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US 2015/0176027 A1Published application
Adeno-associated virus (aav) serotype 8 sequences, vectors containing same, and uses therefor
AssigneeThe Trustees of the University of Pennsylvania
InventorsGuangping Gao, James M. Wilson, Mauricio R. Alvira
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Abstract
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.
Claims24
(canceled)
2:
- A recombinant adeno-associated virus (AAV) production cell culture system comprising:
- (i) a host cell 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 203 to 737 of SEQ ID NO:
- 2, or an amino acid sequence at least 95% identical therewith 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.
5 more elements
3:
- The system according to claim 2, wherein said nucleic acid sequence is stably contained in said host cell.
4:
- The system according to claim 2, wherein said nucleic acid sequence is provided in trans in said host cell.
5:
- The system according to claim 2, wherein a plasmid comprises said nucleic acid sequence and said one or more rep sequences.
6:
- The system according to claim 2, wherein said rep sequences are expressed under the control of a P5 promoter.
7:
- The system according to claim 6, wherein a spacer is interposed between said rep sequences and said P5 promoter.
8:
- The system according to claim 7, wherein said spacer is of sufficient size to reduce expression of the rep78 and rep68 gene products.
9:
- The system according to claim 7, wherein said rep sequences are separated from said P5 promoter by about 100 bp to 8 kbp.
10:
- The system according to claim 2, wherein said nucleic acid (ii) is expressed under the control of a constitutive promoter.
11:
- The system according to claim 10, wherein said constitutive promoter is a cytomegalovirus (CMV) promoter.
12:
- The system according to claim 2, wherein said nucleic acid is expressed under the control of an inducible promoter.
13:
- The system according to claim 2, wherein said rep sequences comprise rep 78 and rep 68.
14:
- The system according to claim 2, wherein said rep sequences comprise rep 52 and rep 40.
15:
- The system according to claim 2, wherein said minigene comprises regulatory sequences operably linked to, and directing expression of, said heterologous gene.
16:
- The system according to claim 15, wherein said heterologous gene is expressed under the control of a constitutive promoter.
17:
- The system according to claim 16, wherein said constitutive promoter is a cytomegalovirus (CMV) promoter.
18:
- The system according to claim 15, wherein said heterologous gene is expressed under the control of an inducible promoter.
19:
- The system according to claim 15, wherein said heterologous gene is expressed under the control of a tissue-specific promoter.
20:
- The system according to claim 19, wherein said tissue-specific promoter is a human thyroid hormone binding globulin (TBG) gene promoter.
21:
- The system according to claim 2, wherein said adenovirus helper sequences comprise sequences encoding E1a, E1b, E2a, or E4 ORF6.
22:
- The system according to claim 2, wherein said nucleic acid sequence and said rep sequences are contained on the same plasmid.
23:
- The system according to claim 2, wherein said minigene and said adenovirus helper sequences are contained in separate plasmids.
24:
- The system according to claim 2, wherein said adenovirus helper sequences are provided by a replication-defective adenovirus.
Dependencies are inferred from the claim text.