METHODS AND COMPOSITIONS FOR TARGETED GENE MODIFICATION

• US 20110059160A1
• Filed: 08/26/2010
• Published: 03/10/2011
• Est. Priority Date: 08/03/2009
• Status: Active Grant

• Current Assignee: Recombinetics, Inc
• Original Assignee: Recombinetics, Inc
• Inventors: Essner, Jeffrey J., Liao, Hsin-Kai, Fahrenkrug, Scott C.
• Granted Patent: US 9,074,224 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2770/14022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

Disclosed herein are methods and compositions for gene targeting utilizing fusion molecules comprising a recombinase domain and a sequence-specific DNA-binding domain.

Claims

1. A method of transfecting a cell comprising

introducing into the cell:

an exogenous nucleic acid and a nucleoprotein filament of a proteinaceous fusion molecule and a nucleic acid probe complementary to a target site of DNA of the cell,

wherein the fusion protein comprises a recombinase domain that contributes to the filament, a DNA-binding domain, and a nuclear localization signal domain,

wherein the exogenous nucleic acid is incorporated into the DNA of the cell and expressed by the cell.

2. The method of claim 1 wherein the exogenous nucleic acid is double-stranded DNA and is free of promoter sequences.

3. The method of claim 2 wherein the nucleic acid probe comprises complementary single strand DNA (cssDNA) and the DNA-binding domain is not specifically bound to DNA at the time of the introduction.

4. The method of claim 3 wherein the exogenous nucleic acid site of insertion is in the target gene.

5. The method of claim 3 wherein efficiency of transfecting the cell is at least about 1%, as measured by an in vitro test with direct injection.

6. The method of claim 3 wherein the recombinase comprises RecA.

7. The method of claim 3 wherein the DNA-binding domain comprises Gal4.

8. The method of claim 1 wherein the recombinase is chosen from the group consisting of Cre recombinase, Hin recombinase, Tre recombinase, flippase recombination enzyme, uvsX, RuvC, DST2, KEM1 and XRN1, STPa/DST1, and HPP-1.

9. The method of claim 1 wherein the DNA-binding domain comprises a polypeptide that specifically binds to DNA and is chosen from the group consisting of minor groove binders, major groove binders, antibiotics, intercalating agents, polyamides, and a polypeptide sequence of a transcription factor, nuclease, zinc finger nucleases, zinc fingers, and helix-turn-helix proteins.

10. The method of claim 1 wherein the fusion molecule is chosen from the group consisting of SEQ ID NO:

3, SEQ ID NO;

5, and conservative substitutions thereof.

11. The method of claim 1 wherein the nuclear localization signal is chosen from the group consisting of SV40 large T antigen, nucleoplasmin, HIV-1 Rev, and M9.

12. The method of claim 1 wherein the fusion molecule comprises a synthetic non-peptide linker.

13. The method of claim 1 wherein the probe is directed to a mutation in the cell DNA, and the exogenous nucleic acid comprises a wild-type sequence corresponding to the mutation.

14. The method of claim 1 wherein the exogenous nucleic acid is non-homologous relative to the cell DNA.

15. A cell transfected by the method of claim 1.

16. The cell of claim 15 chosen from the group consisting of a vertebrate cell, a mammalian cell, a porcine cell, a human cell, a plant cell, and a stem cell.

17. A transgenic animal formed from progeny of a germline cell transfected by the method of claim 1.

18. A purified composition for transfection of exogenous DNA into chromosomal DNA of a cell, the composition comprising a nucleoprotein filament of a probe and a proteinaceous fusion molecule, wherein the probe comprises double-stranded denatured DNA complementary to a chromosomal DNA site, and the fusion molecule comprises a recombinase domain and a DNA-binding domain, wherein the composition is free of DNA sequences that specifically bind to the DNA-binding domain.

19. The composition of claim 18 wherein the fusion molecule further comprises a nuclear localization signal domain.

20. The composition of claim 18 further comprising exogenous double stranded DNA.

21. A method of treating a genetic disease in an animal comprising

introducing into a cell of the animal:

an exogenous nucleic acid and a nucleoprotein filament of a proteinaceous fusion molecule and a nucleic acid probe complementary to a target site of DNA of the cell,

wherein the fusion molecule comprises a recombinase domain that contributes to the filament, and a DNA-binding domain at the time of the introduction,

wherein the exogenous nucleic acid is expressed by the cell to provide a therapeutic protein to the animal to treat the disease, the method is performed without a viral vector and without a transposon vector, and the cell is transfected by a method chosen from the group consisting of in vitro, in vivo, and ex vivo.

22. The method of claim 21 wherein the method of introduction is chosen from the group consisting of electroporation, liposome, nuclear transplantation, Pronuclear microinjection, and somatic cell nuclear transfer.

23. The method of claim 21 wherein the probe is directed to a mutated DNA of the animal that contributes to the disease.

24. The method of claim 21 wherein the fusion molecule further comprises a nuclear localization signal domain.