Site-specific serine recombinases and methods of their use

• US 10,648,003 B2
• Filed: 06/08/2018
• Issued: 05/12/2020
• Est. Priority Date: 02/02/2005
• Status: Active Grant

• Current Assignee: Intrexon Corporation
• Original Assignee: Intrexon Corporation
• Inventors: Padidam, Malla
• Primary Examiner(s): Treptow, Nancy A
• Application Number:
  US16/003,266
• Publication Number:
  US 20180346934A1
• Time in Patent Office: 704 Days
• Field of Search: None
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/22 for herpes viruses
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/907 in mammalian cells
• 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
• CPC Class Codes: C12Y 301/22 : Endodeoxyribonucleases producing 3'-phosphomonoesters (3.1.22)

Abstract

The present invention provides a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of a Listeria monocytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP and when the first recombination attachment site is attP, the second recombination attachment site is attB. The invention also describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors and methods of the present invention are also useful in gene therapy applications.

Claims

1. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising:

providing a eukaryotic cell that comprises a first recombination site and a second recombination site;

a stop sequence positioned between said first recombination site and said second recombination site;

contacting the first and second recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites and deletion of said stop sequence, wherein the recombinase polypeptide can mediate recombination between the first and second recombination sites, the first recombination site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is a Bacillus subtilis SPβ

c2 phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attB.

2. The method of claim 1, wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.

3. The method of claim 1, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide or by expression of a polynucleotide that encodes the recombinase polypeptide.

4. The method of claim 1, wherein the recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide.

5. The method of claim 1, wherein the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA.

14. The method of claim 3, wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.

15. The method of claim 3, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.

17. The method of claim 1, wherein the recombinase polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

6. A method for obtaining a eukaryotic cell having a stably integrated polynucleotide sequence, the method comprising:

introducing a polynucleotide into a eukaryotic cell that comprises a first recombination attB or attP site, wherein the polynucleotide comprises a plurality of nucleic acid sequences of interest and a second recombination attP or attB site, and contacting the first and the second recombination sites with a prokaryotic recombinase polypeptide, wherein the recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and the recombinase is a Bacillus subtilis SPβ

c2 phage recombinase, provided that when the first recombination site is attB, the second recombination site is attP and when the first recombination site is attP, the second recombination site is attB.

7. The method of claim 6, wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.

8. The method of claim 6, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide or by expression of a polynucleotide that encodes the recombinase polypeptide.

9. The method of claim 6, wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of RNA that encodes the recombinase polypeptide.

13. The method of claim 8, wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.

16. The method of claim 8, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.

10. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising:

providing a eukaryotic cell that comprises a first recombination site and a second recombination site with a polynucleotide sequence flanked by a third recombination site and a fourth recombination site;

contacting the recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, wherein the recombinase polypeptide can mediate recombination between the first and third recombination sites and the second and fourth recombination sites, the first and second recombination sites are attP or attB, the third and fourth recombination sites are attB or attP, and the recombinase is a Bacillus subtilis SPβ

c2 phage recombinase, provided that when the first and second recombination attachment sites are attB, the third and fourth recombination attachment sites are attP, and when the first and second recombination attachment sites are attP, the third and fourth recombination attachment sites are attB.

11. The method of claim 10, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide or by expression of a polynucleotide that encodes the recombinase polypeptide.

12. The method of claim 10, wherein the recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide.

18. The method of claim 11, wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.

19. The method of claim 11, wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.