Helper-free, totally defective adenovirus for gene therapy

• US 6,228,646 B1
• Filed: 03/07/1997
• Issued: 05/08/2001
• Est. Priority Date: 03/07/1996
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hardy, Stephen F.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/812,618
• Time in Patent Office: 1,523 Days
• Field of Search: 435/172.3, 435/320.1, 435/235.01, 435/440, 435/325, 435/455, 435/456, 435/457, 514/44
• US Class Current: 435/455
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10362 by genetic engineering
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/38 being a stuffer
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

A method for producing in vivo packaged recombinant adenovirus vectors is provided. The recombinant Ad vectors do not contain any Adenovirus genes and are therefore useful for gene therapy. The recombinant Adenovirus vectors are packaged in vivo using a helper virus which is itself very inefficiently packaged, providing a recombinant viral preparation with very little or no contamination with helper virus. In particular, the method makes use of a helper virus in which the packaging site can be easily excised in vivo by recombination mediated by a recombinase. The helper virus is also useful for the in vivo construction of new recombinant adenovirus vectors containing substitutions in the E1 or other adenoviral region.

Claims

1. A recombinant adenovirus helper-dependent vector comprising:

a) an adenovirus left inverted terminal repeat sequence (ITR);

b) at least one recombination site located 3′

to the adenovirus left ITR sequence;

c) an adenovirus packaging sequence located 3′

to the recombination site of (b);

d) a first foreign DNA sequence located 3′

to the adenovirus packaging sequence; and

e) an adenovirus right ITR sequence located 3′

to the foreign DNA sequence.

2. The helper-dependent vector of claim 1 which further comprises a pair of identical recombination sites in direct orientation with respect to each other that flank the first foreign DNA sequence of (d).

3. The helper-dependent vector of claim 1 which further comprises a second foreign DNA sequence located 3′

to the first foreign DNA sequence of (d).

4. The helper-dependent vector of claim 1 which further comprises a second foreign DNA sequence flanked by a pair of identical recombination sites in direct orientation with respect to each other located 3′

to the first foreign DNA sequence of (d).

5. The helper-dependent vector of claim 1, wherein when more than one recombination site is present, the recombination sites are different.

6. The helper-dependent vector of claim 1, wherein when more than one recombination site is present, the recombination sites are the same.

7. The helper-dependent vector of claim 1, wherein the first and second foreign DNA sequences together range in size from about 31 kb to about 37 kb.

8. The helper-dependent vector of claim 1, wherein the first foreign DNA sequence ranges in size from about 31 kb to about 37 kb.

9. The helper-dependent vector of claim 1, wherein the second foreign DNA sequence ranges in size from about 31 kb to about 37 kb.

10. The helper-dependent vector of claim 1, wherein the first foreign DNA sequence comprises a nucleotide sequence that expresses a gene product.

11. The helper-dependent vector of claim 1, wherein the vector ranges in size from about 32 kb to about 38 kb.

12. The helper-dependent vector of claim 1, wherein the recombination site comprises a lox sequence.

13. A recombinant adenovirus helper vector comprising:

a) an adenovirus left ITR sequence;

b) an adenovirus packaging sequence located 3′

to the adenovirus left ITR sequence;

c) at least one pair of identical recombination sites in direct orientation to each other located 3′

to the adenovirus left ITR sequence, wherein each pair of recombination sites flank the adenovirus packaging sequence;

i. wherein if more than one pair of recombination sites are present then the recombination sites are nested;

d) at least one adenovirus gene sequence located 3′

to one pair of identical recombination sites of, wherein the adenovirus gene sequence encodes an adenovirus gene product; and

e) an adenovirus right ITR sequence located 3′

to the adenovirus gene sequence.

14. The helper vector of claim 13, wherein:

a) at least one adenovirus gene sequence of claim 13 (d) located 3′

to the adenovirus left ITR sequence;

b) the adenovirus packaging sequence of claim 13 (b) located 3′

to the adenovirus gene sequence; and

c) a pair of identical recombination sites in direct orientation to each other of claim 13 (c) located 3′

to the adenovirus gene sequence, wherein each pair of recombination sites flank the adenovirus packaging sequence, and

i. wherein if more than one pair of recombination sites are present then the recombination sites are nested.

15. The helper vector of claim 13, wherein each member of a pair of recombination sites of claim 13 (c) is in phase with each other.

16. The helper vector of claim 13, wherein a pair of recombination sites of claim 13 (c) comprises a pair of lox sequences.

17. The helper vector of claim 13, wherein the packaging sequence of claim 13 (b) is a synthetic packaging sequence.

18. The helper vector of claim 13, wherein the adenovirus packaging sequence of claim 13 (b) is from a different serotype than the encoded adenovirus proteins.

19. The helper vector of claim 13, wherein the adenovirus packaging sequence of claim 13 (b) contains a mutated nucleotide sequence.

20. The adenovirus packaging sequence of claim 19, wherein the mutated nucleotide sequence includes a deletion.

23. A method for generating packaged recombinant adenovirus vectors by a recombination event comprising:

a) introducing a recombinant adenovirus vector comprising an adenovirus left ITR, an adenovirus packaging sequence, a foreign DNA sequence, and at least one recombination site, and recombinant adenovirus helper vector of claim 13 into a recombinase-expressing host cell,

b) propagating the recombinant adenovirus vector and the recombinant adenovirus helper vector in the recombinase-expressing host cell; and

c) isolating the packaged recombinant adenovirus resolved vector generated from the recombination event.

24. A method for generating recombinant adenovirus helper-dependent vectors comprising:

a) introducing a recombinant adenovirus helper vector of claim 13 and a recombinant adenovirus helper-dependent vector into a first recombinase-expressing host cell,

i. wherein the recombinant adenovirus helper-dependent vector comprises an adenovirus left ITR, at least one recombination site, an adenovirus packaging sequence, foreign DNA sequence, an adenovirus right ITR; and

ii. wherein the recombination sites in the recombinant adenovirus helper vector and the recombinant adenovirus helper-dependent vector are identical;

b) propagating the recombinant adenovirus helper vector and recombinant adenovirus helper-dependent vector in the first recombinase-expressing host cell; and

c) isolating a packaged recombinant adenovirus helper-dependent vector.

25. The method of claim 24 further comprising:

a) introducing the packaged recombinant adenovirus helper-dependent vector into a second recombinase-expressing host cell; and

b) isolating packaged recombinant adenovirus helper-dependent vectors.

26. The method of claim 24 or 25, wherein steps (a) and (b) are repeated at least once.

21. A recombinant adenovirus vector comprising:

a) an adenovirus left ITR sequence;

b) an adenovirus packaging sequence located 3′

to the adenovirus left ITR sequence;

c) a foreign DNA sequence located 3′

to the adenovirus packaging sequence; and

d) at least one recombination site located 3′

to a foreign DNA sequence.

22. The recombinant adenovirus vector of claim 21, wherein the recombination site comprises a lox sequence.