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1. WO2019113711 - ENCAPSULATION D'EXOSOMES ET AUTOPHAGIE CIBLÉE

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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WHAT IS CLAIMED IS:

1. A fusion construct comprising a biologically active agent functionally linked with a functional moiety comprising at least one of an LC3 or GABARAP protein or a portion thereof; an LC3 -interacting motif (LIM); an LC3 -interacting region (LIR); Atg4 protein or an LC3 -binding portion thereof; Atg7 protein or an LC3 -binding portion thereof; AtglO protein or an LC3 -binding portion thereof; another LC3 -binding sequence or domain; Atg5 protein or an autophagy-triggering portion thereof; Atgl6Ll or an autophagy-triggering portion thereof; or another autophagy-triggering sequence or domain; or an amino acid sequence having at least 50% sequence identity therewith.

2. The fusion construct of claim 1, wherein the biologically active agent comprises a targeting moiety for binding a cellular target.

3. The fusion construct of claim 2, wherein the biologically active agent comprises an antibody, an antigen-binding portion thereof, or an antibody derivative or mimic; or wherein the biologically active agent comprises a protein or peptide-based binder of the cellular target.

4. The fusion construct of claim 3, wherein the antibody derivative or mimic comprises a single-chain variable fragment (scFv), a nanobody, or an antibody-like molecule from a camel, llama, shark, or other animal, a centyrin, or other protein-based agent with target binding properties.

5. The fusion construct of any one of claims 1-4, wherein the biologically active agent comprises an antibody, antigen-binding fragment thereof, an antibody derivative or mimic, or a protein or peptide which specifically binds a cellular target comprising misfolded proteins, altered or post-translationally modified protein, proteins arranged in a specific conformation, protein inclusions, protein aggregates, lipid droplets, bacteria, virus, cytoplasmic DNA, or mitochondria or other cellular organelle or membraneless structure, or crystals or uric acid.

6. The fusion construct of claim 2 or 5, wherein the cellular target is associated with a neurodegenerative disease, non-alcoholic steatohepatitis, inflammatory disease, autoimmune disease, or another disease or disorder relating to or treatable by autophagy.

7. The fusion construct of claim 5, wherein the misfolded proteins, altered protein, protein inclusions, and/or protein aggregates comprise TDP-43 inclusions found in ALS patients; Tau fibrils found in Alzheimer’s, CTE, Corticobasal Degeneration, Progressive Supranuclear Palsy, and/or other Tauopathies patients; synuclein or Alpha-synuclein or Lewy bodies and/or damaged mitochondria found in Parkinson’s patients; Htt repeats in Huntington’s disease; dipeptide repeats produced by C90RF72 intronic repeats; misfolded SOD1; and/or hyperphosphorylated or fibrillary Tau.

8. The fusion construct of any one of claims 1-7, wherein the biologically active agent is functionally linked with the functional moiety directly, or indirectly via one or more linkers and/or intervening groups.

9. The fusion construct of any one of claims 1-8, wherein the biologically active agent is functionally linked to an N-terminal portion or end of the functional moiety.

10. The fusion construct of any one of claims 1-9, wherein the biologically active agent is functionally linked to a C-terminal portion or end of the functional moiety.

11. The fusion construct of any one of claims 1-10, wherein the functional moiety comprises LC3 protein or a portion thereof.

12. The fusion construct of claim 11, wherein the LC3 protein comprises an LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2, or GABARAPL3 homologue, or an amino acid sequence having at least 50% sequence identity therewith.

13. The fusion construct of claim 12, wherein the LC3 protein comprises LC3A, or an amino acid sequence having at least 50% sequence identity therewith.

14. The fusion construct of claim 12, wherein the LC3 protein comprises LC3B, or an amino acid sequence having at least 50% sequence identity therewith.

15. The fusion construct of any one of claims 1-14, wherein the biologically active agent

comprises anti-TDP43 scFv clone VH7; anti-TDP-43 scFv 4aDl; anti-Tau scFv 4A3; anti- Tau scFv 4E4; adipophilin; perilipin2; a protein which binds to a lipid droplet surface; PINK1; Parkin; or a protein which binds mitochondria.

16. A nucleic acid encoding a fusion construct as defined in any one of claims 1-15.

17. An expression vector for expressing the fusion construct as defined in any one of claims

1-15.

18. A host cell comprising the nucleic acid of claim 16 or the expression vector of claim 17, the host cell expressing the fusion construct of any one of claims 1-15.

19. The host cell of claim 18, wherein the host cell produces exosomes comprising the fusion construct of any one of claims 1-15.

20. The host cell of claim 18 or 19, wherein the host cell has low or reduced levels of Atg7, or another protein required for autophagy such as Atg4, Atgl4, Atg3, Atgl4, or Atgl2.

21. The host cell of claim 20, wherein the host cell is Atg7-/-.

22. The host cell of any one of claims 18-21, wherein the host cell is a 293 cell; a human neonatal fibroblast; an NSC-34 cell; an SH5Y cell; or a BV2 cell.

23. An exosome comprising the fusion construct of any one of claims 1-15.

24. A composition comprising one or more of a fusion construct of any one of claims 1-15; a nucleic acid of claim 16; an expression vector of claim 17; a host cell of any one of claims 18-22; and/or an exosome of claim 23; and, optionally, a pharmaceutically acceptable excipient, carrier, or diluent.

25. The composition of claim 24, further comprising at least one autophagy-activating agent, an siRNA or other gene silencing agent, or both.

26. The composition of claim 25, wherein the autophagy-activating agent comprises an mTOR inhibitor.

27. The composition of claim 25 or 26, wherein the autophagy-activating agent comprises one or more of rapamycin, sirolimus, eversolimus, tacrolimus, INK 128, pp242, starvation, or other mTORCl and/or mTORC inhibitor.

28. The composition of claim 25, wherein the autophagy-activating agent comprises Trehalose and/or Beclinl peptide.

29. A method for packaging a fusion construct as defined in any one of claims 1-15 into an exosome, said method comprising:

expressing the fusion construct in an exosome-producing cell or otherwise introducing the fusion construct into the exosome-producing cell; and

culturing the exosome-producing cell in a cell media under conditions in which the exosome-producing cell generates and secretes exosomes comprising the fusion construct into the cell media; or

administering the exosome-producing cell into a subject in need thereof such that the exosome-producing cell generates and secretes exosomes comprising the fusion construct within the subj ect.

30. The method of claim 29, further comprising a step of obtaining, isolating, or purifying the secreted exosomes comprising the fusion construct from the cell media.

31. The method of claim 29 or 30, wherein the exosome-producing cell has low or reduced levels of Atg7.

32. The method of any one of claims 29-31, wherein the exosome-producing cell is Atg7-/-

33. The method of any one of claims 29-32, wherein the exosome-producing cell is a 293 cell; a human neonatal fibroblast; an NSC-34 cell; an SH5Y cell; or a BV2 cell.

34. The method of any one of claims 29-33, wherein the exosome-producing cell is a host cell as defined in any one of claims 18-22.

35. The method of any one of claims 29-34, wherein the secreted exosomes further comprise an autophagy-activating agent, a gene silencing nucleic acid, or both.

36. An exosome produced by the method of any one of claims 29-35.

37. A method for delivering a biologically active agent into a cell, said method comprising:

expressing or introducing a fusion construct into an exosome-producing cell, the fusion construct comprising the biologically active agent functionally linked to a functional moiety comprising at least one of an LC3 protein or a portion thereof; an LC3 -interacting motif (LIM); an LC3 -interacting region (LIR); Atg4 protein or an LC3 -binding portion thereof; Atg7 protein or an LC3 -binding portion thereof; AtglO protein or an LC3- binding portion thereof; another LC3 -binding sequence or domain; Atg5 protein or an autophagy-triggering portion thereof; Atgl6Ll or an autophagy-triggering portion thereof; or another autophagy-triggering sequence or domain; or an amino acid sequence having at least 50% sequence identity therewith; and

administering the exosome-producing cell into a subject in need thereof such that the exosome-producing cell generates and secretes exosomes comprising the fusion construct within the subject so as to contact the cell with the secreted exosomes;

or

culturing the exosome-producing cell in a cell media under conditions in which the exosome-producing cell generates and secretes exosomes comprising the fusion construct into the cell media;

obtaining, isolating, or purifying the secreted exosomes comprising the fusion construct from the cell media; and

contacting the cell with the secreted exosomes.

38. The method of claim 37, wherein the step of contacting the cell with the secreted

exosomes comprises administering the secreted exosomes to a subject in which the cell is located.

39. The method of claim 37 or 38, wherein the fusion construct comprises a fusion construct as defined in any one of claims 1-15.

40. The method of any one of claims 37-39, wherein the exosome-producing cell has low or reduced levels of Atg7.

41. The method of any one of claims 37-40, wherein the exosome-producing cell is Atg7-/-

42. The method of any one of claims 37-41, wherein the exosome-producing cell is a 293 cell; a human neonatal fibroblast; an NSC-34 cell; an SH5Y cell; or a BV2 cell.

43. The method of claim 42, wherein the exosome-producing cell is a 293 cell and the cell is a liver cell, a fibroblast cell, or a motor neuron.

44. The method of claim 42, wherein the exosome-producing cell is a human neonatal fibroblast, and the cell is a liver cell, a brain cell, a spinal cord cell, or a kidney cell.

45. The method of claim 42, wherein the exosome-producing cell is an NSC-34 cell, and the cell is a liver cell, a small intestine cell, a brain cell, or a spinal cord cell.

46. The method of claim 42, wherein the exosome-producing cell is a SH5Y cell, and the cell is a liver cell, a kidney cell, or a brain cell.

47. The method of any one of claims 37-46, wherein the exosome-producing cell is a host cell as defined in any one of claims 18-22.

48. The method of any one of claims 37-47, wherein the secreted exosomes comprising the fusion construct further comprise an autophagy-activating agent, a gene silencing nucleic acid, or both.

49. Use of the construct of any one of claims 1-15, the nucleic acid of claim 16; the expression vector of claim 17; the host cell of any one of claims 18-22; the exosome of claim 23; the composition of any one of claims 24-28; the exosome of claim 36; or any combination thereof; for treating or preventing a disease or disorder in a subject in need thereof.

50. Use of the construct of any one of claims 1-15, the nucleic acid of claim 16; the expression vector of claim 17; the host cell of any one of claims 18-22; the exosome of claim

23; the composition of any one of claims 24-28; the exosome of claim 36; or any combination thereof; in the manufacture of a medicament for treating or preventing a disease or disorder in a subject in need thereof.

51. The use of claim 49 or 50, wherein the disease or disorder is caused by, or associated with, any one or more of misfolded proteins, altered proteins, protein inclusions, protein aggregates, lipid droplets, bacteria, cytoplasmic DNA, or mitochondria.

52. The use of any one of claims 49-51, wherein the disease or disorder is a neurodegenerative disease or atherosclerosis.

53. The use of any one of claims 49-52, wherein the disease or disorder is Alzheimer’s, CTE, and/or Tauopathy-related disease; Parkinson’s; Frontal Temporal Dementia; and/or

Amyotrophic Lateral Sclerosis (ALS).

54. A method for treating or preventing a disease or disorder in a subject in need thereof, said method comprising:

administering the fusion construct of any one of claims 1-15; the nucleic acid of claim 16; the expression vector of claim 17; the host cell of any one of claims 18-22; the exosome of claim

23; the composition of any one of claims 24-28; the exosome of claim 36; or any combination thereof; to the subject.

55. The method of claim 54, wherein the step of administering results in the fusion construct being present in a cell of the subject which is causing, or affected by, the disease or disorder.

56. The method of claim 54 or 55, wherein the disease or disorder is caused by, or associated with, any one or more of misfolded proteins, altered proteins, protein inclusions, protein

aggregates, lipid droplets, bacteria, cytoplasmic DNA, or mitochondria.

57. The method of any one of claims 54-56, wherein the disease or disorder is a neurodegenerative disease or atherosclerosis or NASH or a viral infection.

58. The method of any one of claims 54-57, wherein the disease or disorder is Alzheimer’s, CTE, and/or Tauopathy -related disease; Parkinson’s; Frontal Temporal Dementia; and/or Amyotrophic Lateral Sclerosis (ALS).

59. The use of any one of claims 49-53, or the method of any one of claims 54-58, wherein the fusion construct of any one of claims 1-15; the nucleic acid of claim 16; the expression vector of claim 17; the host cell of any one of claims 18-22; the exosome of claim 23; the composition of any one of claims 24-28; the exosome of claim 36; or any combination thereof; is for administration in combination with at least one autophagy-activating agent, a gene silencing agent, or both.

60. The use or method of claim 59, wherein the autophagy-activating agent comprises an mTOR inhibitor.

61. The use or method of claim 59 or 60, wherein the autophagy-activating agent comprises one or more of rapamycin, sirolimus, eversolimus, tacrolimus, INK 128, pp242, starvation, or other mTORCl and/or mTORC inhibitor.

62. The use or method of claim 59, wherein the autophagy-activating agent comprises Trehalose and/or Beclinl peptide.

63. The use or method of claim 59, wherein the gene silencing agent targets a cellular target which is also targeted by the biologically active agent of the fusion construct.

64. A virus comprising a nucleic acid of claim 16, or an expression vector of claim 17.

65. The virus of claim 64, for use in expressing a fusion construct of any one of claims 1-15 in an exosome-producing cell, or in a target cell in need thereof.

66. The virus of claim 64 or 65, which is an AAV, lentivirus, or oncolytic virus.

67. A kit comprising at least one of:

the construct of any one of claims 1-15;

the nucleic acid of claim 16;

the expression vector of claim 17;

the host cell of any one of claims 18-22;

the exosome of claim 23;

the composition of any one of claims 24-28;

the exosome of claim 36;

the virus of any one of claims 64-66;

instructions for performing a method of any one of claims 29-35, 37-48, or 54-63; or any combination thereof.