Production of adeno-associated virus in insect cells

• US 6,723,551 B2
• Filed: 08/13/2002
• Issued: 04/20/2004
• Est. Priority Date: 11/09/2001
• Status: Expired due to Term

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Kotin, Robert M., Urabe, Masashi, Ding, Chuan-Tian
• Primary Examiner(s): PARK, HANKYEL
• Application Number:
  US10/216,870
• Publication Number:
  US 20030148506A1
• Time in Patent Office: 616 Days
• Field of Search: 435/5, 435/6, 435/69.1, 435/69.7, 435/320.1, 435/235.1, 536/23.72
• US Class Current: 435/235.1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/16143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 2840/20 translation of more than one cistron

Abstract

A method of producing an adeno-associated virus (AAV) in an insect cell comprising (i) providing at least one insect cell-compatible vector comprising a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence, a second nucleotide sequence containing an open reading frame encoding AAV VP1, VP2, and VP3 capsid proteins, a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence, and a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence, (ii) introducing the at least one insect cell-compatible vector into an insect cell, and (iii) maintaining the insect cell under conditions such that AAV is produced. Also provided are recombinant AAV made in accordance with the method, insect cell-compatible vectors, and insect cells comprising nucleotide sequences for production of AAV in an insect cell.

Claims

1. A method of producing an adeno-associated virus (AAV) in an insect cell, comprising:

(i) providing at least one insect cell-compatible vector comprising

a first nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence,

a second nucleotide sequence comprising an open reading frame (ORF) comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,

a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and

a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell,

(ii) introducing said at least one insect cell-compatible vector into an insect cell, and

(iii) maintaining said insect cell under conditions such that AAV is produced.

2. The method of claim 1, wherein said insect cell is Sf9.

3. The method of claim 1, wherein said at least one vector is a baculoviral vector, a viral vector or a plasmid.

4. The method of claim 1, wherein said vector is a baculoviral vector.

5. The method of claim 1, wherein two insect cell-compatible vectors are provided,

a first vector comprising said first nucleotide sequence comprising at least one AAV ITR nucleotide sequence, and

a second vector comprising said second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, said third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and said fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.

6. The method of claim 1, wherein three insect-compatible vectors are provided,

a first vector comprising said first nucleotide sequence comprising at least one AAV ITR nucleotide sequence,

a second vector comprising said second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell, and

a third vector comprising said third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and said fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.

7. The method of claim 1, wherein said first nucleotide sequence further comprises at least one nucleotide sequence encoding a gene product of interest for expression in a mammalian cell and the at least one nucleotide sequence encoding a gene product of interest becomes incorporated into the genome of an AAV produced in the insect cell.

8. The method of claim 1, wherein said fourth nucleotide sequence comprises a Kozak-like expression control sequence.

9. The method of claim 1, wherein said fourth nucleotide sequence comprises an expression control sequence selected from an immediate early 1 gene (IE-1) promoter, a Δ

IE-1 promoter, a promoter substantially homologous to the IE-1 promoter, and a promoter substantially homologous to the Δ

IE-1 promoter.

10. The method of claim 9, wherein the Δ

IE-1 promoter consists essentially of SEQ. ID. NO;

1.

11. The method of claim 1, wherein said second nucleotide sequence comprising at least one expression control sequence comprises a polyhedron (polh) promoter sequence.

12. The method of claim 1, wherein said second nucleotide sequence comprises at least one expression control sequence comprising

a nine nucleotide sequence of SEQ. ID NO:

4 or a nucleotide sequence substantially homologous to SEQ. ID NO;

4, upstream of the initiation codon of the nucleotide sequence encoding AAV VP1 capsid protein, and

a C at position 2 of the nucleotide sequence encoding AAV VP1 capsid protein.

13. The method of claim 12, wherein said second nucleotide sequence further comprises at least one modification of the nucleotide sequence encoding AAV VP1 capsid protein selected from among a G at nucleotide position 12, an A at nucleotide position 21, and a C at nucleotide position 24.

14. The method of claim 1, wherein an ACG initiation codon is an initiation codon for translation of said AAV VP1 capsid protein.

15. The method of claim 1, wherein said third nucleotide sequence encodes Rep52.

16. The method of claim 1, wherein said fourth nucleotide sequence encodes Rep78.

30. The method of claim 7, wherein the at least one nucleotide sequence encoding a gene product of interest encodes a Rep78 or a Rep68 protein.

31. The method of claim 1, wherein said first nucleotide sequence comprises an AAV1, AAV2, or AAV4 ITR, said third nucleotide sequence comprises an AAV1, AAV2, or AAV4 Rep52 or Rep40 coding sequence, and said fourth nucleotide sequence comprises an AAV1, AAV2, or AAV4 Rep78 or Rep68 coding sequence.

32. The method of claim 1, wherein said second nucleic acid sequence encodes AAV1, AAV2, or AAV4 VP1, VP2, and VP3 capsid proteins.

33. The method of claim 1, wherein said first nucleotide sequence comprises an AAV5 ITR, said third nucleotide sequence comprises an AAV5 Rep52 or Rep40 coding sequence, and said fourth nucleotide sequence comprises an AAV5 Rep78 or Rep68 coding sequence.

17. A method of producing an AAV in an insect cell, comprising:

(i) providing an insect cell comprising

a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence,

a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2 and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,

a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell,

a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and, optionally,

 

at least one insect cell-compatible vector,

wherein at least one of the first, second, third and fourth nucleotide sequences is/are stably integrated in the insect cell and the at least one insect cell-compatible vector, when present, comprises the remainder of the first, second, third and fourth nucleotide sequences which is/are not stably integrated in the insect cell, and

(ii) maintaining the insect cell under conditions such that AAV is produced.

18. The method of claim 17, wherein said nucleotide sequence stably integrated in the insect cell is said second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell.

19. An insect cell-compatible vector comprising a nucleotide sequence encoding Rep78 or Rep68 operably linked to a Δ

IE-1 promoter and a Kozak-like expression control sequence.

20. An insect cell-compatible vector comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence comprising:

a nine nucleotide sequence of SEQ. ID NO;

4 or a nucleotide sequence substantially homologous to SEQ. ID NO;

4, located upstream of the initiation codon of the nucleotide sequence encoding AAV VP1 capsid protein, and

a C at nucleotide position 2 of the nucleotide sequence encoding AAV VP1 capsid protein.

21. The insect cell-compatible vector of claim 20, wherein said AAV VP1, VP2, and VP3 capsid proteins are AAV2 VP1, VP2, and VP3 capsid proteins, and wherein the nucleotide sequence encoding VP1 capsid protein comprises at least one modification selected from a C at nucleotide position 12, an A at nucleotide position 21, and a C at nucleotide position 24.

22. A recombinant AAV comprising a VP1 capsid protein comprising threonine at amino acid position 1.

23. An insect cell comprising:

a first nucleotide sequence comprising at least one AAV ITR nucleotide sequence,

a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,

a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and

a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell.

24. The insect cell of claim 23, wherein said fourth nucleotide sequence comprises a Kozak-like expression control sequence.

25. The insect cell of claim 23, wherein said fourth nucleotide sequence comprises an expression control sequence selected from an IE-1 promoter, a promoter substantially homologous to the IE-1 promoter, a Δ

IE-1 promoter, or a promoter substantially homologous to the Δ

IE-1 promoter.

26. The insect cell of claim 23, wherein said first nucleotide sequence comprises two AAV ITR nucleotide sequences and further comprises at least one nucleotide sequence encoding a gene product of interest for expression in a mammalian cell between the two AAV ITR nucleotide sequences.

27. The insect cell of claim 23, wherein at least one of said first nucleotide sequence, second nucleotide sequence, third nucleotide sequence, and fourth nucleotide sequence is stably integrated in said insect cell.

28. A method of producing an AAV in an insect cell comprising:

(i) providing at least one insect cell-compatible vector comprising

a first nucleotide sequence comprising at least one chimeric ITR nucleotide sequence, the ITR nucleotide sequence comprising an AAV backbone and a specific binding and a nicking site of a Rep protein from a parvovirus other than AAV,

a second nucleotide sequence comprising an ORF comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in an insect cell,

a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and

a fourth nucleotide sequence comprising a nucleotide sequence encoding a parvoviral Rep protein that can specifically bind and nick the site in the ITR nucleotide sequence within the first nucleotide sequence, operably linked to at least one expression control sequence for expression in an insect cell,

(ii) introducing said at least one insect cell-compatible vector into an insect cell, and

(iii) maintaining said insect cell under conditions such that AAV is produced.

29. The method of claim 28, wherein said chimeric ITR nucleotide sequence comprises the specific binding and nicking site of NS-1 and said nucleotide sequence encoding a parvoviral Rep protein is that of NS-1.

34. A method of producing a parvoviral genome in an insect cell, which method comprises:

(i) providing at least one insect cell-compatible vector comprising

a first nucleotide sequence comprising at least one parvoviral ITR,

a second nucleotide sequence comprising an AAV Rep52 or Rep40 coding sequence operably linked to at least one expression control sequence for expression in an insect cell, and

a third nucleotide sequence comprising an AAV Rep78 or Rep68 coding sequence operably linked to at least one expression control sequence for expression in an insect cell,

(ii) introducing said at least one insect cell-compatible vector into an insect cell, and

(iii) maintaining said insect cell under conditions such that a parvovirus genome is produced therein.

35. The method of claim 34, wherein the parvovirus is a dependovirus.

36. The method of claim 35, wherein the dependovirus is a human or simian AAV.