Site specific recombinase based method for producing adenoviral vectors

• US 6,838,285 B2
• Filed: 09/17/2002
• Issued: 01/04/2005
• Est. Priority Date: 09/18/2001
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Becton Dickinson SA (Becton, Dickinson & Co)
• Inventors: Quinn, Thomas Patrick, Farmer, Andrew Alan
• Primary Examiner(s): Leffers, Gerry
• Assistant Examiner(s): Marvich, Maria
• Application Number:
  US10/246,782
• Publication Number:
  US 20030054555A1
• Time in Patent Office: 840 Days
• Field of Search: 435/69.1, 435/320.1, 435/325, 435/172.3, 435/235.1, 435/366, 435/456, 435/455, 435/462, 435/91.4, 435/91.41, 424/93.2, 424/93.21, 514/44
• US Class Current: 435/456
• 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 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• 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

Site-specific recombinase based methods for making a recombinant adenoviral genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from donor and acceptor vectors that each include at least one site recombinase recognition site, where in certain preferred embodiments, one of the donor and acceptor vectors includes a single recombinase recognition site while the other includes two recombinase recognition sites. The acceptor vector includes an adenoviral genome having an E region deletion. The donor vector includes an insertion nucleic acid. In the subject methods, the donor and acceptor vectors are combined in the presence of a recombinase to produce an adenoviral genome that includes the insertion nucleic acid. The subject adenoviral genomes find use in a variety of applications, including as vectors for use in a variety of applications.

Claims

1. A method of preparing a recombinant adenoviral genome having an insertion nucleic acid located in an early (E) gene region of said genome, said method comprising:

(a) providing a donor vector comprising an insertion nucleic acid and a first and second recombinase recognition site that are able to recombine with each other;

(b) providing an acceptor vector comprising an adenoviral genome having an E region deletion and at least one recombinase recognition site located in E region deletion;

(c) combining said donor and acceptor vectors with a recombinase that recognizes said donor and acceptor recombinase recognition sites to produce an adenoviral genome having an insertion nucleic acid located in an E gene region of said genome.

2. The method according to claim 1, wherein said first and second recombinase recognition sites flank said insertion nucleic acid.

3. The method according to claim 1, wherein said acceptor vector comprises a single recombinase recognition site.

4. The method according to claim 1, wherein said donor and acceptor vectors are plasmids.

5. The method according to claim 1, wherein said E region deletion comprises a deletion of at least a portion of at least one of E1, E3 and E4 region.

6. The method according to claim 1, wherein said recombinase is a Cre recombinase.

7. The method according to claim 1, wherein said recombinase recognition sites are lox sites.

8. The method according to claim 1, wherein said adenoviral genome is present on the acceptor plasmid.

9. A method of producing a recombinant adenovirus having a genome having an insertion nucleic acid located in an E gene region of said genome, said method comprising:

(a) preparing a recombinant adenoviral genome having an insertion nucleic acid located in an E gene region of said genome by;

(i) providing a donor vector comprising an insertion nucleic acid and first and second recombinase recognition sites that are able to recombine with each other;

(ii) providing an acceptor vector comprising an adenoviral genome having an E gene deletion and at least one recombinase recognition site; and

(iii) combining said donor and acceptor vectors with a recombinase that recognizes said donor and acceptor recombinase recognition sites to produce an adenoviral genome having an insertion nucleic acid located in an E gene region of said genome;

(b) transforming a cell that is transcomplementing for any inactivated E genes of said E gene region with said adenoviral genome; and

(c) maintaining said transformed cell under conditions sufficient for virus to be produced so as to produce said recombinant adenovirus having a genome having an insertion nucleic acid located in an E gene region of said genome.

10. A kit for use in preparing a recombinant adenovirus, said kit comprising:

(a) a donor vector comprising an insertion nucleic acid and a first and second recombinase recognition site that are able to recombine with each other, and

(b) an acceptor vector comprising an adenoviral genome comprising an E gene deletion region and at least one recombinase recognition site located in said E gene deletion region.

11. The kit according to claim 10, wherein said first and second recombinase recognition sites flank said insertion nucleic acid.

12. The kit according to claim 10, wherein said acceptor vector comprises a single recombinase recognition site.

13. The kit according to claim 10, wherein said donor and acceptor vectors are plasmids.

14. The kit according to claim 13, wherein said kit further comprises a recombinase.

15. The kit according to claim 10, wherein said recombinase is Cre.

16. The kit according to claim 15, wherein said recombinase recognition sites are lox site.

17. The kit according to claim 10, wherein said kit further comprises a cell that is transcomplementing for any inactivated E genes of said E gene region of said adenoviral genome.

18. A system for use in preparing a recombinant adenovirus, said system comprising:

(a) a donor vector comprising an insertion nucleic acid and first and second recombinase recognition sites that are able to recombine with each other;

(b) an acceptor vector comprising an adenoviral genome comprising;

(c) an E gene deletion region and at least one recombinase recognition site located in said E gene deletion region;

(d) a recombinase; and

(e) a cell that is transcomplementing for any inactivated E genes of said E gene region of said adenoviral genome.