells and vectors in production of rAAV particles.
1. A recombinant adeno-associated virus (AAV) production cell culture system comprising:
(i) a host cell in culture for packaging said recombinant AAV;
(ii) a nucleic acid sequence encoding an adeno-associated virus (AAV) 8 capsid, wherein the AAV8 capsid comprises a vp3 capsid protein having the sequence of aa 204 to 738 of SEQ ID NO: 2 , or an amino acid sequence at least 95 % identical to said sequence of aa 204 to 738 of SEQ ID NO: 2 under the control of regulatory sequences which direct expression in said host cell;
(iii) rep sequences necessary for replication;
(iv) a minigene comprising a heterologous gene flanked by 5′ and 3′ AAV inverted terminal repeat (ITR) sequences for packaging into said AAV8 capsid; and
(v) adenovirus helper sequences.
2. The system according to claim 1 , wherein said nucleic acid sequence is stably contained in said host cell.
3. The system according to claim 1 , wherein said nucleic acid sequence is provided in trans to said host cell.
4. The system according to claim 1 , wherein a plasmid comprises said nucleic acid sequence and said one or more rep sequences.
5. The system according to claim 1 , wherein said rep sequences are expressed under the control of a P5 promoter.
6. The system according to claim 5 , wherein a spacer is interposed between said rep sequences and said P5 promoter.
7. The system according to claim 6 , wherein said rep sequences comprise sep 78 and rep 68, wherein said spacer is of sufficient size reduce expression of rep78 and rep68 gene products.
8. The system according to claim 6 , wherein said rep sequences are separated from said P5 promoter by about 100 by to 8 kbp.
9. The system according to claim 1 , wherein said nucleic acid (ii) is expressed under the control of a constitutive promoter.
10. The system according to claim 9 , wherein said constitutive promoter is a cytomegalovirus (CMV) promoter.
11. The system according to claim 1 , wherein said nucleic acid is expressed under the control of an inducible promoter.
12. The system according to claim 1 , wherein said rep sequences comprise rep 78 and rep 68.
13. The system according to claim 1 , wherein said rep sequences comprise rep 52 and rep 40.
14. The system according to claim 1 , wherein said minigene comprises regulatory sequences operably linked to, and directing expression of, said heterologous gene.
15. The system according to claim 14 , wherein said heterologous gene is expressed under the control of a constitutive promoter.
16. The system according to claim 15 , wherein said constitutive promoter is a cytomegalovirus (CMV) promoter.
17. The system according to claim 14 , wherein said heterologous gene is expressed under the control of an inducible promoter.
18. The system according to claim 14 , wherein said heterologous gene is expressed under the control of a tissue-specific promoter.
19. The system according to claim 18 , wherein said tissue-specific promoter is a human thyroid hormone binding globulin (TBG) gene promoter.
20. The system according to claim 1 , wherein said adenovirus helper sequences comprise sequences encoding E1a, E1b, E2a, or E4ORF6.
21. The system according to claim 1 , wherein said nucleic acid sequence and said rep sequences are contained on the same plasmid.
22. The system according to claim 1 , wherein said minigene and said adenovirus helper sequences are contained in separate plasmids.