Nucleic acid ligand conjugates and their use for delivery to cells

Abstract

The present invention provides compositions and methods for using targeting ligands to deliver nucleic acids to cells. [Solution] A conjugate product comprising a polypeptide containing an epidermal growth factor receptor (EGFR) targeting moiety, a linker, and a nucleic acid is provided.

Claims

1. a) Polypeptides containing an epidermal growth factor receptor (EGFR) targeting moiety, b) Linker, and c) Nucleic acid A conjugate product containing the conjugate product.

2. The conjugate product according to claim 1, wherein the polypeptide comprises GE11.

3. The polypeptide has been modified to include a cysteine ​​residue at its C-terminus. The conjugate product described in item 1 or 2.

4. The polypeptide comprises the amino acid sequence of SEQ ID NO: 2, according to any one of claims 1 to 3. The conjugate product described.

5. The linker comprises polyethylene glycol (PEG), any one of claims 1 to 4. The conjugate product described in item 1.

6. Claims 1 to 5, wherein the linker comprises dibenzocyclooctin-PEG4-maleimide The conjugate product described in any one of the items.

7. The linker is conjugated with a severable disulfide bond or an inseparable handle. A gated hexyl amino linker, according to any one of claims 1 to 4. Jugate product.

8. The linker is succinimidyl 3-(2-pyridyldithio)propionate, Sinimidyl-trans-4-(N-maleimidylmethyl)cyclohexane-1-cal The following is a boxylate or triethylene glycol as described in any one of claims 1 to 4. The conjugate product of [the product].

9. The polypeptide is covalently bonded to the linker, according to any one of claims 1 to 8. The conjugate product described.

10. Claim 9, wherein the polypeptide is covalently bonded to the thiol group on the cysteine ​​residue. The conjugate product described above.

11. The conjugate generation according to any one of claims 1 to 10, wherein the nucleic acid is DNA. thing.

12. The conjugate generation according to any one of claims 1 to 10, wherein the nucleic acid is RNA. thing.

13. Conjugate generation according to any one of claims 1 to 12, wherein the nucleic acid is single-stranded. thing.

14. Conjugate generation according to any one of claims 1 to 12, wherein the nucleic acid is double-stranded. thing.

15. The nucleic acids include siRNA, microRNA, shRNA, antisense nucleic acids, and ribonucleotides. Mu, killer tRNA, guide RNA, long non-coding RNA, anti-miRNA oligonucleotides Any one of claims 1 to 14, selected from the group consisting of tides and plasmid DNA. The conjugate product described above.

16. The nucleic acid is KRAS silencing siRNA, any one of claims 1 to 15. The conjugate product described above.

17. The linker is covalently bonded to the nucleic acid, according to any one of claims 1 to 16. Conjugate product.

18. Claim 17, wherein the nucleic acid is modified to provide a binding site to the linker. The conjugate product described above.

19. Claim 18, wherein the nucleic acid is modified to include an azide group as the binding site. The conjugate product described.

20. Composition comprising the conjugate product and carrier according to any one of claims 1 to 19 。

21. The conjugate product according to any one of claims 1 to 19 and pharmaceutically acceptable A pharmaceutical composition containing a carrier.

22. A method for delivering nucleic acids to cells, wherein the cells are in an effective quantity as described in claim 20 or 21. A method comprising the step of bringing into contact with the composition.

23. The method according to claim 22, wherein the cells are cancer cells.

24. The aforementioned cancer cells include non-small cell lung cancer cells, lung cancer cells, colon cancer cells, pancreatic cancer cells, and The method according to claim 23, wherein cells are selected from the group consisting of blood cancer cells.

25. The cells express high levels of EGFR, as described in any one of claims 22 to 24. The method.

26. Any one of claims 22 to 25, which does not involve the use of a transfection reagent. The method described in section [section number].

27. A method of treating a disease in a person who requires treatment for the disease, wherein the treatment is performed on the person. The pharmaceutical composition according to claim 21 is administered to treat the disease. Methods of including.

28. The method according to claim 27, wherein the disease is cancer.

29. Is the aforementioned cancer a group consisting of non-small cell lung cancer, lung cancer, colon cancer, pancreatic cancer, and hematological cancer? The method according to claim 28, which is selected from the above.

30. Any one of claims 27 to 29, which does not involve the use of a transfection reagent. The method described in section [section number].

31. A method for increasing nucleic acid uptake by cells, comprising forming a conjugate product. To do this, the nucleic acid is linked to the polypeptide containing the EGFR targeting portion via a linker. The step includes conjugating the cells, wherein the cells express EGFR, and the cells The incorporation of the nucleic acid by the above means that the polypeptide containing the EGFR targeting portion A method that increases compared to unjugated nucleic acids.

32. The method according to claim 31, wherein the polypeptide comprises GE11.

33. The polypeptide has been modified to include a cysteine ​​residue at its C-terminus. The method described in claim 31 or 32.

34. Any of claims 31 to 33, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1. The method described in paragraph 1.

35. The linker comprises polyethylene glycol (PEG), according to any of claims 31 to 34. The method described in item 1.

36. Claim 31, wherein the linker comprises dibenzocyclooctin-PEG4-maleimide The method described in any one of items ~35.

37. Any of claims 31 to 36, wherein the polypeptide is covalently bonded to the linker. The method described in paragraph 1.

38. The polypeptide is covalently bonded to the thiol group on the cysteine ​​residue, claim Method 37.

39. The method according to any one of claims 31 to 38, wherein the nucleic acid is DNA.

40. The method according to any one of claims 31 to 38, wherein the nucleic acid is RNA.

41. The method according to any one of claims 31 to 40, wherein the nucleic acid is single-stranded.

42. The method according to any one of claims 31 to 40, wherein the nucleic acid is double-stranded.

43. The nucleic acids include siRNA, microRNA, shRNA, antisense nucleic acids, and ribonucleotides. Mu, killer tRNA, guide RNA, long non-coding RNA, anti-miRNA oligonucleotides Selected from the group consisting of cido and plasmid DNA, any one of claims 31 to 42 The method described in section [section number].

44. Any of claims 31 to 43, wherein the nucleic acid is KRAS silencing siRNA. The method described in paragraph 1.

45. The linker is covalently bonded to the nucleic acid, as described in any one of claims 31 to 44. The method.

46. Claim 45, wherein the nucleic acid is modified to provide a binding site to the linker. Methods used.

47. Claim 46, wherein the nucleic acid is modified to include an azide group as the binding site. Method of description.

Images