Methods and compositions for administering otoferlin dual vector systems

Abstract

The disclosure features compositions and methods for the treatment of sensorineural hearing loss and auditory neuropathy, particularly forms of the disease that are associated with a mutation in otoferlin (OTOF), by way of OTOF gene therapy. The disclosure provides a variety of compositions that include a first nucleic acid vector that contains a polynucleotide encoding an N-terminal portion of an OTOF protein (e.g., an OTOF isoform 5 protein) and a second nucleic acid vector that contains a polynucleotide encoding a C-terminal portion of an OTOF protein (e.g., an OTOF isoform 5 protein). These vectors can be used to increase the expression of OTOF in a subject, such as a human subject suffering from sensorineural hearing loss.

Description

This disclosure features compositions and methods for treating sensorineural hearing loss and auditory neuropathy, particularly disease forms associated with mutations in OTOF, via otomorphin (OTOF) gene therapy. The disclosure provides various compositions comprising a first nucleic acid vector containing a polynucleotide encoding the N-terminal portion of an OTOF protein (e.g., OTOF isoform 5 protein) and a second nucleic acid vector containing a polynucleotide encoding the C-terminal portion of an OTOF protein (e.g., OTOF isoform 5 protein). These vectors can be used to increase OTOF expression in subjects (e.g., human subjects) suffering from sensorineural hearing loss. Abstract

Claims

Claims1 . A method of treating a human subject having biallelic otoferlin (OTOF) mutations, the method comprising administering to an inner ear of the subject an amount of 1 x 1013vg / mL to 1 x 1014vg / mL of an OTOF dual vector system in a volume of 200-250 pL by intracochlear injection, wherein the OTOF dual vector system comprises: a first adeno-associated virus (AAV) vector comprising a first inverted terminal repeat (ITR) sequence; a Myosin 15 (Myo15) promoter operably linked to a first coding polynucleotide that encodes an N-terminal portion of an OTOF isoform 5 protein; a splice donor sequence positioned 3' of the first coding polynucleotide; a recombinogenic region positioned 3' of the splice donor sequence; and a second ITR sequence; and a second AAV vector comprising a first ITR sequence; a second recombinogenic region; a splice acceptor sequence positioned 3' of the second recombinogenic region; a second coding polynucleotide that encodes a C-terminal portion of the OTOF isoform 5 protein positioned 3' of the splice acceptor sequence; a poly(A) sequence positioned 3' of the second coding polynucleotide; and a second ITR sequence; wherein the first coding polynucleotide and the second coding polynucleotide that encode the OTOF isoform 5 protein do not overlap, wherein neither the first nor second AAV vector encodes the full- length OTOF isoform 5 protein, and wherein the first AAV vector and the second AAV vector are administered at a ratio of about 3:1 to about 1 :3.

2. A method of improving hearing in a human subject, the method comprising selecting a subject having biallelic OTOF mutations and administering to an inner ear of the subject an amount of 1 x 1013vg / mL to 1 x 1014vg / mL of an OTOF dual vector system in a volume of 200-250 pL by intracochlear injection, wherein the OTOF dual vector system comprises: a first AAV vector comprising a first ITR sequence; a Myo15 promoter operably linked to a first coding polynucleotide that encodes an N-terminal portion of an OTOF isoform 5 protein; a splice donor sequence positioned 3' of the first coding polynucleotide; a recombinogenic region positioned 3' of the splice donor sequence; and a second ITR sequence; and a second AAV vector comprising a first ITR sequence; a second recombinogenic region; a splice acceptor sequence positioned 3' of the second recombinogenic region; a second coding polynucleotide that encodes a C-terminal portion of the OTOF isoform 5 protein positioned 3' of the splice acceptor sequence; a poly(A) sequence positioned 3' of the second coding polynucleotide; and a second ITR sequence; wherein the first coding polynucleotide and the second coding polynucleotide that encode the OTOF isoform 5 protein do not overlap, wherein neither the first nor second AAV vector encodes the full- length OTOF isoform 5 protein, and wherein the first AAV vector and the second AAV vector are administered at a ratio of about 3:1 to 1 :3.

3. The method of claim 1 or 2, wherein the first AAV vector and the second AAV vector comprise an AAV1 capsid.

4. The method of any one of claims 1 -3, wherein the Myo15 promoter comprises a first region having at least 85% sequence identity to SEQ ID NO: 7 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 9 and / or SEQ ID NO: 10 operably linked to a second region having at least 85% sequence identity to SEQ ID NO: 8 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 14 and / or SEQ ID NO: 15.

5. The method of any one of claims 1 -4, wherein the Myo15 promoter comprises the sequence of SEQ ID NO: 21 .

6. The method of claim 5, wherein the Myo15 promoter consists of the sequence of SEQ ID NO: 21 .

7. The method of any one of claims 1 -6, wherein the first recombinogenic region and the second recombinogenic region are each an AP gene fragment.

8. The method of claim 7, wherein the AP gene fragment comprises the sequence of any one of SEQ ID NOs: 48-53.

9. The method of claim 8, wherein the AP gene fragment consists of the sequence of any one of SEQ ID NOs: 48-53.

10. The method of claim 8, wherein the AP gene fragment comprises the sequence of SEQ ID NO: 51 .1 1 . The method of claim 10, wherein the AP gene fragment consists of the sequence of SEQ ID NO: 51 .

12. The method of any one of claims 1 -1 1 , wherein the OTOF isoform 5 protein comprises the sequence of SEQ ID NO: 1 or a variant thereof having one or more conservative amino acid substitutions.

13. The method of claim 12, wherein no more than 10% of the amino acids in the OTOF isoform 5 protein variant are conservative amino acid substitutions.

14. The method of claim 12, wherein the OTOF isoform 5 protein consists of the sequence of SEQ ID NO: 1 .

15. The method of any one of claims 1 -12 and 14, wherein the OTOF isoform 5 protein is encoded by the sequence of SEQ ID NO: 2.

16. The method of any one of claims 1 -12 and 14, wherein the OTOF isoform 5 protein is encoded by the sequence of SEQ ID NO: 3.

17. The method of any one of claims 1 -16, wherein the first coding polynucleotide encodes amino acids 1 -802 of SEQ ID NO: 1 and the second coding polynucleotide encodes amino acids 803-1997 of SEQ ID NO: 1.

18. The method of any one of claims 1 -17, wherein the N-terminal portion of the OTOF isoform 5 protein comprises the sequence of SEQ ID NO: 58 or a variant thereof having one or more conservative amino acid substitutions.

19. The method of claim 18, wherein no more than 10% of the amino acids in the N-terminal portion of the OTOF isoform 5 protein variant are conservative amino acid substitutions.

20. The method of claim 18, wherein the N-terminal portion of the OTOF isoform 5 protein consists of the sequence of SEQ ID NO: 58.21 . The method of any one of claims 1 -18 and 20, wherein the N-terminal portion of the OTOF isoform 5 protein is encoded by the sequence of SEQ ID NO: 56.

22. The method of any one of claims 1 -21 , wherein the C-terminal portion of the OTOF isoform 5 protein comprises the sequence of SEQ ID NO: 59 or a variant thereof having one or more conservative amino acid substitutions.

23. The method of claim 22, wherein no more than 10% of the amino acids in the C-terminal portion of the OTOF isoform 5 protein variant are conservative amino acid substitutions.

24. The method of claim 22, wherein the C-terminal portion of the OTOF isoform 5 protein consists of the sequence of SEQ ID NO: 59.

25. The method of any one of claims 1 -22 and 24, wherein the C-terminal portion of the OTOF isoform 5 protein is encoded by the sequence of SEQ ID NO: 57.

26. The method of any one of claims 1 -25, wherein the ITRs in the first vector and second vector have at least 80% sequence identity to AAV2 ITRs.

27. The method of any one of claims 1 -26, wherein the poly(A) sequence is a bovine growth hormone (bGH) poly(A) sequence.

28. The method of any one of claims 1 -27, wherein the splice donor sequence in the first vector comprises the sequence of SEQ ID NO: 54.

29. The method of claim 28, wherein the splice donor sequence in the first vector consists of the sequence of SEQ ID NO: 54.

30. The method of any one of claims 1 -29, wherein the splice acceptor sequence in the second vector comprises the sequence of SEQ ID NO: 55.31 . The method of claim 30, wherein the splice acceptor sequence in the second vector consists of the sequence of SEQ ID NO: 55.

32. The method of any one of claims 1 -31 , wherein the first AAV vector comprises a Kozak sequence 3’ of the Myo15 promoter and 5’ of the first coding polynucleotide that encodes the N-terminal portion of the OTOF isoform 5 protein.

33. The method of claim 1 , wherein the first AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 235 to 4004 of SEQ ID NO: 66.

34. The method of claim 1 or 33, wherein the second AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 229 to 4438 of SEQ ID NO: 67.

35. The method of claim 1 , wherein the first AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 2272 to 6041 of SEQ ID NO: 60.

36. The method of claim 1 , wherein the first AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 182 to 3949 of SEQ ID NO: 62.

37. The method of claim 1 or 35, wherein the second AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 2267 to 6476 of SEQ ID NO: 61 .

38. The method of claim 1 or 36, wherein the second AAV vector comprises a polynucleotide sequence comprising the sequence of nucleotides 187 to 4396 of SEQ ID NO: 63.

39. The method of any one of claims 1 -38, wherein the subject is less than 18 years of age.

40. The method of claim 39, wherein the subject is at least 7 years of age.41 . The method of claim 39, wherein the subject is older than 24 months of age and younger than 7 years of age.

42. The method of claim 39, wherein the subject is 24 months of age or younger.

43. The method of any one of claims 1 -42, wherein the subject has profound sensorineural hearing loss.

44. The method of any one of claims 1 -43, wherein the subject has behavioral open-set word detection scores of < 30% in the ear(s) to be treated.

45. The method of any one of claims 1 -44, wherein the subject has congenital auditory neuropathy.

46. The method of any one of claims 1 -45, wherein the subject has present outer hair cell function.

47. The method of any one of claims 1 -46, wherein the subject has detectable otoacoustic emissions.

48. The method of any one of claims 1 -47, wherein a cochlear microphonic is present in the ear(s) to be treated.

49. The method of any one of claims 1 -48, wherein the administering to the inner ear comprises intracochlear injection via insertion of a catheter through the round window membrane into the inner ear perilymph.

50. The method of any one of claims 1 -49, wherein the administering further comprises creating a fenestration in the lateral semicircular canal.51 . The method of any one of claims 1 -50, wherein the injection is performed using a syringe and syringe pump.

52. The method of any one of claims 1 -51 , wherein the administering is at a rate of 0.9 mL / hr.

53. The method of any one of claims 1 -52, wherein the administering occurs once per ear.

54. The method of any one of claims 1 -53, wherein the first vector and the second vector are administered at a ratio of about 1 :1 .

55. The method of any one of claims 1 -54, wherein the dual vector system is formulated as an aqueous suspension.

56. The method of claim 55, wherein the first vector and second vector are matched in titer and mixed at an approximately equal ratio.

57. The method of claim 55 or 56, wherein the suspension comprises 10 mM sodium phosphate or disodium phosphate, 180 mM sodium chloride, 5% (w / v) sucrose, and 0.001 % (w / v) poloxamer 188 at a pH of 7.4.

58. The method of claim 57, wherein the suspension comprises 10 mM sodium phosphate.

59. The method of claim 57, wherein the suspension comprises 10 mM disodium phosphate.

60. The method of any one of claims 1 -59, wherein the method further comprises administering a corticosteroid to the subject for the first four weeks after the administering of the OTOF dual vector system.61 . The method of claim 60, wherein the method comprises administering 1 mg / kg of the corticosteroid daily during the first two weeks after the administering of the OTOF dual vector system, administering 0.5 mg / kg daily during the third week after the administering of the OTOF dual vector system, and administering 0.25 mg / kg daily during the fourth week after the administering of the OTOF dual vector system.

62. The method of any one of claims 1 -61 , wherein the OTOF dual vector system is administered in an amount of 1 .0 x 1013vg / mL to 5.0 x 1013vg / mL in a volume of 200-250 pL.

63. The method of claim 62, wherein the OTOF dual vector system is administered in an amount of 2.0 x 1013vg / mL to 5.0 x 1013vg / mL in a volume of 200-250 pL.

64. The method of claim 63, wherein the OTOF dual vector system is administered in an amount of 3.0 x 1013vg / mL to 5.0 x 1013vg / mL in a volume of 200-250 pL.

65. The method of claim 64, wherein the OTOF dual vector system is administered in an amount of 4.0 x 1013vg / mL to 5.0 x 1013vg / mL in a volume of 200-250 pL.

66. The method of claim 63, wherein the OTOF dual vector system is administered in an amount of 3.0 x 1013vg / mL in a volume of 240 pL.

67. The method of any one of claims 1 -61 , wherein the OTOF dual vector system is administered in an amount of 5.0 x 1013vg / mL to 1 .0 x 1014vg / mL in a volume of 200-250 pL.

68. The method of claim 67, wherein the OTOF dual vector system is administered in an amount of 5.0 x 1013vg / mL to 9.0 x 1013vg / mL in a volume of 200-250 pL.

69. The method of claim 68, wherein the OTOF dual vector system is administered in an amount of 5.0 x 1013vg / mL to 8.0 x 1013vg / mL in a volume of 200-250 pL.

70. The method of claim 69, wherein the OTOF dual vector system is administered in an amount of 5.0 x 1013vg / mL to 7.0 x 1013vg / mL in a volume of 200-250 pL.71 . The method of claim 70, wherein the OTOF dual vector system is administered in an amount of 5.0 x 1013vg / mL to 6.0 x 1013vg / mL in a volume of 200-250 pL.

72. The method of claim 69, wherein the OTOF dual vector system is administered in an amount of 7.3 x 1013vg / mL in a volume of 240 pL.

73. The method of any one of claims 1 -72, wherein the method improves one or more parameters selected from the subject's auditory brainstem response (ABR), behavioral audiometry, and score in one or more hearing questionnaires or behavioral tasks.

74. The method of claim 73, wherein the hearing questionnaires and behavioral tasks include one or more of the Auditory Skills Checklist, Open & Closed set task, Early Speech Perception test, Pediatric Speech Intelligibility test, Lexical Neighborhood Test Multisyllabic Lexical Neighborhood Test, Consonant- Nucleus-Consonant test, Bamford-Kowal-Bench sentence test, LittlEARS® Auditory Questionnaire, MacArthur-Bates Communicative Development Inventories Words and Gestures, Quality of Life-Cochlear Implant, Pediatric Quality of Life Inventory, Hearing Environments and Reflection on Quality of Life (HEAR-QL)-26, HEAR-QL-28, The Health Utilities Index 3, Vanderbilt Fatigue Scales, and AzBio test.

75. The method of claim 74, wherein the method improves the subject’s score in the LittlEARS® Auditory Questionnaire.

76. The method of claim 73, wherein the method improves hearing thresholds by at least 55 decibels hearing level with air conduction as assessed by behavioral pure tone audiometry.

77. The method of claim 73, wherein the method results in a positive ABR wave V amplitude response.

78. An aqueous suspension comprising an OTOF dual vector system, 10 mM sodium phosphate or disodium phosphate, 180 mM sodium chloride, 5% (w / v) sucrose, and 0.001% (w / v) poloxamer 188 at a pH of 7.4, wherein the OTOF dual vector system comprises: a first AAV vector comprising a first ITR sequence; a Myosin 15 (Myo15) promoter operably linked to a first coding polynucleotide that encodes an N-terminal portion of an OTOF isoform 5 protein; a splice donor sequence positioned 3' of the first coding polynucleotide; a recombinogenic region positioned 3' of the splice donor sequence; and a second ITR sequence; and a second AAV vector comprising a first ITR sequence; a second recombinogenic region; a splice acceptor sequence positioned 3' of the second recombinogenic region; a second coding polynucleotide that encodes a C-terminal portion of the OTOF isoform 5 protein positioned 3' of the splice acceptor sequence; a poly(A) sequence positioned 3' of the second coding polynucleotide; and a second ITR sequence; wherein the first coding polynucleotide and the second coding polynucleotide that encode the OTOF isoform 5 protein do not overlap, and wherein neither the first nor second AAV vector encodes the full-length OTOF isoform 5 protein.

79. The suspension of claim 78, wherein the suspension comprises 10 mM sodium phosphate.

80. The suspension of claim 78, wherein the suspension comprises 10 mM disodium phosphate.81 . The suspension of any one of claims 78-80, wherein the ratio of the first vector to the second vector is about 3:1 to about 1 :3.

82. The suspension of any one of claims 78-81 , wherein the first vector and second vector are matched in titer and mixed at an approximately equal ratio.

83. The suspension of claim 82, wherein the ratio of the first vector to the second vector is about 1 :1 .

84. The suspension of any one of claims 78-83, wherein the suspension has a titer of 1 x 1013vg / mL to 1 x 1014vg / mL.

85. The suspension of claim 84, wherein the suspension has a titer of 1 x 1013vg / mL to 5 x 1013vg / mL.

86. The suspension of claim 85, wherein the suspension has a titer of 3 x 1013vg / mL.

87. The suspension of claim 84, wherein the suspension has a titer of 5 x 1013vg / mL to 1 x 1014vg / mL.

88. The suspension of claim 87, wherein the suspension has a titer of 7.3 x 1013vg / mL.

89. The suspension of any one of claims 78-88, wherein the suspension is formulated for intracochlear injection.

90. A kit comprising the suspension of any one of claims 78-89.91 . The kit of claim 90, further comprising one or more of a syringe, syringe pump, and catheter.

92. A syringe comprising the suspension of any one of claims 78-89.

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