Site-specific serine recombinases and methods of their use

• US 9,034,650 B2
• Filed: 02/02/2005
• Issued: 05/19/2015
• Est. Priority Date: 02/02/2005
• Status: Active Grant

• Current Assignee: Intrexon Corporation
• Original Assignee: Intrexon Corporation
• Inventors: Padidam, Malla
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US11/049,552
• Publication Number:
  US 20060172377A1
• Time in Patent Office: 3,758 Days
• Field of Search: None
• US Class Current: 435/462
• 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;

contacting the first and second recombination sites with a prokaryotic serine recombinase polypeptide, resulting in recombination between the recombination sites,

wherein the serine 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 serine recombinase is a Streptococcus pyogenes SF370.1 phage serine 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.

3. The method of claim 1 or 2, wherein the serine recombinase polypeptide is encoded by a polynucleotide operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.

4. The method of claim 1 or 2, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the serine recombinase polypeptide.

5. The method of claim 1 or 2, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.

6. The method of claim 1 or 2, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the serine recombinase polypeptide.

7. The method of claim 1 or 2, wherein the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA.

22. The method of claim 1 or claim 2, wherein the site-specific recombination is irreversible.

2. 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;

contacting the first and second recombination sites with a prokaryotic serine recombinase polypeptide, resulting in recombination between the recombination sites,

wherein the serine recombinase polypeptide can mediate recombination between the first and second recombination sites,

the first recombination site is attP or attB,

the second recombination site is a pseudo attachment site, and

the serine recombinase is a Streptococcus pyogenes SF370.1 phage serine recombinase.

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

providing a eukaryotic cell that comprises a first recombination attB or attP site,

introducing a polynucleotide into the eukaryotic cell, wherein the polynucleotide comprises a nucleic acid sequence and a second recombination attP or attB site, and

contacting the first and the second recombination sites with a prokaryotic serine recombinase polypeptide,

wherein the serine recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and

the serine recombinase is a Streptococcus pyogenes SF370.1 phage serine 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.

10. The method of claim 8 or 9, wherein the serine recombinase polypeptide is encoded by a polynucleotide operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.

11. The method of claim 8 or 9, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the serine recombinase polypeptide.

12. The method of claim 8 or 9, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.

13. The method of claim 8 or 9, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell by expression of RNA that encodes the serine recombinase polypeptide.

20. The method of claim 8, wherein the first attB or attP site is located in the chromosome of the eukaryotic cell.

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

providing a eukaryotic cell that comprises a first recombination pseudo attachment site,

introducing a polynucleotide into the eukaryotic cell,

wherein the polynucleotide comprises a nucleic acid sequence and a second recombination attP or attB site, and

contacting the first and the second recombination sites with a prokaryotic serine recombinase polypeptide, wherein the serine recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and

the serine recombinase is a Streptococcus pyogenes SF370.1 phage serine recombinase.

21. The method of claim 9, wherein the first recombination pseudo attachment site is located in the chromosome of the eukaryotic cell.

14. 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 serine recombinase polypeptide, resulting in recombination between the recombination sites,

wherein the serine 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 serine recombinase is a Streptococcus pyogenes SF370.1 phage serine 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.

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

16. The method of claim 14, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.

17. The method of claim 14, wherein the serine recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the serine recombinase polypeptide.

23. The method of any one of claims 14 and 18 wherein the site specific recombination is irreversible.

18. A method for site-specific recombination, the method comprising:

providing a first recombination site and a second recombination site;

contacting the first and second recombination sites with a prokaryotic serine recombinase polypeptide, resulting in recombination between the recombination sites,

wherein the serine recombinase polypeptide can mediate recombination between the first and second recombination sites,

the first recombination site is attP or attB, the second recombination site is attB or attP, and

the serine recombinase is a Streptococcus pyogenes SF370.1 phage serine 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.

19. A method for the site-specific integration of a polynucleotide of interest into the genome of a transgenic subject,

wherein said genome comprises a first recombination attB or attP site or pseudo attB or pseudo attP site,

the method comprising:

introducing a nucleic acid that comprises the polynucleotide of interest and a second recombination attP or attB site;

contacting the first and the second recombination sites with a prokaryotic serine recombinase polypeptide,

wherein the serine recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and

the serine recombinase is a Streptococcus pyogenes SF370.1 phage serine recombinase,

provided that when the first recombination site is attB or pseudo attB, the second recombination site is attP and when the first recombination site is attP or pseudo attP, the second recombination site is attB.

24. The method of claim 19, wherein the site-specific integration is irreversible.