Compositions and methods for screening agents targeting Tau protein 4R

Claims

1. A cell containing a tau protein isoform with four repeats (4R) associated with a first reporter protein and a tau protein isoform with three repeats (3R) associated with a second reporter protein that differs from the first reporter protein.

2. The cell of claim 1, comprising a first fusion protein comprising the 4R tau protein isoform fused to a first reporter protein, and a second fusion protein comprising the 3R tau protein isoform fused to a second reporter protein.

3. The cell of claim 2, comprising a first nucleic acid encoding a first fusion protein and a second nucleic acid encoding a second fusion protein, wherein the cell expresses the first fusion protein and the second fusion protein.

4. The cell of claim 1, comprising a first nucleic acid comprising a coding sequence for the 4R tau protein isoform and a coding sequence for a first reporter protein, and a second nucleic acid comprising a coding sequence for the 3R tau protein isoform and a coding sequence for a second reporter protein, wherein the cell expresses the 4R tau protein isoform, the 3R tau protein isoform, the first reporter protein and the second reporter protein.

5. The cell according to claim 4, wherein the coding sequence of the 4R tau protein isoform and the coding sequence of the first reporter protein are separated by the coding sequence of the first 2A peptide, and the coding sequence of the 3R tau protein isoform and the coding sequence of the second reporter protein are separated by the coding sequence of the second 2A peptide.

6. The cell of claim 5, wherein the first peptide 2A is a first peptide P2A, and the second peptide 2A is a second peptide P2A.

7. The cell of any one of claims 3 to 6, wherein the first nucleic acid and the second nucleic acid are integrated into the genome of the cell.

8. A cell according to any one of claims 3-7, comprising a viral vector comprising a first nucleic acid and a second nucleic acid.

9. The cell of claim 8, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

10. A cell according to any one of claims 3-7, comprising a first viral vector comprising a first nucleic acid, and a second viral vector comprising a second nucleic acid.

11. The cell of claim 10, wherein the first viral vector and the second viral vector are lentiviral vectors or adeno-associated virus (AAV) vectors.

12. The cell of any one of the preceding claims, wherein the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein.

13. The cell of any one of the preceding claims, wherein the first reporter protein is eYFP and the second reporter protein is mCherry.

14. A cell according to any one of the preceding claims, wherein the 4R tau isoform and the 3R tau isoform are human.

15. A cell according to any one of the preceding claims, wherein the 4R tau isoform is the 2N4R tau isoform.

16. A cell according to any one of the preceding claims, wherein the 3R tau isoform is the 2N3R tau isoform.

17. The cell of any one of claims 1 to 14, wherein the 4R tau protein isoform is 2N4R tau protein, and wherein the 3R tau protein isoform is 2N3R tau protein.

18. The cell according to any of the preceding claims, wherein the 4R tau protein isoform comprises the sequence presented in SEQ ID NO: 13, and the 3R tau protein isoform comprises the sequence presented in SEQ ID NO:

14.

19. The cell of any one of claims 1-14, wherein the 4R tau protein isoform is 1N4R tau protein isoform.

20. The cell of any one of claims 1-14 and 19, wherein the 3R tau protein isoform is the 1N3R tau protein isoform.

21. The cell of any one of claims 1-14, wherein the 4R tau protein isoform is 1N4R tau protein, and wherein the 3R tau protein isoform is 1N3R tau protein.

22. The cell according to any one of claims 1-14 and 19-21, wherein the 4R tau protein isoform comprises the sequence presented in SEQ ID NO: 23, 27, 31 or 47, and the 3R tau protein isoform comprises the sequence presented in SEQ ID NO: 24, 28, 32 or 49.

23. A cell according to any preceding claim that is a mammalian cell.

24. A cell according to any preceding paragraph that is a human cell.

25. A cell according to any preceding claim that is an immortalized cell.

26. The cell according to any preceding paragraph, which is a HEK293 cell.

27. A population of cells containing a plurality of cells of any preceding item.

28. A non-human animal comprising a four-repeat (4R) tau isoform linked to a first reporter protein and a three-repeat (3R) tau isoform linked to a second reporter protein that is different from the first reporter protein.

29. The non-human animal of claim 28, comprising a first fusion protein comprising a 4R tau isoform fused to a first reporter protein, and a second fusion protein comprising a 3R tau isoform fused to a second reporter protein.

30. The non-human animal of claim 29, comprising a first nucleic acid encoding a first fusion protein and a second nucleic acid encoding a second fusion protein, wherein the non-human animal expresses the first fusion protein and the second fusion protein.

31. The non-human animal of claim 28, comprising a first nucleic acid comprising a coding sequence for a 4R tau isoform and a coding sequence for a first reporter protein, and a second nucleic acid comprising a coding sequence for a 3R tau isoform and a coding sequence for a second reporter protein, wherein the non-human animal expresses a 4R tau isoform, a 3R tau isoform, a first reporter protein, and a second reporter protein.

32. The non-human animal of claim 31, wherein the coding sequence of the 4R tau protein isoform and the coding sequence of the first reporter protein are separated by the coding sequence of the first 2A peptide, and the coding sequence of the 3R tau protein isoform and the coding sequence of the second reporter protein are separated by the coding sequence of the second 2A peptide.

33. The non-human animal of claim 32, wherein the first peptide 2A is a first peptide P2A and the second peptide 2A is a second peptide P2A.

34. The non-human animal of any one of paragraphs 30-33, wherein the first nucleic acid and the second nucleic acid are integrated into the genome of the non-human animal.

35. The non-human animal of any one of claims 30 to 34, comprising a viral vector comprising a first nucleic acid and a second nucleic acid.

36. The non-human animal of claim 35, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

37. The non-human animal of any one of claims 30 to 34, comprising a first viral vector comprising a first nucleic acid and a second viral vector comprising a second nucleic acid.

38. The non-human animal of claim 37, wherein the first viral vector and the second viral vector are lentiviral vectors or adeno-associated virus (AAV) vectors.

39. The non-human animal of any one of claims 28-38, wherein the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein.

40. The non-human animal of any one of claims 28 to 39, wherein the first reporter protein is eYFP and the second reporter protein is mCherry.

41. A non-human animal according to any one of claims 28 to 40, wherein the 4R tau protein isoform and the 3R tau protein isoform are human, and optionally each 4R tau protein isoform and 3R tau protein isoform comprises the R5L mutation, the L237V mutation, or the G243V mutation, or optionally each 4R tau protein isoform and 3R tau protein isoform comprises the N279K mutation, the L284R mutation, or the S285R mutation.

42. The non-human animal of any one of claims 28 to 41, wherein the 4R tau isoform is the 2N4R tau isoform.

43. The non-human animal of any one of claims 28 to 42, wherein the 3R tau isoform is the 2N3R tau isoform.

44. The non-human animal of any one of claims 28 to 41, wherein the 4R tau isoform is the 2N4R tau isoform, and wherein the 3R tau isoform is the 2N3R tau isoform.

45. The non-human animal of any one of claims 28-44, wherein the 4R tau protein isoform comprises the sequence presented in SEQ ID NO: 13, and the 3R tau protein isoform comprises the sequence presented in SEQ ID NO:

14.

46. ​​The non-human animal of any one of claims 28 to 41, wherein the 4R tau isoform is the 1N4R tau isoform.

47. The non-human animal of any one of claims 28 to 41 and 46, wherein the 3R tau isoform is the 1N3R tau isoform.

48. The non-human animal of any one of claims 28 to 41, wherein the 4R tau isoform is the 1N4R tau isoform, and wherein the 3R tau isoform is the 1N3R tau isoform.

49. The non-human animal of any one of claims 28-41 and 46-48, wherein the 4R tau protein isoform comprises the sequence presented in SEQ ID NO: 23, 27, 31 or 47, and the 3R tau protein isoform comprises the sequence presented in SEQ ID NO: 24, 28, 32 or 49.

50. A non-human animal according to any of paragraphs 28 to 49 that is a mammal.

51. A non-human animal according to any of paragraphs 28 to 50 that is a rodent.

52. The non-human animal according to any of paragraphs 28 to 51, which is a mouse.

53. The non-human animal according to any of paragraphs 28 to 51, which is a rat.

54. The non-human animal of any one of claims 28 to 53, wherein the 4R tau isoform, the first reporter protein, the 3R tau isoform, and the second reporter protein are expressed in neurons of the central nervous system of the non-human animal.

55. A non-human animal according to any one of paragraphs 28 to 54, comprising filamentous inclusion bodies of tau protein.

56. A method for assessing the activity of a reagent targeting tau protein, comprising: (a) introducing a tau protein-targeting reagent into a cell according to any one of claims 1 to 26; and (b) assessment of the activity of the tau-targeting reagent in the cell.

57. The method of claim 56, wherein the activity of the tau-targeting reagent is assessed in comparison to a control cell into which the tau-targeting reagent is not administered, or is assessed in comparison to a cell before the administration of the tau-targeting reagent.

58. The method of claim 56 or 57, wherein the assessing comprises measuring one or more of the expression of 4R tau protein messenger RNA, the expression of the messenger RNA of the first reporter protein, and the expression of the messenger RNA of the second reporter protein.

59. The method according to any one of paragraphs 56-58, in which the assessment comprises measuring the expression of messenger RNA of the 4R tau protein isoform and the expression of messenger RNA of a second reporter protein, wherein a greater relative decrease in the expression of the messenger RNA of the 4R tau protein isoform compared to the expression of the messenger RNA of the second reporter protein after introducing the tau protein targeting reagent into the cell indicates that the tau protein targeting reagent is a 4R-preferential tau protein targeting reagent, wherein optionally a decrease in the expression of the messenger RNA of the 4R tau protein isoform by at least 70% and a decrease in the expression of the messenger RNA of the second reporter protein by no more than 30% after introducing the tau protein targeting reagent into the cell indicates that the tau protein targeting reagent is a 4R-preferential tau protein targeting reagent.

60. The method according to any one of paragraphs 56-59, wherein the evaluation comprises measuring the expression of messenger RNA of the first reporter protein and the expression of messenger RNA of the second reporter protein, wherein a greater relative decrease in the expression of the messenger RNA of the first reporter protein compared to the expression of the messenger RNA of the second reporter protein after the introduction of the tau protein-targeting reagent into the cell indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent, wherein optionally a decrease in the expression of the messenger RNA of the first reporter protein by at least 70% and a decrease in the expression of the messenger RNA of the second reporter protein by no more than 30% after the introduction of the tau protein-targeting reagent into the cell indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent.

61. The method of any one of claims 56-60, wherein the assessing comprises measuring one or more of the expression of the first reporter protein and the expression of the second reporter protein.

62. The method according to any one of paragraphs 56-61, wherein the assessing comprises measuring the expression of the first reporter protein and the expression of the second reporter protein, wherein a greater relative decrease in the expression of the first reporter protein compared to the expression of the second reporter protein after introducing the tau protein-targeting reagent into the cell indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent, wherein optionally a decrease in the expression of the first reporter protein by at least 70% and a decrease in the expression of the second reporter protein by no more than 30% after introducing the tau protein-targeting reagent into the cell indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent.

63. The method according to any one of claims 56-62, wherein the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the assessment in step (b) comprises immunofluorescence staining or flow cytometry.

64. The method according to any one of claims 56-63, wherein the assessment in step (b) comprises assessing tau protein hyperphosphorylation or tau protein aggregation.

65. The method of any one of claims 56-64, wherein the tau protein targeting reagent is an RNAi agent or an antisense oligonucleotide.

66. The method according to any one of claims 56-64, wherein the tau protein-targeting reagent is an intrabody.

67. The method according to any one of claims 56-64, wherein the tau protein targeting reagent is a nuclease agent.

68. The method of claim 67, wherein the nuclease agent comprises a Cas protein and a guide RNA designed to target a target sequence of the guide RNA in a sequence encoding a tau protein.

69. A method for assessing the activity of a tau-targeting reagent in vivo, comprising: (a) administering a tau-targeting reagent to a non-human animal according to any one of paragraphs 28 to 55; and (b) evaluation of the activity of a tau-targeting reagent in a non-human animal.

70. The method of claim 69, wherein the activity of the tau-targeting reagent is assessed in comparison to a non-human control animal that is not administered the tau-targeting reagent, or is assessed in comparison to a state prior to administration of the tau-targeting reagent.

71. The method according to 69 or 70, wherein the assay comprises measuring one or more of the expression of 4R tau protein messenger RNA, the expression of the messenger RNA of the first reporter protein, and the expression of the messenger RNA of the second reporter protein.

72. The method according to any one of paragraphs 69-71, in which the assessment comprises measuring the expression of messenger RNA of the 4R tau protein isoform and the expression of messenger RNA of a second reporter protein, wherein a greater relative reduction in the expression of the 4R tau isoform messenger RNA compared to the expression of the second reporter protein messenger RNA after administration of the tau protein targeting reagent to a non-human animal indicates that the tau protein targeting reagent is a 4R-preferential tau protein targeting reagent, wherein optionally a reduction in the expression of the 4R tau isoform messenger RNA by at least 70% and a reduction in the expression of the second reporter protein messenger RNA by no more than 30% after administration of the tau protein targeting reagent to a non-human animal indicates that the tau protein targeting reagent is a 4R-preferential tau protein targeting reagent.

73. The method according to any one of paragraphs 69-72, wherein the evaluation comprises measuring the expression of messenger RNA of the first reporter protein and the expression of messenger RNA of the second reporter protein, wherein a greater relative decrease in the expression of the messenger RNA of the first reporter protein compared to the expression of the messenger RNA of the second reporter protein after administration of the tau protein-targeting reagent to a non-human animal indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent, wherein optionally a decrease in the expression of the messenger RNA of the first reporter protein by at least 70% and a decrease in the expression of the messenger RNA of the second reporter protein by no more than 30% after administration of the tau protein-targeting reagent to a non-human animal indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent.

74. The method of any one of claims 69-73, wherein the assessing comprises measuring one or more of the expression of the first reporter protein and the expression of the second reporter protein.

75. The method according to any one of paragraphs 69-74, wherein the assessing comprises measuring the expression of the first reporter protein and the expression of the second reporter protein, wherein a greater relative decrease in the expression of the first reporter protein compared to the expression of the second reporter protein after administration of the tau protein-targeting reagent to a non-human animal indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent, wherein optionally a decrease in the expression of the first reporter protein by at least 70% and a decrease in the expression of the second reporter protein by no more than 30% after administration of the tau protein-targeting reagent to a non-human animal indicates that the tau protein-targeting reagent is a 4R-preferential tau protein-targeting reagent.

76. The method according to any one of claims 69-75, wherein the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein, and the assessment in step (b) comprises immunofluorescence staining or flow cytometry.

77. The method according to any one of claims 69-76, wherein the assessment in step (b) comprises assessing tau protein hyperphosphorylation or tau protein aggregation.

78. The method according to any one of claims 69-77, wherein the reagent targeting the tau protein is an RNAi agent or an antisense oligonucleotide.

79. The method according to any one of claims 69-77, wherein the tau protein-targeting reagent is an intrabody.

80. The method according to any one of claims 69 to 77, wherein the tau protein targeting reagent is a nuclease agent.

81. The method of claim 80, wherein the nuclease agent comprises a Cas protein and a guide RNA designed to target a target sequence of the guide RNA in a sequence encoding a tau protein.

82. The method according to any one of claims 69-81, wherein the assessment is carried out in neurons of the central nervous system of a non-human animal.

83. A composition comprising: (a) a four-repeat (4R) tau protein isoform associated with a first reporter protein and a three-repeat (3R) tau protein isoform associated with a second reporter protein that is different from the first reporter protein; or (b) a first nucleic acid encoding the 4R tau protein isoform associated with a first reporter protein and a second nucleic acid encoding the 3R tau protein isoform associated with a second reporter protein.

84. The composition of claim 83, which comprises a first fusion protein comprising a 4R tau protein isoform fused to a first reporter protein and a second fusion protein comprising a 3R tau protein isoform fused to a second reporter protein, or wherein the first nucleic acid encodes the first fusion protein and the second nucleic acid encodes the second fusion protein.

85. The composition of claim 83, wherein the first nucleic acid comprises a coding sequence for the 4R tau protein isoform and a coding sequence for a first reporter protein, separated by a coding sequence for the first 2A peptide, and wherein the second nucleic acid comprises a coding sequence for the 3R tau protein isoform and a coding sequence for a second reporter protein, separated by a coding sequence for the second 2A peptide.

86. The composition of claim 85, wherein the first peptide 2A is a first peptide P2A, and the second peptide 2A is a second peptide P2A.

87. The composition of any one of claims 83-86, wherein the first nucleic acid and the second nucleic acid are in a viral vector.

88. The composition of claim 87, wherein the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

89. The composition of any one of claims 83-86, wherein the first nucleic acid is in a first viral vector and the second nucleic acid is in a second viral vector.

90. The composition of claim 89, wherein the first viral vector and the second viral vector are lentiviral vectors or adeno-associated virus (AAV) vectors.

91. The composition of any one of claims 83-90, wherein the first reporter protein is a first fluorescent reporter protein and the second reporter protein is a second fluorescent reporter protein.

92. The composition of any one of claims 83-91, wherein the first reporter protein is eYFP and the second reporter protein is mCherry.

93. The composition of any one of claims 83-92, wherein the 4R tau protein isoform and the 3R tau protein isoform are human.

94. The composition of any one of claims 83 to 93, wherein the 4R tau protein isoform is 2N4R tau protein isoform.

95. The composition of any one of claims 83-94, wherein the 3R tau protein isoform is 2N3R tau protein isoform.

96. The composition of any one of claims 83-93, wherein the 4R tau protein isoform is 2N4R tau protein, and wherein the 3R tau protein isoform is 2N3R tau protein.

97. The composition according to any one of claims 83-96, wherein the 4R tau protein isoform comprises the sequence presented in SEQ ID NO: 13, and the 3R tau protein isoform comprises the sequence presented in SEQ ID NO:

14.

98. The composition of any one of claims 83 to 93, wherein the tau protein isoform 4R is tau protein isoform 1N4R.

99. The composition according to any one of claims 83-93 and 98, wherein the 3R tau protein isoform is the 1N3R tau protein isoform.

100. The composition of any one of claims 83 to 93, wherein the 4R tau protein isoform is 1N4R tau protein, and wherein the 3R tau protein isoform is 1N3R tau protein.

101. The composition according to any one of claims 83-93 and 98-100, wherein the tau protein isoform 4R comprises the sequence presented in SEQ ID NO: 23, 27, 31 or 47, and the tau protein isoform 3R comprises the sequence presented in SEQ ID NO: 24, 28, 32 or 49.

102. A cell comprising a composition according to any one of claims 83-101.

103. A non-human animal comprising a composition according to any one of paragraphs 83-101.

104. A method for producing a cell according to any one of claims 1-26 and 102, comprising introducing into the cell an isoform of tau protein with four repeats (4R) linked to a first reporter protein and an isoform of tau protein with three repeats (3R) linked to a second reporter protein, or introducing into the cell a first nucleic acid encoding an isoform of tau protein 4R linked to a first reporter protein and a second nucleic acid encoding an isoform of tau protein 3R linked to a second reporter protein.

105. A method for producing a non-human animal according to any one of claims 28 to 55 and 103, comprising administering to the non-human animal a 4R tau protein isoform linked to a first reporter protein and a 3R tau protein isoform linked to a second reporter protein, or administering to the non-human animal a first nucleic acid encoding a 4R tau protein isoform linked to a first reporter protein and a second nucleic acid encoding a 3R tau protein isoform linked to a second reporter protein.