Claims
- 1. A method for targeting and altering, by homologous recombination, a pre-selected target DNA sequence in a eukaryotic cell to make a targeted sequence modification, said method comprising introducing into at least one eukaryotic cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each having a homology clamp that substantially corresponds to or is substantially complementary to a preselected target DNA sequence.
- 2. A method according to claim 1 further comprising identifying a target cell having a targeted DNA sequence modification at a preselected target DNA sequence.
- 3. A method according to claim 1, wherein said targeting polynucleotides are coated with said recombinase.
- 4. A method according to claim 1, wherein said eucaryotic cell is a plant cell.
- 5. A method according to claim 1, wherein said eucaryotic cell is a mammalian cell.
- 6. A method according to claim 1, wherein said eucaryotic cell is a zygote.
- 7. A method according to claim 1, wherein said eucaryotic cell is an embryonic stem cell.
- 8. A method according to claim 1, wherein said eucaryotic cell is an avian cell.
- 9. A method according to claim 1, wherein said recombinase is a species of prokaryotic recombinase.
- 10. A method according to claim 8, wherein said prokaryotic recombinase is a species of prokaryotic recA protein.
- 11. A method according to claim 10, wherein said recA protein species is E. coli recA.
- 12. A method according to claim 1, wherein said recombinase is a species of eukaryotic recombinase.
- 13. A method according to claim 12, wherein said recombinase is a Rad5l recombinase.
- 14. A method according to claim 12, wherein said eukaryotic recombinase is a complex of recombinase proteins.
- 15. A method according to claim 1, wherein said targeting polynucleotide is conjugated to a cell-uptake component.
- 16. A method according to claim 15, wherein said cell-uptake component is conjugated to said targeting polynucleotide by noncovalent binding.
- 17. A method according to claim 15, wherein the cell-uptake component comprises an asialoglycoprotein.
- 18. A method according to claim 15, wherein the cell-uptake component comprises a protein-lipid complex.
- 19. A method according to claim 15, wherein said targeting polynucleotide is conjugated to a cell-uptake component and to a recombinase, forming a cell targeting complex.
- 20. A method according to claim 1, wherein the targeted sequence modification comprises a deletion of at least one additional nucleotide.
- 21. A method according to claim 1, wherein the targeted sequence modification comprises the addition of at least one additional nucleotide.
- 22. A method according to claim 20 or 21, wherein said complementary single stranded targeting polynucleotides comprise an internal homology clamp.
- 23. A method according to claim 1, wherein the targeted sequence modification comprises the substitution of at least one nucleotide.
- 24. A method according to claim 23, wherein the targeted sequence modification comprises a plurality of substitutions.
- 25. A method according to claim 1, wherein the targeted sequence modification corrects a disease allele in a cell.
- 26. A method according to claim 25, wherein said cell is a human cell and the disease allele is a CFTR allele associated with cystic fibrosis.
- 27. A method according to claim 25, wherein said cell is a mammalian cell and the 10disease allele is an OTC allele.
- 28. A method according to claim 1, wherein the recombinase and the targeting polynucleotides are introduced simultaneously.
- 29. A method according to claim 28, wherein the recombinase and the targeting polynucleotide are introduced into the target cell by a method selected from the group consisting of: microinjection, electroporation, laser poration, biolistics, or contacting of the cell with a lipid-protein-targeting polynucleotide complex.
- 30. A method according to claim 1, wherein the targeted sequence modification creates a sequence that encodes a polypeptide having a biological activity.
- 31. A method according to claim 30, wherein the biological activity is an enzymatic activity.
- 32. A method according to claim 30 or 31, wherein the targeted sequence modification is in a human cell and encodes a human polypeptide.
- 33. A method according to claim 32, wherein the targeted sequence modification is in a human oncogene or tumor suppressor gene sequence.
- 34. A method according to claim 33, wherein the targeted sequence modification is in a human p53 sequence.
- 35. A method according to claim 1, wherein each targeting polynucleotide comprises a homology clamp that is less than 1200 nucleotides long.
- 36. A method according to claim 1, wherein the targeting polynucleotide is less than 1200 nucleotides long.
- 37. A method according to claim 1, wherein the targeted sequence modification corrects a gene in a cell.
- 38. A method according to claim 1, wherein the targeted sequence modification adds a gene to a cell.
- 39. A method according to claim 1, wherein the targeted sequence modification disrupts a gene in a cell.
- 40. A method according to claim 1, wherein the targeted sequence modification modifies a gene in a cell.
- 41. A method according to claim 40, wherein the gene is the gal T gene associated with xenoreactivity in humans.
- 42. A method according to claim 1, wherein at least one of said complementary single stranded nucleic acids further comprise a chemical substituent.
- 43. A method according to claim 42, wherein said chemical substitutent is covalently attached to said nucleic acid.
- 44. A composition for producing a targeted modification of an endogenous DNA sequence, comprising two substantially complementary single-stranded targeting polynucleotides and at least one recombinase.
- 45. A composition according to claim 44, further comprising a cell-uptake component.
- 46. A composition for producing a targeted sequence modification of a disease allele, comprising two substantially complementary single-stranded targeting polynucleotides, at least one of which contains a corrected sequence, and a recombinase.
- 47. A kit for therapy, monitoring, or prophylaxis of a gene comprising at least one recombinase and two substantially complementary single-stranded targeting polynucleotides.
- 48. A method for treating a disease of a animal harboring a disease allele, comprising administering to the animal a composition consisting essentially of two substantially complementary single-stranded targeting polynucleotides, at least one of which corrects the disease allele, and at least one recombinase.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuing application of U.S. Serial No. 60/041,173, filed Mar. 21, 1997, and of Ser. No. 08/385,713, filed Feb. 8, 1995 and of Ser. No. 08/275,916, filed Jul. 14, 1994, and of Ser. No. 07/939,767, filed Sep. 2, 1992, abandoned, and of Ser. No. 07/873,438 filed Apr. 24, 1992, abandoned.
Provisional Applications (1)
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60041173 |
Mar 1997 |
US |
Continuations (3)
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08910415 |
Aug 1997 |
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Child |
09990433 |
Nov 2001 |
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Parent |
09927160 |
Aug 2001 |
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Child |
09990433 |
Nov 2001 |
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Parent |
09079877 |
May 1998 |
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Child |
09927160 |
Aug 2001 |
US |
Continuation in Parts (1)
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08910415 |
Aug 1997 |
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09079877 |
May 1998 |
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