Altered recombinases for genome modification

• US 6,808,925 B2
• Filed: 02/16/2001
• Issued: 10/26/2004
• Est. Priority Date: 02/18/2000
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Calos, Michele P.
• Primary Examiner(s): Mckelvey, Terry
• Assistant Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US09/788,297
• Publication Number:
  US 20020094516A1
• Time in Patent Office: 1,348 Days
• Field of Search: 435/462, 435/320.1, 435/183, 435/468, 800/278, 536/23.72
• US Class Current: 435/462
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• 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 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

Claims

1. A method of site-specifically integrating a nucleic acid into a genome of a cell of a multicellular organism, said method comprising:

introducing a targeting construct comprising said nucleic acid and a vector attachment site and an altered, unidirectional site-specific bacteriophage integrase into a cell present in vitro and maintaining said cell under conditions sufficient for said construct to integrate into an attachment site of said genome of said cell by a recombination event mediated by said altered, unidirectional site-specific bacteriophage integrase;

wherein said integrase is at least 95% identical to SEQ ID NO;

21 and has integrase activity.

2. The method according to claim 1, wherein said nucleic acid comprises a coding sequence.

3. The method according to claim 2, wherein said coding sequence is present in an expression cassette.

5. The method of claim 1, wherein said genome attachment site is a pre-selected site in said genome.

6. The method of claim 1, wherein said nucleic acid comprises a control region.

7. The method of claim 6, wherein said control region is a promoter.

8. The method of claim 1, wherein said cell is a mammalian cell.

9. The method of claim 8, wherein said cell is a human cell.

4. A kit for use in integrating a nucleic acid into a genome of a cell of a multicellular organism, said kit comprising:

(a) a targeting construct comprising a vector attachment site; and

(b) an altered, unidirectional site-specific bacteriophage integrase or nucleic acid encoding the same,

wherein said integrase is at least 95% identical to SEQ ID NO;

21 and has integrase activity.

10. The kit of claim 4, further comprising instructions for using said targeting construct and said integrase or nucleic acid encoding the same in a method of modifying a genome of a cell of a multicellular organism.

11. The kit of claim 4, wherein said targeting construct further comprises a polylinker for cloning of DNA into the targeting construct at a specific position.