High throughput system for producing recombinant viruses using site-specific recombination

• US 7,319,001 B2
• Filed: 03/07/2003
• Issued: 01/15/2008
• Est. Priority Date: 03/09/2002
• Status: Active Grant

• Current Assignee: Neurogenex Co., Ltd.
• Original Assignee: Neurogenex Co., Ltd.
• Inventors: Kim, Do-Hui, Kim, Min-Sung, Yi, Yong-Weon, Jung, Neon-C, Kim, Kyung-Jin, Seen, Dong-Seung, Choi, Eun-Wook, Son, Ho-Sun
• Primary Examiner(s): Guzo; David
• Application Number:
  US10/383,838
• Publication Number:
  US 20040185565A1
• Time in Patent Office: 1,775 Days
• Field of Search: None
• US Class Current: 435/5
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/14143 viral genome or elements thereof as genetic vector

Abstract

Disclosed are the methods for producing recombinant viruses using site-specific recombination in vitro. In the present invention, circular viral genomic DNAs are digested with restriction enzymes to generate a linear form viral genomic DNAs flanked by site-specific recombination sites, and then are subjected to site-specific recombination with the desired genomic materials flanked by site-specific recombination sites in vitro. According to the present invention, since the site-specific recombination mixture can be applied to host cells without further procedures of selecting the desired recombinant viral genomic DNAs, it is possible to obtain numerous recombinant viruses rapidly at the same time. Thus, the present invention can be used as a high throughput system for generating and screening hundreds or thousands of recombinant viruses.

Claims

1. A method for preparing expressive circular recombinant viral vectors, comprising:

i) providing a plurality of linearized viral DNA fragments wherein each fragment contains at least two site-specific recombination sites and at least one restriction enzyme recognition site;

ii) providing a plurality of viral vector carriers wherein each viral vector carrier contains a gene cassette having a desired genomic material with a desired nucleic acid sequence flanked with at least two site-specific sites for homologous recombination with the linearized DNA fragments at the two site-specific recombination sites; and

iii) reacting the linearized viral DNA fragments and the viral vector carriers to create homologous recombinations in vitro wherein a plurality of recombinant viral vectors carrying the desired genomic material is formed in vitro therefrom;

iv) wherein non-recombined linearized viral DNA fragments, non-recombined viral vector carriers, and non-linearized viral DNA do not express a gene for a protein responsible for viral replication and viral particle formation.

2. The method for preparing expressive circular recombinant viral vectors as claimed in claim 1, wherein one or more viral DNA fragments are digested with restriction enzymes at one or more sites to generate the linearized viral DNA fragments.

3. The method for preparing expressive circular recombinant viral vectors as claimed in claim 1, wherein the recombinant viral vectors are obtained from the group consisting of baculovirus, poliomavirus, papillomavirus, and hepatitis B virus (HBV).

4. The method for preparing expressive circular recombinant viral vectors as claimed in claim 2, wherein said one or more sites comprise the nucleotide sequence of CCTNAGG.

5. A method of preparing recombinant viruses having circular genomic DNA, comprising:

i) providing a plurality of linearized viral DNA fragments wherein each fragment contains at least two site-specific recombination sites and at least one restriction enzyme recognition site;

ii) providing a plurality of viral vector carriers wherein each viral vector carrier contains a gene cassette having a desired genomic material with a desired nucleic acid sequence flanked with at least two site-specific sites for homologous recombination with the linearized DNA fragments at the two site-specific recombination sites; and

iii) reacting the linearized viral DNA fragments and the viral vector carriers to create homologous recombinations in vitro to produce a reaction mixture including expressive circular recombinant viral vectors carrying the desired genomic material formed in vitro therefrom;

iv) wherein non-recombined linearized viral DNA fragments, non-recombined viral vector carriers and non-linearized viral DNA do not express a gene for a protein responsible for viral replication and viral particle formation; and

v) transferring the reaction mixture to host cells in a multi-well plate.

6. The method of preparing recombinant viruses having circular genomic DNA as claimed in claim 5, wherein the transfer of the reaction mixture to host cells is carried out without a need for a selection process to isolate the circular genomic DNA.

7. The method of preparing recombinant viruses having circular genomic DNA as claimed in claim 5, wherein viral genomic DNA of i) are digested with a restriction enzyme at one or more sites to generate the linearized viral DNA fragments.

8. The method of preparing recombinant viruses having circular genomic DNA as claimed in claim 5, wherein the recombinant viral vectors are obtained from the group consisting of baculovirus, poliomavirus, papillomavirus, and hepatitis B virus (HBV).

9. The method of preparing recombinant viruses having circular genomic DNA as claimed in claim 7, wherein said one or more sites comprise the nucleotide sequence of CCTNAGG.