MYOSIN 15 PROMOTERS AND USES THEREOF

Abstract

The disclosure provides polynucleotides containing regions of the Myosin 15 (Myo15) promoter, as well as vectors containing the same, that can be used to promote expression of a transgene specifically in hair cells. The polynucleotides described herein may be operably linked to a transgene, such as a transgene encoding a therapeutic protein, so as to promote hair cell-specific expression of the transgene. The polynucleotides described herein may be operably linked to a therapeutic transgene and used for the treatment of subjects having or at risk of developing hearing loss or vestibular dysfunction.

Claims

1. A polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 1 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4 operably linked to a second region having at least 85% sequence identity to SEQ ID NO: 2 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 9, optionally comprising a linker comprising one to one hundred nucleotides between the first region and the second region.

2. The polynucleotide of claim 1, wherein the first region comprises or consists of the sequence of SEQ ID NO: 1.

3. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3.

4. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4.

5. The polynucleotide of claim 1, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4.

6. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5.

7. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6.

8. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7.

9. The polynucleotide of claim 5, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27.

10. The polynucleotide of any one of claims 1-9, wherein the second region comprises or consists of the sequence of SEQ ID NO: 2.

11. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8.

12. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9.

13. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9.

14. The polynucleotide of claim 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10.

15. The polynucleotide of claim 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11.

16. The polynucleotide of claim 13, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12.

17. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28.

18. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29.

19. The polynucleotide of any one of claims 1-9, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29.

20. The polynucleotide of claim 19, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32.

21. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 13.

22. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 15.

23. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 16.

24. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 36.

25. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 37.

26. A polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 2 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 9 operably linked to a second region having at least 85% sequence identity to SEQ ID NO: 1 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4, optionally comprising a linker comprising one to one hundred nucleotides between the first region and the second region.

27. The polynucleotide of claim 26, wherein the first region comprises or consists of the sequence of SEQ ID NO: 2.

28. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8.

29. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9.

30. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9.

31. The polynucleotide of claim 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10.

32. The polynucleotide of claim 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11.

33. The polynucleotide of claim 30, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12.

34. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28.

35. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29.

36. The polynucleotide of claim 26, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29.

37. The polynucleotide of claim 36, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32.

38. The polynucleotide of any one of claims 26-37, wherein the second region comprises or consists of the sequence of SEQ ID NO: 1.

39. The polynucleotide of any one of claims 26-37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3.

40. The polynucleotide of any one of claims 26-37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4.

41. The polynucleotide of any one of claims 26-37, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4.

42. The polynucleotide of claim 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5.

43. The polynucleotide of claim 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6.

44. The polynucleotide of claim 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7.

45. The polynucleotide of claim 41, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27.

46. The polynucleotide of claim 26, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 14.

47. A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 1 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4.

48. The polynucleotide of claim 47, wherein the first region comprises or consists of the sequence of SEQ ID NO: 1.

49. The polynucleotide of claim 47, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3.

50. The polynucleotide of claim 47, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 4.

51. The polynucleotide of claim 47, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4.

52. The polynucleotide of claim 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 5.

53. The polynucleotide of claim 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 6.

54. The polynucleotide of claim 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 7.

55. The polynucleotide of claim 51, wherein the functional portion of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 27.

56. A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 2 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 9.

57. The polynucleotide of claim 56, wherein the first region comprises or consists of the sequence of SEQ ID NO: 2.

58. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8.

59. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 9.

60. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 8 and the sequence of SEQ ID NO: 9.

61. The polynucleotide of claim 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 10.

62. The polynucleotide of claim 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11.

63. The polynucleotide of claim 60, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12.

64. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28.

65. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 29.

66. The polynucleotide of claim 56, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 28 and the sequence of SEQ ID NO: 29.

67. The polynucleotide of claim 66, wherein the functional portion of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 32.

68. A polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 17 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 19 operably linked to a second region having at least 85% sequence identity to SEQ ID NO: 18 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 20 and/or SEQ ID NO: 21, optionally comprising a linker comprising one to four hundred nucleotides between the first region and the second region.

69. The polynucleotide of claim 68, wherein the first region comprises or consists of the sequence of SEQ ID NO: 17.

70. The polynucleotide of claim 68, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19.

71. The polynucleotide of any one of claims 68-70, wherein the second region comprises or consists of the sequence of SEQ ID NO: 18.

72. The polynucleotide of any one of claims 68-70, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20.

73. The polynucleotide of any one of claims 68-70, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21.

74. The polynucleotide of any one of claims 68-70, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21.

75. The polynucleotide of claim 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22.

76. The polynucleotide of claim 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23.

77. The polynucleotide of claim 74, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24.

78. The polynucleotide of claim 68, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 25.

79. The polynucleotide of claim 68, wherein the polynucleotide comprises or consists of the sequence of SEQ ID NO: 26.

80. A polynucleotide comprising a first region having at least 85% sequence identity to SEQ ID NO: 18 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 20 and/or SEQ ID NO: 21 operably linked to a second region having at least 85% sequence identity to SEQ ID NO: 17 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 19, optionally comprising a linker comprising one to four hundred nucleotides between the first region and the second region.

81. The polynucleotide of claim 80, wherein the first region comprises or consists of the sequence of SEQ ID NO: 18.

82. The polynucleotide of claim 80, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20.

83. The polynucleotide of claim 80, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21.

84. The polynucleotide of claim 80, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21.

85. The polynucleotide of claim 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22.

86. The polynucleotide of claim 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23.

87. The polynucleotide of claim 84, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24.

88. The polynucleotide of any one of claims 80-87, wherein the second region comprises or consists of the sequence of SEQ ID NO: 17.

89. The polynucleotide of any one of claims 80-87, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19.

90. A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 17 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 19.

91. The polynucleotide of claim 90, wherein the region comprises or consists of the sequence of SEQ ID NO: 17.

92. The polynucleotide of claim 90, wherein the functional portion of SEQ ID NO: 17 comprises the sequence of SEQ ID NO: 19.

93. A polynucleotide comprising a region having at least 85% sequence identity to SEQ ID NO: 18 or a functional portion or derivative thereof comprising the sequence of SEQ ID NO: 20 and/or SEQ ID NO: 21.

94. The polynucleotide of claim 93, wherein the region comprises or consists of the sequence of SEQ ID NO: 18.

95. The polynucleotide of claim 93, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20.

96. The polynucleotide of claim 93, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 21.

97. The polynucleotide of claim 93, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 20 and the sequence of SEQ ID NO: 21.

98. The polynucleotide of claim 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 22.

99. The polynucleotide of claim 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 23.

100. The polynucleotide of claim 97, wherein the functional portion of SEQ ID NO: 18 comprises the sequence of SEQ ID NO: 24.

101. A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 28.

102. A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 32.

103. A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 33.

104. A polynucleotide having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 34.

105. The polynucleotide of any one of claims 1-104, wherein the polynucleotide induces transgene expression when operably linked to a transgene and introduced into a hair cell.

106. A nucleic acid vector comprising the polynucleotide of any one of claims 1-105.

107. The nucleic acid vector of claim 106, wherein the polynucleotide is operably linked to a transgene.

108. The nucleic acid vector of claim 107, wherein the transgene comprises a nucleic acid sequence encoding a therapeutic protein.

109. The nucleic acid vector of claim 108, wherein the polynucleotide is capable of directing hair cell-specific expression of the therapeutic protein from the nucleic acid sequence in a mammalian hair cell.

110. The nucleic acid vector of claim 109, wherein the hair cell is a cochlear hair cell.

111. The nucleic acid vector of claim 110, wherein the cochlear hair cell is an inner hair cell and/or an outer hair cell.

112. The nucleic acid vector of claim 109, wherein the hair cells is a vestibular hair cell.

113. The nucleic acid vector of any one of claims 106-112, wherein the therapeutic protein is selected from the group consisting of ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

114. The nucleic acid vector of any one of claims 106-113, wherein the nucleic acid vector is a plasmid, cosmid, artificial chromosome, or viral vector.

115. The nucleic acid vector of claim 114, wherein the nucleic acid vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, and a lentivirus.

116. The nucleic acid vector of claim 115, wherein the viral vector is an AAV vector.

117. The nucleic acid vector of claim 116, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, and PHP.S.

118. A composition comprising the nucleic acid vector of any one of claims 106-117.

119. The composition of claim 118, further comprising a pharmaceutically acceptable excipient.

120. A method of increasing expression of a therapeutic protein in a mammalian hair cell, comprising contacting the mammalian hair cell with the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

121. The method of claim 120, wherein expression of the therapeutic protein is specifically increased in hair cells.

122. The method of claim 120 or 121, wherein the mammalian hair cell is a human hair cell.

123. The method of any one of claims 120-122, wherein the mammalian hair cell is a cochlear hair cell.

124. The method of claim 123, wherein the cochlear hair cell is an inner hair cell.

125. The method of claim 123, wherein the cochlear hair cell is an outer hair cell.

126. The method of any one of claims 120-122, wherein the mammalian hair cell is a vestibular hair cell.

127. The method of any one of claims 120-126, wherein expression of the therapeutic protein is not substantially increased in inner ear cells that are not hair cells.

128. A method of treating a subject having or at risk of developing hearing loss, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

129. The method of claim 128, wherein the hearing loss is genetic hearing loss.

130. The method of claim 129, wherein the genetic hearing loss is autosomal dominant hearing loss, autosomal recessive hearing loss, or X-linked hearing loss.

131. The method of claim 128, wherein the hearing loss is acquired hearing loss.

132. The method of claim 131, wherein the acquired hearing loss is noise-induced hearing loss, age-related hearing loss, disease or infection-related hearing loss, head trauma-related hearing loss, or ototoxic drug-induced hearing loss.

133. A method of treating a subject having or at risk of developing vestibular dysfunction, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

134. The method of claim 133, wherein the vestibular dysfunction is vertigo, dizziness, or imbalance.

135. A method of promoting hair cell regeneration in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

136. The method of claim 135, wherein the hair cell is a cochlear hair cell.

137. The method of claim 135, wherein the hair cell is a vestibular hair cell.

138. A method of preventing or reducing ototoxic drug-induced hair cell damage or death, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

139. The method of claim 132 or 138, wherein the ototoxic drug is selected from the group consisting of aminoglycosides, antineoplastic drugs, ethacrynic acid, furosemide, salicylates, and quinine.

140. A method of treating a subject having tinnitus, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

141. A method of preventing or reducing hair cell damage or death in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

142. A method of increasing hair cell survival in a subject in need thereof, comprising administering to the subject an effective amount of the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

143. The method of any one of claims 128-132, 135, 136, and 138-142, wherein the method further comprises evaluating the hearing of the subject prior to administering the nucleic acid vector or composition.

144. The method of any one of claims 128-132, 135, 136, and 138-143, wherein the method further comprises evaluating the hearing of the subject after administering the nucleic acid vector or composition.

145. The method of any one of claims 133-135, 137-139, 141, and 142, wherein the method further comprises evaluating the vestibular function of the subject prior to administering the nucleic acid vector or composition.

146. The method of any one of claims 133-135, 137-139, 141, 142, and 145, wherein the method further comprises evaluating the vestibular function of the subject prior to administering the nucleic acid vector or composition.

147. The method of any one of claims 128-146, wherein the nucleic acid vector or composition is locally administered.

148. The method of any one of claims 128-147, wherein the nucleic acid vector or composition is administered in an amount sufficient to prevent or reduce hearing loss, prevent or reduce vestibular dysfunction, prevent or reduce tinnitus, delay the development of hearing loss, delay the development of vestibular dysfunction, slow the progression of hearing loss, slow the progression of vestibular dysfunction, improve hearing, improve vestibular function, improve hair cell function, prevent or reduce hair cell damage, prevent or reduce hair cell death, or increase hair cell numbers.

149. The method of any one of claims 128-148, wherein the subject is a human.

150. A kit comprising the nucleic acid vector of any one of claims 106-117 or the composition of claim 118 or 119.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIGS. 1A-1B are a series of fluorescent images of mouse cochlea transduced with either an adeno-associated virus (AAV) vector expressing GFP under the control of the cytomegalovirus (CMV) promoter (FIG. 1A) or an AAV vector expressing GFP under control of the murine Myo15 promoter (SEQ ID NO: 13, FIG. 1B). AAV-Myo15-GFP virus was infused via posterior semi-circular canal to 6-8-week-old C57Bl/6J male mice. Mice recovered from surgery and were euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bone was harvested and decalcified in 8% EDTA for 3 days. The cochlea was dissected from the de-calcified temporal bone and mounted on a slide for confocal imaging. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia (FIG. 1A). Using the hair cell-specific promoter, AAV-Myo15-GFP induced expression exclusively in the inner and outer hair cells (FIG. 1B).

[0100] FIG. 2 is a fluorescent image of regions of the mouse vestibular system (utricle, saccule, posterior crista (PC), anterior crista (AC), and horizontal crista (HC)) transduced with an AAV vector expressing GFP under control of the murine Myo15 promoter (SEQ ID NO: 13, FIG. 2). AAV-Myo15-GFP virus was infused via posterior semi-circular canal to 6-8-week-old C57Bl/6J male mice. Mice recovered from surgery and were euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bone was harvested and decalcified in 8% EDTA for 3 days. The vestibular organs were dissected from the de-calcified temporal bone and mounted on a slide for imaging. Using the hair cell-specific promoter, AAV-Myo15-GFP induced expression exclusively in vestibular hair cells (FIG. 2).

[0101] FIGS. 3A-3B are series of fluorescent images of mouse cochlea transduced with an AAV 2/9 viral vector expressing GFP under the control of the murine Myo15 1 kb promoter (SEQ ID NO: 15, FIG. 3A) or the Myo15 mini intron promoter (SEQ ID NO: 16, FIG. 3B). As shown in FIGS. 3A-3B, both the murine Myo15 1 kb (SEQ ID NO: 15) and murine Myo15 mini intron (SEQ ID NO: 16) promoters induced expression of GFP specifically in hair cells.

[0102] FIGS. 4A-4G are a series of images of mouse cochlea showing the specificity of murine and human Myo15 promoters. Neonatal mouse cochlear explants were treated in culture with AAV1 vectors encoding GFP under control of either the 0.6 kb human Myo15 promoter (SEQ ID NO: 26; FIG. 4A), 1.2 kb human Myo15 promoter (SEQ ID NO: 25; FIG. 4B) or the 1 kb mouse Myo15 promoter (SEQ ID NO: 15; FIG. 4C) (scale bars=100 μm). GFP signal, as detected with anti-GFP antibody, is shown. In a separate experiment, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb murine Myo15-GFP was delivered to adult mouse cochlea via round window injection. Whole mount images (FIGS. 4D-4E) or sections (FIGS. 4F-4G) show hair cell-specific signal in the apex, middle, and base of the cochlea, as detected with anti-GFP antibody. Arrows in FIG. 4F point to GFP-labeled cochlear hair cells. As shown in FIGS. 4F and 4G, expression with both the 1.2 kb human Myo15 promoter and 1 kb murine Myo15 promoter was restricted primarily to inner hair cells.

[0103] FIGS. 5A-5C is a series of fluorescent images of non-human primate cochlea showing that the Myo15 promoter restricts GFP expression to hair cells in the cochlea of a non-human primate. FIG. 5A is a confocal image of the cochlea from a non-human primate that received a local injection of AAV1-CMV-GFP through the round window membrane. Tissue was harvested 28 days after injection. Native GFP fluorescence is shown. GFP expression was detected in a broad array of cell types throughout the organ.

[0104] FIG. 5B is a confocal image of the cochlea from a non-human primate that was injected with AAV1-Myo15 (SEQ ID NO: 13)-GFP and processed in the same way as the cochlea in FIG. 5A. GFP expression was restricted to hair cells. FIG. 5C is a magnified view of the hair cells in the box region shown in FIG. 5B.

[0105] FIG. 6 is a graph showing that the 1.6 kb Myo15 promoter (SEQ ID NO: 13) enhanced biological efficacy of an AAV-mouse TMC1 vector in Tmc1 knockout (KO) mice compared to the ubiquitous CMV promoter. Tmc1 KO mice were injected at postnatal day 2 (P2) and auditory brainstem response (ABR) was assessed at the indicated ages. ABR thresholds were plotted as a function of stimulus frequency for naïve homozygous (light gray line with open circles) and heterozygous (dark gray line through black circles) Tmc1 KO mice as well as homozygous Tmc1 KO mice injected with AAV-CMV-mouse TMC1 (CMV_mTmc1; dark gray line with open circles) or AAV-Myo15 (SEQ ID NO:13)-mouse TMC1 (PdBx_mTmc1; P23, light gray line through light gray circles; P28, dark gray line with filled circles). Restoration of ABR thresholds was greatly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 compared to AAV-CMV-mouse TMC1.

[0106] FIGS. 7A-7B are a series of fluorescence images of the inner ear of a cynomolgus monkey treated with an AAV1 vector expressing GFP under control of the 1.2 kb human myosin 15 promoter (SEQ ID NO: 25). Bilateral (n=1 animal) injections were used to administer 30 μL of AAV1-1.2 kb Myosin 15-GFP (viral titer at 1.06×10.sup.13 genome copies/mL) to the round window of the inner ear at a flow rate of 15 μL/min. Four weeks post-injection, inner ears were removed, and a surface preparation of the basilar membranes was made. GFP expression was used to monitor Myosin 15 promoter activity (FIG. 7A, one of the representative ears is shown). AAV1-1.2 kb Myosin 15-GFP transduction of the inner ear of cynomolgus monkeys resulted in GFP expression across the entire baso-apical axis of the cochlea with high frequency at base and low frequency at apex (FIG. 7A, frequencies at kHz). High magnification images at 2.8 kHz showed GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHC) (FIG. 7B, upper panel). Immunohistochemistry for Myo7A was used to visualize hair cells and nuclei were stained with DAPI (FIG. 7B, middle and lower panels).

[0107] FIGS. 8A-8B are a series of fluorescence images of the inner ear of a cynomolgus monkey treated with an AAV1 vector expressing GFP under control of the 0.6 kb human myosin 15 promoter (SEQ ID NO: 26). Bilateral (n=1 animal) injections were used to administer 30 μL of AAV1-0.6 kb Myosin 15-GFP (viral titer at 1.22×10.sup.13 genome copies/mL) to the round window of the inner ear at a flow rate of 15 μL/min. Four weeks post-injection, inner ears were removed, and a surface preparation of the basilar membranes was made. GFP expression was used to monitor Myosin 15 promoter activity (FIG. 8A, one of the representative ears is shown). AAV1-0.6 kb Myosin 15-GFP transduction of the inner ear of cynomolgus monkeys resulted in GFP expression across the entire baso-apical axis of the cochlea with high frequency at base and low frequency at apex (FIG. 8A, frequencies at kHz). High magnification images at 2.8 kHz showed GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHC) (FIG. 8B, upper panel). Immunohistochemistry for Myo7A was used to visualize hair cells and nuclei were stained with DAPI (FIG. 8B, middle and lower panels).

[0108] FIG. 9 is a bar plot showing quantification of GFP expression from three different murine Myo15 (mMyo15) promoters with hair cell specific expression using quantitative PCR (qPCR). Transcript levels were normalized to the hair cell specific gene Myo7a. Expression levels of the GFP reporter from the mMyo15 1.6 kb (SEQ ID NO: 13), 1 kb (SEQ ID NO: 15), and 0.5 kb (SEQ ID NO: 16) promoters were comparable to each other and robust.

[0109] FIG. 10 is a graph showing the results of quantitative image analysis of GFP fluorescence in myosin 7a (Myo7a)-positive hair cells driven by a variety of different promoters including a truncated chimeric CMV-chicken β-actin (smCBA) promoter, 1.6 kb mMyo15 promoter (SEQ ID NO: 13), 1 kb mMyo15 promoter (SEQ ID NO: 15), 0.5 kb mMyo15 promoter (SEQ ID NO: 16), 1.2 kb hMyo15 promoter (SEQ ID NO: 25), and 0.6 kb hMyo15 promoter (SEQ ID NO: 26). Expression levels of GFP protein per cell indicate differences in expression between Myo15 promoters, with the mMyo15 1 kb promoter yielding the highest levels of GFP protein per cell.

[0110] FIGS. 11A-11J are a series of whole mount fluorescence images of neonatal cochlear explant cultures infected with viral vectors containing the Myo15 promoter variants of SEQ ID NOs: 16 and 27-35. The promoter sequence of SEQ ID NO: 27 (Myo15 A) was neither sufficient nor necessary for general promoter activity or hair cell specificity as shown in FIGS. 11B (SEQ ID NO: 27), 11E (SEQ ID NO: 30), 11F (SEQ ID NO: 31), and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 27 did provide a splice donor site that increased expression levels when paired with a splice acceptor site as shown in FIGS. 11A (SEQ ID NO: 16) and 11H (SEQ ID NO: 33), thus introducing an intron into the promoter sequence. The promoter sequence of SEQ ID NO: 28 (Myo15 B) alone was found to be a core promoter that conferred general promoter activity as shown in FIG. 11C (SEQ ID NO: 28), but did not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO: 29 (Myo15 C) as shown in FIGS. 11C (SEQ ID NO: 29) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 29 (Myo15 C) was found to be an enhancer that restricted activity of the core promoter (SEQ ID NO: 28) to inner and outer hair cells as shown in FIG. 11G (SEQ ID NO: 32), but was not sufficient for general promoter activity in the absence of the core promoter as shown in FIG. 11D (SEQ ID NO: 29). The promoter sequences of SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29 were reordered into various combinations and still resulted in hair cell specific promoter activity as shown in FIGS. 11H (SEQ ID NO: 33) and 11I (SEQ ID NO: 34), although not all rearrangements were tolerated, as shown in FIG. 11J (SEQ ID NO: 35).

[0111] FIG. 12 is a bar plot showing the quantification of GFP expression from promoter variants with hair cell specific expression as measured by qPCR. Transcript levels were normalized to the hair cell specific gene Myo7a. Expression levels of the GFP reporter from the Myo15 0.5 kb (SEQ ID NO: 16), Myo15 B-C(SEQ ID NO: 32), and Myo15 B-A-C(SEQ ID NO: 33) promoters were comparable to each other and robust. The Myo15 C-A-B promoter (SEQ ID NO: 34) was generated by rearranging positions of the splice donor and acceptor sites, resulting in cryptic splicing into the middle of the GFP cDNA as evidenced by the higher expression levels detected with GFP probes located in the 3′ end of the transcript versus the 5′ end or middle of the transcript.

[0112] FIGS. 13A-13B are a series of graphs showing quantitative image analysis of GFP expression levels in Myo7a-positive hair cells. Expression levels of GFP protein per cell showed differences in expression between Myo15 promoters (FIGS. 13A-13B), including the 0.5 kb mMyo15 promoter (also referred to as a Myo15 A-B-C promoter; SEQ ID NO: 16), Myo15 A-B promoter (SEQ ID NO: 30), Myo15 A-C promoter (SEQ ID NO: 31), Myo15 B-C promoter (SEQ ID NO: 32), Myo15 A promoter (SEQ ID NO: 27), Myo15 B promoter (SEQ ID NO: 28), Myo15 C promoter (SEQ ID NO: 29), Myo15 B-A-C promoter (SEQ ID NO: 33), Myo15 C-A-B promoter (SEQ ID NO: 34), and the Myo15 B-C-A promoter (SEQ ID NO: 35). The Myo15 A-B-C promoter (SEQ ID NO: 16) yielded the highest expression of GFP per cell (FIG. 13A). Background levels of fluorescence were determined per dataset based on autofluorescence of tissues (<2000 units for FIG. 13A and <6000 units for FIG. 13B).

DETAILED DESCRIPTION

[0113] Described herein are compositions and methods for inducing transgene expression specifically in hair cells (e.g., cochlear and/or vestibular hair cells). The invention features polynucleotides containing regions of the Myosin 15 (Myo15) promoter that are capable of expressing a transgene specifically in cochlear hair cells. The invention also features nucleic acid vectors containing these promoters operably linked to polynucleotides encoding polypeptides. The compositions and methods described herein can be used to express polynucleotides encoding hair cell proteins specifically in cochlear and vestibular hair cells, and, therefore, the compositions described herein can be administered to a subject (such as a mammalian subject, for instance, a human) to treat disorders caused by dysfunction of hair cells, such as hearing loss or vestibular dysfunction.

Hair Cells

[0114] Hair cells are sensory cells of the auditory and vestibular systems that reside in the inner ear. Cochlear hair cells are the sensory cells of the auditory system and are made up of two main cell types: inner hair cells, which are responsible for sensing sound, and outer hair cells, which are thought to amplify low-level sound. Vestibular hair cells are located in the semicircular canals and otolith organs of the inner ear and are involved in the sensation of movement that contributes to the sense of balance and spatial orientation. Hair cells are named for the stereocilia that protrude from the apical surface of the cell, forming a hair cell bundle. Deflection of the stereocilia (e.g., by sound waves in cochlear hair cells, or by rotation or linear acceleration in vestibular hair cells) leads to the opening of mechanically gated ion channels, which allows hair cells to release neurotransmitters to activate nerves, thereby converting mechanical sound or motion signals into electrical signals that can be transmitted to the brain. Cochlear hair cells are essential for normal hearing, and damage to cochlear hair cells and genetic mutations that disrupt cochlear hair cell function are implicated in hearing loss and deafness. Damage to vestibular hair cells and genetic mutations that disrupt vestibular hair cell function are implicated in vestibular dysfunction, such as loss of balance and vertigo (e.g., dizziness). Gene therapy has recently emerged as an attractive therapeutic approach for treating hearing loss and vestibular dysfunction; however, the field lacks methods for specifically targeting the nucleic acid vectors used in gene therapy to hair cells.

Myosin 15

[0115] Myo15 is an unconventional actin-based molecular motor that regulates stereocilia development. Mice carrying mutations in Myo15 have been found to have short stereocilia and profound hearing loss and vestibular dysfunction, and mutations in the human ortholog, Myo15A, cause non-syndromic autosomal recessive deafness, DFNB3. Myo15 has been observed to localize to stereocilia and is indispensable for stereocilia development and maintenance. The pattern of localization indicates that Myo15 may be specifically expressed in hair cells. However, the Myo15 promoter has not previously been isolated and characterized. We identified evolutionarily conserved blocks in orthologous genomic sequences that may constitute regulatory elements of the promoter and found that they are located more than 7200 base pairs (bp) upstream of the translation start site. This genomic region is too large to be used in conjunction with adeno-associated virus (AAV) vectors to deliver transgenes of interest for gene therapy, which have a maximum packaging capacity of 4.7 kb.

[0116] The present invention is based, in part, on the discovery of regions upstream of the Myo15 translation start site that can be used to promote expression of a transgene specifically in hair cells (e.g., cochlear and/or vestibular hair cells). The compositions and methods described herein can, thus, be used to express a gene of interest in hair cells (e.g., a gene implicated in hair cell development, function, cell fate specification, regeneration, survival, or maintenance, or a gene known to be disrupted, e.g., mutated, in subjects with hearing loss or vestibular dysfunction) to treat subjects having or at risk of developing hearing loss (e.g., sensorineural hearing loss) and/or vestibular dysfunction (e.g., vertigo, dizziness, or loss of balance).

[0117] Murine Myosin 15 Promoters

[0118] The polynucleotides of the compositions and methods described herein include nucleic acid sequences from regions of the murine Myo15 locus that are capable of expressing a transgene specifically in hair cells, or variants thereof, such as a nucleic acid sequences that have at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to regions of the murine Myo15 locus that are capable of expressing a transgene specifically in hair cells. These regions include nucleic acid sequences immediately preceding the murine Myo15 translation start site and an upstream regulatory element that is located over 5 kb from murine the Myo15 translation start site. The polynucleotides of the compositions and methods described herein can optionally include a linker operably linking the regions of the murine Myo15 locus that are capable of expressing a transgene specifically in hair cells, or the regions of the murine Myo15 locus can be joined directly without an intervening linker.

[0119] In some embodiments, the polynucleotides described herein contain a first region (an upstream regulatory element) having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acids from −6755 to −7209 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 1) or a functional portion or derivative thereof joined (e.g., operably linked) to a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence immediately preceding the translation start site of murine Myo15 (nucleic acids from −1 to −1157 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 2) or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 1 may have the sequence of nucleic acids from −7166 to −7091 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 3) and/or the sequence of nucleic acids from −7077 to −6983 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 4). The first region may contain the nucleic acid sequence of SEQ ID NO: 3 fused to the nucleic acid sequence of SEQ ID NO: 4 with no intervening nucleic acids, as set forth in SEQ ID NO: 5, or the first region may contain the nucleic acid sequence of SEQ ID NO: 4 fused to the nucleic acid sequence of SEQ ID NO: 3 with no intervening nucleic acids, as set forth in SEQ ID NO: 6. Alternatively, the first region may contain the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 joined by the endogenous intervening nucleic acid sequence (e.g., the first region may have or include the sequence of nucleic acids from −7166 to −6983 with respect to the murine Myo15 translation start site, as set forth in SEQ ID NO: 7 and SEQ ID NO: 27) or a nucleic acid linker. In polynucleotides in which the first region contains both SEQ ID NO: 3 and SEQ ID NO: 4, the two sequences can be included in any order (e.g., SEQ ID NO: 3 may be joined to (e.g., precede) SEQ ID NO: 4, or SEQ ID NO: 4 may be joined to (e.g., precede) SEQ ID NO: 3). The functional portion of SEQ ID NO: 2 may have the sequence of nucleic acids from −590 to −509 with respect to the Myo15 translation start site (set forth in SEQ ID NO: 8) and/or the sequence of nucleic acids from −266 to −161 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 9). In some embodiments, the sequence containing SEQ ID NO: 8 has the sequence of SEQ ID NO: 28. In some embodiments, the sequence containing SEQ ID NO: 9 has the sequence of SEQ ID NO: 29. The second region may contain the nucleic acid sequence of SEQ ID NO: 8 fused to the nucleic acid sequence of SEQ ID NO: 9 with no intervening nucleic acids, as set forth in SEQ ID NO: 10, or the second region may contain the nucleic acid sequence of SEQ ID NO: 9 fused to the nucleic acid sequence of SEQ ID NO: 8 with no intervening nucleic acids, as set forth in SEQ ID NO: 11. The second region may contain the nucleic acid sequence of SEQ ID NO: 28 fused to the nucleic acid sequence of SEQ ID NO: 29 with no intervening nucleic acids, as set forth in SEQ ID NO: 32, or the second region may contain the nucleic acid sequence of SEQ ID NO: 29 fused to the nucleic acid sequence of SEQ ID NO: 28 with no intervening nucleic acids. Alternatively, the second region may contain the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 joined by the endogenous intervening nucleic acid sequence (e.g., the second region may have the sequence of nucleic acids from −590 to −161 with respect to the murine Myo15 translation start site, as set forth in SEQ ID NO: 12) or a nucleic acid linker. In polynucleotides in which the second region contains both SEQ ID NO: 8 and SEQ ID NO: 9, the two sequences can be included in any order (e.g., SEQ ID NO: 8 may be joined to (e.g., precede) SEQ ID NO: 9, or SEQ ID NO: 9 may be joined to (e.g., precede) SEQ ID NO: 8).

[0120] The first region and the second region of the polynucleotide can be joined directly or can be joined by a nucleic acid linker. For example, the polynucleotide can contain the sequence of SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs 3 and 4) fused to the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs 8 and 9) with no intervening nucleic acids. For example, the nucleic acid sequence of the polynucleotide that results from direct fusion of SEQ ID NO: 1 to SEQ ID NO: 2 is set forth in SEQ ID NO: 13. Alternatively, a linker can be used to join the sequence of SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs 3 and 4) to the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs 8 and 9). Exemplary polynucleotides containing functional portions of both SEQ ID NO: 1 and SEQ ID NO: 2 are provided in SEQ ID NOs: 15, 16, 30, and 31.

[0121] The length of a nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5, kb, 4, kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15, bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp, or less). Nucleic acid linkers that can be used in the polynucleotides described herein do not disrupt the ability of the polynucleotides of the invention to induce transgene expression in hair cells.

[0122] In some embodiments, the sequence of SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs 3 and 4) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs 8 and 9), and, in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, 28, 29, and 32, e.g., SEQ ID NOs 8 and 9) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7 and 27, e.g., SEQ ID NOs 3 and 4)). For example, the nucleic acid sequence of the polynucleotide that results from direct fusion of SEQ ID NO: 2 to SEQ ID NO: 1 is set forth in SEQ ID NO: 14. An example of a polynucleotide in which a functional portion or derivative of SEQ ID NO: 2 precedes a functional portion or derivative of SEQ ID NO: 1 is provided in SEQ ID NO: 35. Regardless of order, the sequence of SEQ ID NO: 1 or a functional portion or derivative thereof and the sequence of SEQ ID NO: 2 or a functional portion or derivative thereof can be joined by direct fusion or a nucleic acid linker, as described above.

[0123] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acids from −6755 to −7209 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 1) or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 1 may have the sequence of nucleic acids from −7166 to −7091 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 3) and/or the sequence of nucleic acids from −7077 to −6983 with respect to the Myo15 translation start site (set forth in SEQ ID NO: 4). The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 3 fused to the nucleic acid sequence of SEQ ID NO: 4 with no intervening nucleic acids, as set forth in SEQ ID NO: 5, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 4 fused to the nucleic acid of SEQ ID NO: 3 with no intervening nucleic acids, as set forth in SEQ ID NO: 6. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 joined by the endogenous intervening nucleic acid sequence (e.g., the first region may have or include the sequence of nucleic acids from −7166 to −6983 with respect to the murine Myo15 translation start site, as set forth in SEQ ID NO: 7 and SEQ ID NO: 27) or a nucleic acid linker. In polynucleotides that contain both SEQ ID NO: 3 and SEQ ID NO: 4, the two sequences can be included in any order (e.g., SEQ ID NO: 3 may be joined to (e.g., precede) SEQ ID NO: 4, or SEQ ID NO: 4 may be joined to (e.g., precede) SEQ ID NO: 3).

[0124] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence immediately upstream of the murine Myo15 translation start site (nucleic acids from −1 to −1157 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 2) or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 2 may have the sequence of nucleic acids from −590 to −509 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 8) and/or the sequence of nucleic acids from −266 to −161 with respect to the murine Myo15 translation start site (set forth in SEQ ID NO: 9). In some embodiments, the sequence containing SEQ ID NO: 8 has the sequence of SEQ ID NO: 28. In some embodiments, the sequence containing SEQ ID NO: 9 has the sequence of SEQ ID NO: 29. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 8 fused to the nucleic acid sequence of SEQ ID NO: 9 with no intervening nucleic acids, as set forth in SEQ ID NO: 10, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 9 fused to the nucleic acid sequence of SEQ ID NO: 8 with no intervening nucleic acids, as set forth in SEQ ID NO: 11. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 28 fused to the nucleic acid sequence of SEQ ID NO: 29 with no intervening nucleic acids, as set forth in SEQ ID NO: 32, or the second region may contain the nucleic acid sequence of SEQ ID NO: 29 fused to the nucleic acid sequence of SEQ ID NO: 28 with no intervening nucleic acids. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 joined by the endogenous intervening nucleic acid sequence (e.g., the second region may have the sequence of nucleic acids from −590 to −161 with respect to the murine Myo15 translation start site, as set forth in SEQ ID NO: 12) or a nucleic acid linker. In polynucleotides that contain both SEQ ID NO: 8 and SEQ ID NO: 9, the two sequences can be included in any order (e.g., SEQ ID NO: 8 may be joined to (e.g., precede) SEQ ID NO: 9, or SEQ ID NO: 9 may be joined to (e.g., precede) SEQ ID NO: 8).

[0125] In some embodiments, the polynucleotides described herein contain a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to a region containing the first non-coding exon of the Myo15 gene (nucleic acids from −6755 to −7209 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 1) flanked on either side by a portion or derivative of a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence immediately upstream of the murine Myo15 translation start site (nucleic acids from −1 to −1157 with respect to the murine Myo15 translation start site, the sequence of which is set forth in SEQ ID NO: 2). For example, a functional portion or derivative of SEQ ID NO: 2, such as SEQ ID NO: 8 or 28 may be directly fused or joined by a nucleic acid linker to a portion of SEQ ID NO: 1, such as any one of SEQ ID NOs: 3-7 and 27, which is directly fused or joined by a nucleic acid linker to a different functional portion of SEQ ID NO: 2, such as SEQ ID NO: 9 or 29. In other embodiments, a functional portion or derivative of SEQ ID NO: 2, such as SEQ ID NO: 9 or 29 may be directly fused or joined by a nucleic acid linker to a portion of SEQ ID NO: 1, such as any one of SEQ ID NOs: 3-7 and 27, which is directly fused or joined by a nucleic acid linker to a different functional portion of SEQ ID NO: 2, such as SEQ ID NO: 8 or 28. For example, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence of SEQ ID NOs: 28, 27, and 29 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, polynucleotides having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence of SEQ ID NOs: 29, 27, and 28 can be fused to produce a polynucleotide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequence of SEQ ID NO: 34.

[0126] Human Myosin 15 Promoters

[0127] The polynucleotides of the compositions and methods described herein may also include nucleic acid sequences from regions of the human Myo15 locus that are capable of expressing a transgene specifically in hair cells, or variants thereof, such as a nucleic acid sequences that have at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to regions of the human Myo15 locus that are capable of expressing a transgene specifically in hair cells. The polynucleotides of the compositions and methods described herein can optionally include a linker operably linking the regions of the human Myo15 locus that are capable of expressing a transgene specifically in hair cells, or the regions of the human Myo15 locus can be joined directly without an intervening linker.

[0128] In some embodiments, the polynucleotides described herein contain a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the sequence set forth in SEQ ID NO: 17 or a functional portion or derivative thereof joined (e.g., operably linked) to a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the sequence set forth in SEQ ID NO: 18 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 17 may have the sequence set forth in SEQ ID NO: 19. The functional portion of SEQ ID NO: 18 may have the sequence set forth in SEQ ID NO: 20 and/or the sequence set forth in SEQ ID NO: 21. The second region may contain the nucleic acid sequence of SEQ ID NO: 20 fused to the nucleic acid sequence of SEQ ID NO: 21 with no intervening nucleic acids, as set forth in SEQ ID NO: 22, or the second region may contain the nucleic acid sequence of SEQ ID NO: 21 fused to the nucleic acid sequence of SEQ ID NO: 20 with no intervening nucleic acids, as set forth in SEQ ID NO: 23. Alternatively, the second region may contain the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 joined by the endogenous intervening nucleic acid sequence (as set forth in SEQ ID NO: 24) or a nucleic acid linker. In polynucleotides in which the second region contains both SEQ ID NO: 20 and SEQ ID NO: 21, the two sequences can be included in any order (e.g., SEQ ID NO: 20 may be joined to (e.g., precede) SEQ ID NO: 21, or SEQ ID NO: 21 may be joined to (e.g., precede) SEQ ID NO: 20).

[0129] The first region and the second region of the polynucleotide can be joined directly or can be joined by a nucleic acid linker. For example, the polynucleotide can contain the sequence of SEQ ID NO: 17 or a functional portion or derivative thereof (e.g., SEQ ID NO: 19) fused to the sequence of SEQ ID NO: 18 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and/or 21) with no intervening nucleic acids. Alternatively, a linker can be used to join the sequence of SEQ ID NO: 17 or a functional portion or derivative thereof (e.g., SEQ ID NO: 19) to the sequence of SEQ ID NO: 18 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and/or 21). Exemplary polynucleotides containing functional portions of both SEQ ID NO: 17 and SEQ ID NO: 18 are provided in SEQ ID NOs: 25 and 26.

[0130] In some embodiments, the sequence of SEQ ID NO: 17 or a functional portion or derivative thereof (e.g., SEQ ID NO: 19) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 18 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and 21), and, in some embodiments, the order of the regions is reversed (e.g., the sequence of SEQ ID NO: 18 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 20-24, e.g., SEQ ID NOs: 20 and/or 21) is joined (e.g., operably linked) to the sequence of SEQ ID NO: 17 or a functional portion or derivative thereof (e.g., SEQ ID NO: 19). Regardless of order, the sequence of SEQ ID NO: 17 or a functional portion or derivative thereof and the sequence of SEQ ID NO: 18 or a functional portion or derivative thereof can be joined by direct fusion or a nucleic acid linker, as described above.

[0131] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to a region containing the sequence set forth in SEQ ID NO: 17 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 17 may have the sequence of nucleic acids set forth in SEQ ID NO: 19.

[0132] In some embodiments, the polynucleotides described herein contain a region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the sequence set forth in SEQ ID NO: 18 or a functional portion or derivative thereof. The functional portion of SEQ ID NO: 18 may have the sequence set forth in SEQ ID NO: 20 and/or the sequence set forth in SEQ ID NO: 21. The polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 20 fused to the nucleic acid sequence of SEQ ID NO: 21 with no intervening nucleic acids, as set forth in SEQ ID NO: 22, or the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 21 fused to the nucleic acid sequence of SEQ ID NO: 20 with no intervening nucleic acids, as set forth in SEQ ID NO: 23. Alternatively, the polynucleotide may contain the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 joined by the endogenous intervening nucleic acid sequence (e.g., as set forth in SEQ ID NO: 24) or a nucleic acid linker. In polynucleotides that contain both SEQ ID NO: 20 and SEQ ID NO: 21, the two sequences can be included in any order (e.g., SEQ ID NO: 20 may be joined to (e.g., precede) SEQ ID NO: 21, or SEQ ID NO: 21 may be joined to (e.g., precede) SEQ ID NO: 20).

[0133] The length of a nucleic acid linker for use in the polynucleotides described herein can be about 5 kb or less (e.g., about 5 kb, 4.5, kb, 4, kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 90 bp, 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15, bp, 10 bp, 5 bp, 4 bp, 3 bp, 2 bp, or less). Nucleic acid linkers that can be used in the polynucleotides described herein do not disrupt the ability of the polynucleotides of the invention to induce transgene expression in hair cells.

[0134] The foregoing nucleic acid sequences are summarized in Table 2, below.

TABLE-US-00002 TABLE 2 Exemplary nucleotide sequences for use in the polynucleotides described herein SEQ ID Description of nucleic NO. acid sequence Nucleic Acid Sequence 1 Region containing non- CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT coding exon 1 of murine TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA Myo15 (−6755 to −7209) ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTAAGCTTCT GCCACTGGCTCCGGCATTGCAGAGAGAAGAGAAGGGGCGG CAGAGCTGAACCTTAGCCTTGCCTTCCTGGGTACCCTTCTG AGCCTCACTGTCTTCTGTGAGATGGGCAAAGTGCGGGTGTG ACTCCTTGGCAACGGTGTTACACCAGGGCAGGTAAAGTTGT AGTTATTTGTGGGGTACACCAGGACTGTTAAAGGTGTAACTA T 2 Region immediately GGTCTCACCCAGCATTTTCACTTCTAATAAGTTCAAATGTGA preceding the translation TACGGCACCTTTCTAAAAATTAGTTTTCAGGGAAATAGGGTT start site of murine CAAAACTGGTAGTGGTAGGGTCCATTCTCACGACCCCCAGG Myo15 (−1 to −1157) CCTGCTAACCCTGACCAAGCTACCTATTACTTACCCTCCTCT TTCTCCTCCTCCTCTTTCTCCTTCTCCTGCTTCCCCTCTTCCT TCTCCCTCCCTTCCTCTCCCTCCTCCCCCTCCTTGGCTGTGA TCAGATCCAGAGCCTGAATGAGCCTCCTGACCCCACACCCC CACTAGCATGGGCCTGCAAGTGCCCAGAAGTCCCTCCTGCC TCCTAAACTGCCCAGCCGATCCATTAGCTCTTCCTTCTTCCC AGTGAAAGAAGCAGGCACAGCCTGTCCCTCCCGTTCTACAG AAAGGAAGCTACAGCACAGGGAGGGCCAAAGGCCTTCCTG GGACTAGACAGTTGATCAACAGCAGGACTGGAGAGCTGGG CTCCATTTTTGTTCCTTGGTGCCCTGCCCCTCCCCATGACCT GCAGAGACATTCAGCCTGCCAGGCTTTATGAGGTGGGAGCT GGGCTCTCCCTGATGTATTATTCAGCTCCCTGGAGTTGGCC AGCTCCTGTTACACTGGCCACAGCCCTGGGCATCCGCTTCT CACTTCTAGTTTCCCCTCCAAGGTAATGTGGTGGGTCATGAT CATTCTATCCTGGCTTCAGGGACCTGACTCCACTTTGGGGC CATTCGAGGGGTCTAGGGTAGATGATGTCCCCCTGTGGGGA TTAATGTCCTGCTCTGTAAAACTGAGCTAGCTGAGATCCAGG AGGGCTTGGCCAGAGACAGCAAGTTGTTGCCATGGTGACTT TAAAGCCAGGTTGCTGCCCCAGCACAGGCCTCCCAGTCTAC CCTCACTAGAAAACAACACCCAGGCACTTTCCACCACCTCTC AAAGGTGAAACCCAAGGCTGGTCTAGAGAATGAATTATGGA TCCTCGCTGTCCGTGCCACCCAGCTAGTCCCAGCGGCTCAG ACACTGAGGAGAGACTGTAGGTTCAGCTACAAGCAAAAAGA CCTAGCTGGTCTCCAAGCAGTGTCTCCAAGTCCCTGAACCT GTGACACCTGCCCCAGGCATCATCAGGCACAGAGGGCCAC C 3 Portion of SEQ ID NO: 1 CCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAA (−7166 to −7091) TAATAGATGTCATTAAATATACATTGGGCCCCAGG 4 Portion of SEQ ID NO: 1 AGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGACATAGG (−7077 to −6983) ACCCCCAACAAACAGCATGCAGGTTGGGAGCCAGCCACAG GACCCAGGTAAGGG 5 Portion of SEQ ID NO: 1 CCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAA (SEQ ID NO: 3 fused to TAATAGATGTCATTAAATATACATTGGGCCCCAGGAGCCTGA SEQ ID NO: 4) GCCTCCTTTCCATCTCTGTGGAGGCAGACATAGGACCCCCA ACAAACAGCATGCAGGTTGGGAGCCAGCCACAGGACCCAG GTAAGGG 6 Portion of SEQ ID NO: 1 AGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGACATAGG (SEQ ID NO: 4 fused to ACCCCCAACAAACAGCATGCAGGTTGGGAGCCAGCCACAG SEQ ID NO: 3) GACCCAGGTAAGGGCCCATGTCAGCTGCTTGTGCTTTCCAG AGACAAAACAGGAATAATAGATGTCATTAAATATACATTGGG CCCCAGG 7 Portion of SEQ ID NO: 1 CCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAA (−7166 to −6983) TAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCAA TGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGA CATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGG 8 Portion of SEQ ID NO: 2 TGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTC (−590 to −509) CCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTG 9 Portion of SEQ ID NO: 2 CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA (−266 to −161) GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG TCTAGAGAATGAATTATGGATCCT 10 Portion of SEQ ID NO: 2 TGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTC (SEQ ID NO: 8 fused to CCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTG SEQ ID NO: 9) CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG TCTAGAGAATGAATTATGGATCCT 11 Portion of SEQ ID NO: 2 CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA (SEQ ID NO: 9 fused to GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG SEQ ID NO: 8) TCTAGAGAATGAATTATGGATCCTTGAGGTGGGAGCTGGGC TCTCCCTGATGTATTATTCAGCTCCCTGGAGTTGGCCAGCTC CTGTTACACTGGCCACAGCCCTG 12 Portion of SEQ ID NO: 2 TGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTC (−590 to −161) CCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTG GGCATCCGCTTCTCACTTCTAGTTTCCCCTCCAAGGTAATGT GGTGGGTCATGATCATTCTATCCTGGCTTCAGGGACCTGAC TCCACTTTGGGGCCATTCGAGGGGTCTAGGGTAGATGATGT CCCCCTGTGGGGATTAATGTCCTGCTCTGTAAAACTGAGCT AGCTGAGATCCAGGAGGGCTTGGCCAGAGACAGCAAGTTG TTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAGCACA GGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCAGGCA CTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGGTCTA GAGAATGAATTATGGATCCT 13 SEQ ID NO: 1 fused to CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT SEQ ID NO: 2 TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTAAGCTTCT GCCACTGGCTCCGGCATTGCAGAGAGAAGAGAAGGGGCGG CAGAGCTGAACCTTAGCCTTGCCTTCCTGGGTACCCTTCTG AGCCTCACTGTCTTCTGTGAGATGGGCAAAGTGCGGGTGTG ACTCCTTGGCAACGGTGTTACACCAGGGCAGGTAAAGTTGT AGTTATTTGTGGGGTACACCAGGACTGTTAAAGGTGTAACTA TGGTCTCACCCAGCATTTTCACTTCTAATAAGTTCAAATGTG ATACGGCACCTTTCTAAAAATTAGTTTTCAGGGAAATAGGGT TCAAAACTGGTAGTGGTAGGGTCCATTCTCACGACCCCCAG GCCTGCTAACCCTGACCAAGCTACCTATTACTTACCCTCCTC TTTCTCCTCCTCCTCTTTCTCCTTCTCCTGCTTCCCCTCTTCC TTCTCCCTCCCTTCCTCTCCCTCCTCCCCCTCCTTGGCTGTG ATCAGATCCAGAGCCTGAATGAGCCTCCTGACCCCACACCC CCACTAGCATGGGCCTGCAAGTGCCCAGAAGTCCCTCCTGC CTCCTAAACTGCCCAGCCGATCCATTAGCTCTTCCTTCTTCC CAGTGAAAGAAGCAGGCACAGCCTGTCCCTCCCGTTCTACA GAAAGGAAGCTACAGCACAGGGAGGGCCAAAGGCCTTCCT GGGACTAGACAGTTGATCAACAGCAGGACTGGAGAGCTGG GCTCCATTTTTGTTCCTTGGTGCCCTGCCCCTCCCCATGACC TGCAGAGACATTCAGCCTGCCAGGCTTTATGAGGTGGGAGC TGGGCTCTCCCTGATGTATTATTCAGCTCCCTGGAGTTGGC CAGCTCCTGTTACACTGGCCACAGCCCTGGGCATCCGCTTC TCACTTCTAGTTTCCCCTCCAAGGTAATGTGGTGGGTCATGA TCATTCTATCCTGGCTTCAGGGACCTGACTCCACTTTGGGG CCATTCGAGGGGTCTAGGGTAGATGATGTCCCCCTGTGGG GATTAATGTCCTGCTCTGTAAAACTGAGCTAGCTGAGATCCA GGAGGGCTTGGCCAGAGACAGCAAGTTGTTGCCATGGTGA CTTTAAAGCCAGGTTGCTGCCCCAGCACAGGCCTCCCAGTC TACCCTCACTAGAAAACAACACCCAGGCACTTTCCACCACCT CTCAAAGGTGAAACCCAAGGCTGGTCTAGAGAATGAATTAT GGATCCTCGCTGTCCGTGCCACCCAGCTAGTCCCAGCGGC TCAGACACTGAGGAGAGACTGTAGGTTCAGCTACAAGCAAA AAGACCTAGCTGGTCTCCAAGCAGTGTCTCCAAGTCCCTGA ACCTGTGACACCTGCCCCAGGCATCATCAGGCACAGAGGG CCACC 14 SEQ ID NO: 2 fused to GGTCTCACCCAGCATTTTCACTTCTAATAAGTTCAAATGTGA SEQ ID NO: 1 TACGGCACCTTTCTAAAAATTAGTTTTCAGGGAAATAGGGTT CAAAACTGGTAGTGGTAGGGTCCATTCTCACGACCCCCAGG CCTGCTAACCCTGACCAAGCTACCTATTACTTACCCTCCTCT TTCTCCTCCTCCTCTTTCTCCTTCTCCTGCTTCCCCTCTTCCT TCTCCCTCCCTTCCTCTCCCTCCTCCCCCTCCTTGGCTGTGA TCAGATCCAGAGCCTGAATGAGCCTCCTGACCCCACACCCC CACTAGCATGGGCCTGCAAGTGCCCAGAAGTCCCTCCTGCC TCCTAAACTGCCCAGCCGATCCATTAGCTCTTCCTTCTTCCC AGTGAAAGAAGCAGGCACAGCCTGTCCCTCCCGTTCTACAG AAAGGAAGCTACAGCACAGGGAGGGCCAAAGGCCTTCCTG GGACTAGACAGTTGATCAACAGCAGGACTGGAGAGCTGGG CTCCATTTTTGTTCCTTGGTGCCCTGCCCCTCCCCATGACCT GCAGAGACATTCAGCCTGCCAGGCTTTATGAGGTGGGAGCT GGGCTCTCCCTGATGTATTATTCAGCTCCCTGGAGTTGGCC AGCTCCTGTTACACTGGCCACAGCCCTGGGCATCCGCTTCT CACTTCTAGTTTCCCCTCCAAGGTAATGTGGTGGGTCATGAT CATTCTATCCTGGCTTCAGGGACCTGACTCCACTTTGGGGC CATTCGAGGGGTCTAGGGTAGATGATGTCCCCCTGTGGGGA TTAATGTCCTGCTCTGTAAAACTGAGCTAGCTGAGATCCAGG AGGGCTTGGCCAGAGACAGCAAGTTGTTGCCATGGTGACTT TAAAGCCAGGTTGCTGCCCCAGCACAGGCCTCCCAGTCTAC CCTCACTAGAAAACAACACCCAGGCACTTTCCACCACCTCTC AAAGGTGAAACCCAAGGCTGGTCTAGAGAATGAATTATGGA TCCTCGCTGTCCGTGCCACCCAGCTAGTCCCAGCGGCTCAG ACACTGAGGAGAGACTGTAGGTTCAGCTACAAGCAAAAAGA CCTAGCTGGTCTCCAAGCAGTGTCTCCAAGTCCCTGAACCT GTGACACCTGCCCCAGGCATCATCAGGCACAGAGGGCCAC CCTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAG TTCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTAAGCTTCT GCCACTGGCTCCGGCATTGCAGAGAGAAGAGAAGGGGCGG CAGAGCTGAACCTTAGCCTTGCCTTCCTGGGTACCCTTCTG AGCCTCACTGTCTTCTGTGAGATGGGCAAAGTGCGGGTGTG ACTCCTTGGCAACGGTGTTACACCAGGGCAGGTAAAGTTGT AGTTATTTGTGGGGTACACCAGGACTGTTAAAGGTGTAACTA T 15 Portion of SEQ ID NO: 1 CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT that contains SEQ ID TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA NO: 3 and SEQ ID NO: ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA 4 fused to portion of ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG SEQ ID NO: 2 that ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG contains SEQ ID NO: 8 CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTAAGCTTCT and SEQ ID NO: 9 GCCACTGGCTCCGGCATTGCAGAGAGAAGAGAAGGGGCGG CAGACTGGAGAGCTGGGCTCCATTTTTGTTCCTTGGTGCCC TGCCCCTCCCCATGACCTGCAGAGACATTCAGCCTGCCAGG CTTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCA GCTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGC CCTGGGCATCCGCTTCTCACTTCTAGTTTCCCCTCCAAGGTA ATGTGGTGGGTCATGATCATTCTATCCTGGCTTCAGGGACC TGACTCCACTTTGGGGCCATTCGAGGGGTCTAGGGTAGATG ATGTCCCCCTGTGGGGATTAATGTCCTGCTCTGTAAAACTGA GCTAGCTGAGATCCAGGAGGGCTTGGCCAGAGACAGCAAG TTGTTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAGC ACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCAG GCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGGT CTAGAGAATGAATTATGGATCCTCGCTGTCCGTGCCACCCA GCTAGTCCCAGCGGCTCAGACACTGAGGAGAGACTGTAGG TTCAGCTACAAGCAAAAAGACCTAGCTGGTCTCCAAGCAGT GTCTCCAAGTCCCTGAACCTGTGACACCTGCCCCAGGCATC ATCAGGCACAGAGGGCCACC 16 Portion of SEQ ID NO: 1 CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT that contains SEQ ID TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA NO: 3 and SEQ ID NO: ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA 4 fused to portion of ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG SEQ ID NO: 2 that ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG contains SEQ ID NO: 8 CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTTTTATGAG and SEQ ID NO: 9 GTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTCCCTG GAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTGGGCA TCCGCTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAG CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG TCTAGAGAATGAATTATGGATCCTCGCTGTCCGTGCCACCC AGCTAGTCCCAGCGGCTCAGACACTG 17 Region 1 of the human GTATGCCTTTTGAGATGGATGCAGCAGGTTCTGTGAGGCTG Myo15 promoter CCAGGAGGGGTAGAGTTCCCGGGGGCCTCGGGCCCCGCT GGAGTGTGGAGCAGGCCCATGCTCAGCTCTCCAGGCTGTT CGTGGCTCCCCTGTCAGCTGCTCACTCCTTTCCAGAGACAA AACAGGAATAATAGACATCATTAAATATACATAGGGCCCCAG GCGGTCGGCGTGGTGGGCTGGGCCTCCCTTCC 18 Region 2 of human TGCCCTGCCTTCTGAGCCGGCAGCCTGGCTCCCCACCCCAT Myo15 promoter GTATTATTCAGCTCCTGAGAGCCAGCCAGCTCCTGTTACACT GACCGCAGCCCAGCACCTGCTCTGCCCATTCCCCTCCTCCC TTGCCTAGGACCTAGAGGGTTCAAAGTTCTCCTCCAAGATG ACTTGGTGGGCTTTGGCCATCCCACCCTAGGCCCCACTTCT GGCCCAGTGCAGGTGTGCTGGTGATTTAGGGCAGGTGGCA TTCCATCTCTGTGGCTCAATGTCTTCCTCTGTGAAGCCGAAG TGACCCAAGGGCTCCCTTCATGGGGTTGAGCCAGCTGTGG CCCAGGGAGGGCCTAACCAGGATGAGCACTGATGTTGCCAT GACGACTCCGAGGCCAGAATGTCTCCCCCAGCACAGGCCT CATAGGCAGGCTTCCCCATCCTGGTAAACAACACCCACACA CTTTCTACTACTGCTCTAGGGTGAAACCCAAGGCGCTCTAG AGGAGATGAATTATGGATCCGCCCTCCCGGAATCCTGGCTC GGCCCTCCCCACGCCACCCAGGGCCAGTCGGGTCTGCTCA CAGCCCGAGGAGGCCGCGTGTCCAGCCGCGGGCAAGAGA CAGAGCAGGTCCCTGTGTCTCCAAGTCCCTGAGCCCGTGAC ACCGGCCCCAGGCCCTGTAGAGAGCAGGCAGCCACC 19 Portion of SEQ ID NO: CCCCTGTCAGCTGCTCACTCCTTTCCAGAGACAAAACAGGA 17 ATAATAGACATCATTAAATATACATAGGGCCCCAGG 20 Portion of SEQ ID NO: TGAGCCGGCAGCCTGGCTCCCCACCCCATGTATTATTCAGC 18 TCCTGAGAGCCAGCCAGCTCCTGTTACACTGACCGCAGCCC 21 Portion of SEQ ID NO: CACAGGCCTCATAGGCAGGCTTCCCCATCCTGGTAAACAAC 18 ACCCACACACTTTCTACTACTGCTCTAGGGTGAAACCCAAG GCGCTCTAGAGGAGATGAATTATGGATCC 22 Portion of SEQ ID NO: TGAGCCGGCAGCCTGGCTCCCCACCCCATGTATTATTCAGC 18 TCCTGAGAGCCAGCCAGCTCCTGTTACACTGACCGCAGCCC (SEQ ID NO: 20 fused to CACAGGCCTCATAGGCAGGCTTCCCCATCCTGGTAAACAAC SEQ ID NO: 21) ACCCACACACTTTCTACTACTGCTCTAGGGTGAAACCCAAG GCGCTCTAGAGGAGATGAATTATGGATCC 23 Portion of SEQ ID NO: CACAGGCCTCATAGGCAGGCTTCCCCATCCTGGTAAACAAC 18 ACCCACACACTTTCTACTACTGCTCTAGGGTGAAACCCAAG (SEQ ID NO: 21 fused to GCGCTCTAGAGGAGATGAATTATGGATCCTGAGCCGGCAGC SEQ ID NO: 20) CTGGCTCCCCACCCCATGTATTATTCAGCTCCTGAGAGCCA GCCAGCTCCTGTTACACTGACCGCAGCCC 24 Portion of SEQ ID NO: TGAGCCGGCAGCCTGGCTCCCCACCCCATGTATTATTCAGC 18 (contiguous TCCTGAGAGCCAGCCAGCTCCTGTTACACTGACCGCAGCCC sequence including SEQ AGCACCTGCTCTGCCCATTCCCCTCCTCCCTTGCCTAGGAC ID NO: 20 and SEQ ID CTAGAGGGTTCAAAGTTCTCCTCCAAGATGACTTGGTGGGC NO: 21) TTTGGCCATCCCACCCTAGGCCCCACTTCTGGCCCAGTGCA GGTGTGCTGGTGATTTAGGGCAGGTGGCATTCCATCTCTGT GGCTCAATGTCTTCCTCTGTGAAGCCGAAGTGACCCAAGGG CTCCCTTCATGGGGTTGAGCCAGCTGTGGCCCAGGGAGGG CCTAACCAGGATGAGCACTGATGTTGCCATGACGACTCCGA GGCCAGAATGTCTCCCCCAGCACAGGCCTCATAGGCAGGC TTCCCCATCCTGGTAAACAACACCCACACACTTTCTACTACT GCTCTAGGGTGAAACCCAAGGCGCTCTAGAGGAGATGAATT ATGGATCC 25 Polynucleotide GTATGCCTTTTGAGATGGATGCAGCAGGTTCTGTGAGGCTG containing SEQ ID NO: CCAGGAGGGGTAGAGTTCCCGGGGGCCTCGGGCCCCGCT 17 and SEQ ID NO: 18 GGAGTGTGGAGCAGGCCCATGCTCAGCTCTCCAGGCTGTT CGTGGCTCCCCTGTCAGCTGCTCACTCCTTTCCAGAGACAA AACAGGAATAATAGACATCATTAAATATACATAGGGCCCCAG GCGGTCGGCGTGGTGGGCTGGGCCTCCCTTCCCCATAACA CTGAGCTGCTCTGCTGGGCCAACCGTGCTCCTGGGCCAGC CAGAGGACCCCCATGAGGCGGCATGCAGGCGGGGAGCAG GCCACAGAACGCAGGTAAGGAGACCTTAGCCTAGAGTCCTT GGGGTCTGTCACTGGCCACCCTCGCATCCCAGGCTGCAGG AAACTGAGGCCCAGAGAGGACAAGGACTTTCCTGGACCCAC ACAGCCAGTCAGTGACAGAGCCTAGGGTCTGAGCCAGGCC TGACCCAACCTCCATTTCTGCCTCTCTACCCCTGCCCCCGC CCCAACACACACACACACACAAGTGGAGTTCCACTGAAACG CCCCTCCTTGCCCTGCCTTCTGAGCCGGCAGCCTGGCTCCC CACCCCATGTATTATTCAGCTCCTGAGAGCCAGCCAGCTCC TGTTACACTGACCGCAGCCCAGCACCTGCTCTGCCCATTCC CCTCCTCCCTTGCCTAGGACCTAGAGGGTTCAAAGTTCTCC TCCAAGATGACTTGGTGGGCTTTGGCCATCCCACCCTAGGC CCCACTTCTGGCCCAGTGCAGGTGTGCTGGTGATTTAGGGC AGGTGGCATTCCATCTCTGTGGCTCAATGTCTTCCTCTGTGA AGCCGAAGTGACCCAAGGGCTCCCTTCATGGGGTTGAGCC AGCTGTGGCCCAGGGAGGGCCTAACCAGGATGAGCACTGA TGTTGCCATGACGACTCCGAGGCCAGAATGTCTCCCCCAGC ACAGGCCTCATAGGCAGGCTTCCCCATCCTGGTAAACAACA CCCACACACTTTCTACTACTGCTCTAGGGTGAAACCCAAGG CGCTCTAGAGGAGATGAATTATGGATCCGCCCTCCCGGAAT CCTGGCTCGGCCCTCCCCACGCCACCCAGGGCCAGTCGGG TCTGCTCACAGCCCGAGGAGGCCGCGTGTCCAGCCGCGGG CAAGAGACAGAGCAGGTCCCTGTGTCTCCAAGTCCCTGAGC CCGTGACACCGGCCCCAGGCCCTGTAGAGAGCAGGCAGCC ACC 26 Polynucleotide GCAGGCCCATGCTCAGCTCTCCAGGCTGTTCGTGGCTCCCC containing SEQ ID NO: TGTCAGCTGCTCACTCCTTTCCAGAGACAAAACAGGAATAAT 19, SEQ ID NO: 20, and AGACATCATTAAATATACATAGGGCCCCAGGCGGTCGGCGT SEQ ID NO: 21 GGTGGGCTGGGCCTCCCTTCCCCATAACACTGAGCTGCTCT GCTGGGCCAACCGTGCTCCTGGGCCAGCCAGAGGACCCCC ATGAGGCGGCATGCAGGCGGGGAGCAGGCCACAGAACGCA GGTAAGGAGACCTTGCCTTCTGAGCCGGCAGCCTGGCTCC CCACCCCATGTATTATTCAGCTCCTGAGAGCCAGCCAGCTC CTGTTACACTGACCGCAGCCCAGCACCTGCTCTGCCCATTC CCCTCCTCCCTTGCCTAGGACCTAGAGGGTTCAAAGTTCTC CTCCAAGATGACTTGGTGGGCTTTGGCCATCGGGCCTAACC AGGATGAGCACTGATGTTGCCATGACGACTCCGAGGCCAGA ATGTCTCCCCCAGCACAGGCCTCATAGGCAGGCTTCCCCAT CCTGGTAAACAACACCCACACACTTTCTACTACTGCTCTAGG GTGAAACCCAAGGCGCTCTAGAGGAGATGAATTATGGATCC GCCCTCCCGGAATCCTGGCTCGGCCCTCCCCACGC 27 Portion of SEQ ID NO: 1 CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT that contains SEQ ID TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA NO: 3 and SEQ ID NO: ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA 4 ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTT 28 Portion of SEQ ID NO: 2 TTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAG that contains SEQ ID CTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCC NO: 8 CTGGGCATCCGC 29 Portion of SEQ ID NO: 2 TGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAGCACAG that contains SEQ ID GCCTCCCAGTCTACCCTCACTAGAAAACAACACCCAGGCAC NO: 9 TTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGGTCTAG AGAATGAATTATGGATCCTCGCTGTCCGTGCCACCCAGCTA GTCCCAGCGGCTCAGACACTG 30 SEQ ID NO: 27 fused to CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT SEQ ID NO: 28 TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTTTTATGAG GTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTCCCTG GAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTGGGCA TCCGC 31 SEQ ID NO: 27 fused to CTGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGT SEQ ID NO: 29 TCCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGA ATAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCA ATGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAG ACATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTTGCCATG GTGACTTTAAAGCCAGGTTGCTGCCCCAGCACAGGCCTCCC AGTCTACCCTCACTAGAAAACAACACCCAGGCACTTTCCACC ACCTCTCAAAGGTGAAACCCAAGGCTGGTCTAGAGAATGAA TTATGGATCCTCGCTGTCCGTGCCACCCAGCTAGTCCCAGC GGCTCAGACACTG 32 SEQ ID NO: 28 fused to TTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAG SEQ ID NO: 29 CTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCC CTGGGCATCCGCTGCCATGGTGACTTTAAAGCCAGGTTGCT GCCCCAGCACAGGCCTCCCAGTCTACCCTCACTAGAAAACA ACACCCAGGCACTTTCCACCACCTCTCAAAGGTGAAACCCA AGGCTGGTCTAGAGAATGAATTATGGATCCTCGCTGTCCGT GCCACCCAGCTAGTCCCAGCGGCTCAGACACTG 33 SEQ ID NO: 28 fused to TTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAG SEQ ID NO: 27, which is CTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCC fused to SEQ ID NO: 29 CTGGGCATCCGCCTGCAGCTCAGCCTACTACTTGCTTTCCA GGCTGTTCCTAGTTCCCATGTCAGCTGCTTGTGCTTTCCAGA GACAAAACAGGAATAATAGATGTCATTAAATATACATTGGGC CCCAGGCGGTCAATGTGGCAGCCTGAGCCTCCTTTCCATCT CTGTGGAGGCAGACATAGGACCCCCAACAAACAGCATGCAG GTTGGGAGCCAGCCACAGGACCCAGGTAAGGGGCCCTGGG TCCTTTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAG CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG TCTAGAGAATGAATTATGGATCCTCGCTGTCCGTGCCACCC AGCTAGTCCCAGCGGCTCAGACACTG 34 SEQ ID NO: 29 fused to TGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAGCACAG SEQ ID NO: 27, which is GCCTCCCAGTCTACCCTCACTAGAAAACAACACCCAGGCAC fused to SEQ ID NO: 28 TTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGGTCTAG AGAATGAATTATGGATCCTCGCTGTCCGTGCCACCCAGCTA GTCCCAGCGGCTCAGACACTGCTGCAGCTCAGCCTACTACT TGCTTTCCAGGCTGTTCCTAGTTCCCATGTCAGCTGCTTGTG CTTTCCAGAGACAAAACAGGAATAATAGATGTCATTAAATAT ACATTGGGCCCCAGGCGGTCAATGTGGCAGCCTGAGCCTC CTTTCCATCTCTGTGGAGGCAGACATAGGACCCCCAACAAA CAGCATGCAGGTTGGGAGCCAGCCACAGGACCCAGGTAAG GGGCCCTGGGTCCTTTTTATGAGGTGGGAGCTGGGCTCTCC CTGATGTATTATTCAGCTCCCTGGAGTTGGCCAGCTCCTGTT ACACTGGCCACAGCCCTGGGCATCCGC 35 SEQ ID NO: 28 fused to TTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCAG SEQ ID NO: 29, which is CTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGCC fused to SEQ ID NO: 27 CTGGGCATCCGCTGCCATGGTGACTTTAAAGCCAGGTTGCT GCCCCAGCACAGGCCTCCCAGTCTACCCTCACTAGAAAACA ACACCCAGGCACTTTCCACCACCTCTCAAAGGTGAAACCCA AGGCTGGTCTAGAGAATGAATTATGGATCCTCGCTGTCCGT GCCACCCAGCTAGTCCCAGCGGCTCAGACACTGCTGCAGC TCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGTTCCCATG TCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAATAATAGA TGTCATTAAATATACATTGGGCCCCAGGCGGTCAATGTGGC AGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGACATAGG ACCCCCAACAAACAGCATGCAGGTTGGGAGCCAGCCACAG GACCCAGGTAAGGGGCCCTGGGTCCTT 36 Portion of SEQ ID NO: 1 TGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGTT that contains SEQ ID CCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAA NO: 3 and SEQ ID NO: TAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCAA 4 fused to portion of TGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGA SEQ ID NO: 2 that CATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG contains SEQ ID NO: 8 CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTAAGCTTCT and SEQ ID NO: 9 GCCACTGGCTCCGGCATTGCAGAGAGAAGAGAAGGGGCGG CAGACTGGAGAGCTGGGCTCCATTTTTGTTCCTTGGTGCCC TGCCCCTCCCCATGACCTGCAGAGACATTCAGCCTGCCAGG CTTTATGAGGTGGGAGCTGGGCTCTCCCTGATGTATTATTCA GCTCCCTGGAGTTGGCCAGCTCCTGTTACACTGGCCACAGC CCTGGGCATCCGCTTCTCACTTCTAGTTTCCCCTCCAAGGTA ATGTGGTGGGTCATGATCATTCTATCCTGGCTTCAGGGACC TGACTCCACTTTGGGGCCATTCGAGGGGTCTAGGGTAGATG ATGTCCCCCTGTGGGGATTAATGTCCTGCTCTGTAAAACTGA GCTAGCTGAGATCCAGGAGGGCTTGGCCAGAGACAGCAAG TTGTTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAGC ACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCAG GCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGGT CTAGAGAATGAATTATGGATCCTCGCTGTCCGTGCCACCCA GCTAGTCCCAGCGGCTCAGACACTGAGGAGAGACTGTAGG TTCAGCTACAAGCAAAAAGACCTAGCTGGTCTCCAAGCAGT GTCTCCAAGTCCCTGAACCTGTGACACCTGCCCCAGGCATC ATCAGGCACAGAGGGCCACC 37 Portion of SEQ ID NO: 1 TGCAGCTCAGCCTACTACTTGCTTTCCAGGCTGTTCCTAGTT that contains SEQ ID CCCATGTCAGCTGCTTGTGCTTTCCAGAGACAAAACAGGAA NO: 3 and SEQ ID NO: TAATAGATGTCATTAAATATACATTGGGCCCCAGGCGGTCAA 4 fused to portion of TGTGGCAGCCTGAGCCTCCTTTCCATCTCTGTGGAGGCAGA SEQ ID NO: 2 that CATAGGACCCCCAACAAACAGCATGCAGGTTGGGAGCCAG contains SEQ ID NO: 8 CCACAGGACCCAGGTAAGGGGCCCTGGGTCCTTTTTATGAG and SEQ ID NO: 9 GTGGGAGCTGGGCTCTCCCTGATGTATTATTCAGCTCCCTG GAGTTGGCCAGCTCCTGTTACACTGGCCACAGCCCTGGGCA TCCGCTGCCATGGTGACTTTAAAGCCAGGTTGCTGCCCCAG CACAGGCCTCCCAGTCTACCCTCACTAGAAAACAACACCCA GGCACTTTCCACCACCTCTCAAAGGTGAAACCCAAGGCTGG TCTAGAGAATGAATTATGGATCCTCGCTGTCCGTGCCACCC AGCTAGTCCCAGCGGCTCAGACACTG

[0135] Additional polynucleotides useful in conjunction with the compositions and methods described herein include nucleic acid molecules that have at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to the nucleic acid sequences set forth in Table 2 as well as functional portions or derivatives of the nucleic acid sequences set forth in Table 2.

[0136] The foregoing polynucleotides can be included in a nucleic acid vector and operably linked to a transgene to express the transgene specifically in hair cells (e.g., cochlear hair cells and/or vestibular hair cells). In some embodiments, the transgene encodes a protein that is implicated in hair cell function, hair cell development, hair cell fate specification, hair cell regeneration, hair cell survival, or hair cell maintenance, or the transgene is the wild-type version of a gene that has been found to be mutated in subjects having hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction (e.g., vertigo, dizziness, or loss of balance). According to the methods described herein, a subject can be administered a composition containing one or more of the foregoing polynucleotides (e.g., one or more of the polynucleotides listed in Table 2) operably linked to a transgene encoding a therapeutic protein for the treatment of hearing loss and/or vestibular dysfunction. In some embodiments, the transgene encodes a protein selected from the group consisting of Actin Gamma 1 (ACTG1), Fascin Actin-Bundling Protein 2, Retinal (FSCN2), Radixin (RDX), POU Class 4 Homeobox 3 (POU4F3), TRIO and F-Actin Binding Protein (TRIOBP), Taperin (TPRN), Xin Actin Binding Repeat Containing 2 (XIRP2), Atonal BHLH Transcription Factor 1 (ATOH1), Growth Factor Independent 1 Transcriptional Repressor (GFI1), Cholinergic Receptor Nicotinic Alpha 9 Subunit (CHRNA9), Calcium and Integrin Binding Family Member 3 (CIB3), Cadherin 23 (CDH23), Protocadherin 15 (PCDH15), Kinocilin (KNCN), Pejvakin (DFNB59), Otoferlin (OTOF), MKRN2 Opposite Strand (MKRN2OS), LIM Homeobox Protein 3 (LHX3), Transmembrane Channel Like 1 (TMC1), Myosin 15 (MYO15), Myosin 7A (MYO7A), Myosin 6 (MYO6), Myosin IIIA (MYO3A), Myosin IIIB (MYO3B), Glutaredoxin Domain Containing Cysteine-Rich Protein 1 (GRXCR1), Protein Tyrosine Phosphatase, Receptor Type Q (PTPRQ), Late Cornified Envelope 6A (LCE6A), Lipoxygenase Homology Domain-containing Protein 1 (LOXHD1), ADP-Ribosyltransferase 1 (ART1), ATPase Plasma Membrane Ca2+ Transporting 2 (ATP2B2), Calcium and Integrin Binding Family Member 2 (CIB2), Calcium Voltage-Gated Channel Auxiliary Subunit Alpha2delta 4(CACNA2D4), Calcium Binding Protein 2 (CABP2), Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8), EPS8 Like 2 (EPS8L2), Espin (ESPN), Espin Like (ESPNL), Peripherin 2 (PRPH2), Stereocilin (STRC), Solute Carrier Family 8 Member A2 (SLC8A2), Zinc Finger CCHC-Type Containing Protein 12 (ZCCHC12), Leucine Rich Transmembrane and O-methyltransferase Domain Containing (LRTOMT2, LRTOMT1), USH1 Protein Network Component Harmonin (USH1 C), Extracellular Leucine Rich Repeat and Fibronectin Type III Domain Containing 1 (ELFN1), Tetratricopeptide Repeat Protein 24 (TTC24), Dystrotelin (DYTN), Kielin/Chordin-Like Protein (KCP), Coiled-coil Glutamate Rich Protein 2 (CCER2), Leucine-rich Repeat and Transmembrane Domain-containing protein 2 (LRTM2), Potassium Voltage-Gated Channel Subfamily A Member 10 (KCNA10), Neurotrophin 3 (NTF3), Clarin 1 (CLRN1), Clarin 2 (CLRN2), SKI Family Transcriptional Corepressor 1 (SKOR1), Tctexl Domain Containing Protein 1 (TCTEX1 D1), Fc Receptor Like B (FCRLB), Solute Carrier Family 17 Member 8 (SLC17A8), Glutaredoxin Domain Containing Cysteine-Rich Protein 2 (GRXCR2), Brain-derived Neurotrophic Factor (BDNF), Serpin Family E Member 3 (SERPINE3), Nescient Helix-loop Helix 1 (NHLH1), Heat Shock Protein 70 (HSP70), Heat Shock Protein 90 (HSP90), Activating Transcription Factor 6 (ATF6), Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (PERK), Serine/Threonine-Protein Kinase/Endoribonuclease IRE1 (IRE1), and Binding Immunoglobulin Protein (BIP).

Expression of Exogenous Nucleic Acids in Mammalian Cells

[0137] Mutations in a variety of genes, such as MYO7A, POU4F3, SLC17A8, and TMC1, have been linked to sensorineural hearing loss, and some of these mutations, such as mutations in MYO7A, are also associated with vestibular dysfunction. The compositions and methods described herein can be used to induce or increase the expression of proteins encoded by genes of interest (e.g., the wild-type form of genes implicated in hearing loss and/or vestibular dysfunction, or genes involved in hair cell development, function, cell fate specification, regeneration, survival, or maintenance) specifically in hair cells (e.g., cochlear and/or vestibular hair cells) by administering a nucleic acid vector that contains a Myo15 promoter (e.g., a polynucleotide that contains a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, optionally containing a linker joining the first region and the second region) operably linked to a nucleic acid sequence that encodes a protein of interest. A wide array of methods has been established for the delivery of proteins to mammalian cells and for the stable expression of genes encoding proteins in mammalian cells.

[0138] Proteins that can be expressed in connection with the compositions described herein (e.g., when the transgene encoding the protein is operably linked to a polynucleotide that contains a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof) are proteins that are expressed in healthy hair cells (e.g., cochlear and/or vestibular hair cells, e.g., proteins that play a role in hair cell development, function, regeneration, cell fate specification, survival, or maintenance, or proteins that are deficient in subjects with sensorineural hearing loss or vestibular dysfunction) or other therapeutic proteins of interest. Proteins that can be expressed in hair cells using the compositions and methods described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

Polynucleotides Encoding Proteins of Interest

[0139] One platform that can be used to achieve therapeutically effective intracellular concentrations of proteins of interest in mammalian cells is via the stable expression of the gene encoding the protein of interest (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell, or by episomal concatemer formation in the nucleus of a mammalian cell). The gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. In order to introduce exogenous genes into a mammalian cell, genes can be incorporated into a vector. Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposome. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described in more detail, for example, in Green, et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel, et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0140] Proteins of interest can also be introduced into a mammalian cell by targeting a vector containing a gene encoding a protein of interest to cell membrane phospholipids. For example, vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Such a construct can be produced using methods well known to those of skill in the field.

[0141] Recognition and binding of the polynucleotide encoding a protein of interest by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site. Such sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Examples of mammalian promoters have been described in Smith, et al., Mol. Sys. Biol., 3:73, online publication, the disclosure of which is incorporated herein by reference. The promoter used in the methods and compositions described herein is a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region.

[0142] Once a polynucleotide encoding a protein of interest has been incorporated into the nuclear DNA of a mammalian cell, the transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of a transcription factor and/or RNA polymerase to the mammalian promoter and thus regulates gene expression. The chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, e.g., by removing a repressor protein that has bound the promoter. Alternatively, the chemical reagent can serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of the gene located downstream of the promoter is increased in the presence of the chemical reagent. Examples of chemical reagents that potentiate polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to a mammalian cell in order to promote gene expression according to established protocols.

[0143] Other DNA sequence elements that may be included in polynucleotides for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide comprising the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Thus, polynucleotides for use in the compositions and methods described herein include those that encode a protein of interest and additionally include a mammalian enhancer sequence. Many enhancer sequences are now known from mammalian genes, and examples include enhancers from the genes that encode mammalian globin, elastase, albumin, α-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and RSV enhancer. An enhancer may be spliced into a vector containing a polynucleotide encoding a protein of interest, for example, at a position 5′ or 3′ to this gene. In a preferred orientation, the enhancer is positioned at the 5′ side of the promoter, which in turn is located 5′ relative to the polynucleotide encoding a protein of interest.

[0144] The nucleic acid vectors containing a Myo15 promoter described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cell. The addition of the WPRE to a vector can result in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo.

[0145] In some embodiments, the nucleic acid vectors containing a Myo15 promoter described herein include a reporter sequence, which can be useful in verifying the expression of a gene operably linked to a Myo15 promoter, for example, in cells and tissues (e.g., in hair cells, such as cochlear and/or vestibular hair cells). Reporter sequences that may be provided in a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with regulatory elements that drive their expression, such as a Myo15 promoter, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.

Methods for the Delivery of Exogenous Nucleic Acids to Target Cells

[0146] Techniques that can be used to introduce a transgene, such as a transgene operably linked to a Myo15 promoter described herein, into a target cell (e.g., a mammalian cell) are well known in the art. For instance, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.

[0147] Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0148] Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for instance, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for instance, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane include activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for instance, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for instance, in US 2010/0227406, the disclosure of which is incorporated herein by reference.

[0149] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

[0150] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0151] Magnetofection can also be used to deliver nucleic acids to target cells. The magnetofection principle is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0152] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0153] Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For instance, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

Vectors for Delivery of Exogenous Nucleic Acids to Target Cells

[0154] In addition to achieving high rates of transcription and translation, stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide comprising the gene into the nuclear genome of the mammalian cell. A variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Examples of expression vectors are described in, e.g., Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, M A, 2006). Expression vectors for use in the compositions and methods described herein contain a Myo15 promoter (e.g., a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region) operably linked to a polynucleotide sequence that encodes a protein of interest, as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Vectors that can contain a Myo15 promoter operably linked to a transgene encoding a protein of interest include plasmids (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmids (e.g., pWE or sCos vectors), artificial chromosomes (e.g., a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC)), and viral vectors. Certain vectors that can be used for the expression of a protein of interest include plasmids that contain regulatory sequences, such as enhancer regions, which direct gene transcription. Other useful vectors for expression of a protein of interest contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

Viral Vectors for Nucleic Acid Delivery

[0155] Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

AAV Vectors for Nucleic Acid Delivery

[0156] In some embodiments, polynucleotides of the compositions and methods described herein are incorporated into rAAV vectors and/or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a Myo15 promoter described herein (e.g., a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region), (2) a heterologous sequence to be expressed, and (3) viral sequences that facilitate stability and expression of the heterologous genes. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. In typical applications, the transgene encodes a therapeutic protein that can promote hair cell development, hair cell function, hair cell regeneration, hair cell fate specification, hair cell survival, or hair cell maintenance, or a wild-type form of a hair cell protein that is mutated in subjects with forms of hereditary hearing loss or vestibular dysfunction that may be useful for improving hearing or vestibular function in subjects carrying mutations that have been associated with hearing loss, deafness, or vestibular dysfunction (e.g., dizziness, vertigo, or imbalance). Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs can be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0157] The polynucleotides and vectors described herein (e.g., a Myo15 promoter operably linked to a transgene encoding a protein of interest) can be incorporated into a rAAV virion in order to facilitate introduction of the polynucleotide or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, for instance, in U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0158] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, and PHP.S. For targeting hair cells, AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV9, Anc80, Anc80L65, DJ/9, 7m8, and PHP.B may be particularly useful. Serotypes evolved for transduction of the retina may also be used in the methods and compositions described herein. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for instance, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0159] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0160] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

Pharmaceutical Compositions

[0161] The polynucleotides described herein (e.g., a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region) may be operably linked to a transgene (e.g., a transgene encoding a protein of interest) and incorporated into a vehicle for administration into a patient, such as a human patient suffering from sensorineural hearing loss and/or vestibular dysfunction. Pharmaceutical compositions containing vectors, such as viral vectors, that contain a polynucleotide described herein operably linked to a therapeutic transgene can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

[0162] Mixtures of nucleic acid vectors (e.g., viral vectors) containing a polynucleotide described herein (e.g., a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region) operably linked to a therapeutic transgene may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in U.S. Pat. No. 5,466,468, the disclosure of which is incorporated herein by reference). In any case the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0163] For example, a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. For local administration to the inner ear, the composition may be formulated to contain a synthetic perilymph solution. An exemplary synthetic perilymph solution includes 20-200 mM NaCl, 1-5 mM KCl, 0.1-10 mM CaCl.sub.2), 1-10 mM glucose, and 2-50 mM HEPEs, with a pH between about 6 and 9 and an osmolality of about 300 mOsm/kg. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

Methods of Treatment

[0164] The compositions described herein may be administered to a subject with sensorineural hearing loss and/or vestibular dysfunction by a variety of routes, such as local administration to the inner ear (e.g., administration into the perilymph or endolymph, e.g., through the oval window, round window, or a semicircular canal (e.g., the horizontal canal), e.g., administration to a cochlear or vestibular hair cell), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route for administration in any given case will depend on the particular composition administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patients age, body weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. Compositions may be administered once, or more than once (e.g., once annually, twice annually, three times annually, bi-monthly, or monthly).

[0165] Subjects that may be treated as described herein are subjects having or at risk of developing sensorineural hearing loss and/or vestibular dysfunction (e.g., subjects having or at risk of developing hearing loss, vestibular dysfunction, or both). The compositions and methods described herein can be used to treat subjects having or at risk of developing damage to cochlear hair cells (e.g., damage related to acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging), subjects having or at risk of developing damage to vestibular hair cells (e.g., damage related to disease or infection, head trauma, ototoxic drugs, or aging), subjects having or at risk of developing sensorineural hearing loss, deafness, or auditory neuropathy, subjects having or at risk of developing vestibular dysfunction (e.g., dizziness, vertigo, or imbalance), subjects having tinnitus (e.g., tinnitus alone, or tinnitus that is associated with sensorineural hearing loss or vestibular dysfunction), subjects having a genetic mutation associated with hearing loss and/or vestibular dysfunction, or subjects with a family history of hereditary hearing loss, deafness, auditory neuropathy, tinnitus, or vestibular dysfunction. In some embodiments, the subject has hearing loss and/or vestibular dysfunction that is associated with or results from loss of hair cells (e.g., cochlear or vestibular hair cells). The methods described herein may include a step of screening a subject for mutations in genes known to be associated with hearing loss or vestibular dysfunction prior to treatment with or administration of the compositions described herein. A subject can be screened for a genetic mutation using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein may also include a step of assessing hearing and/or vestibular function in a subject prior to treatment with or administration of the compositions described herein. Hearing can be assessed using standard tests, such as audiometry, auditory brainstem response (ABR), electrocochleography (ECOG), and otoacoustic emissions. Vestibular function may be assessed using standard tests, such as eye movement testing (e.g., electronystagmogram (ENG) or videonystagmogram (VNG)), posturography, rotary-chair testing, ECOG, vestibular evoked myogenic potentials (VEMP), and specialized clinical balance tests, such as those described in Mancini and Horak, Eur J Phys Rehabil Med, 46:239 (2010). The compositions and methods described herein may also be administered as a preventative treatment to patients at risk of developing hearing loss and/or vestibular dysfunction, e.g., patients who have a family history of hearing loss or vestibular dysfunction (e.g., inherited hearing loss or vestibular dysfunction), patients carrying a genetic mutation associated with hearing loss or vestibular dysfunction who do not yet exhibit hearing impairment or vestibular dysfunction or patients exposed to risk factors for acquired hearing loss (e.g., disease or infection, head trauma, ototoxic drugs, or aging) or vestibular dysfunction (e.g., acoustic trauma, disease or infection, head trauma, ototoxic drugs, or aging).

[0166] The compositions and methods described herein can be used to promote or induce hair cell regeneration in a subject (e.g., cochlear and/or vestibular hair cell regeneration). Subjects that may benefit from compositions that promote or induce hair cell regeneration include subjects suffering from hearing loss or vestibular dysfunction as a result of loss of hair cells (e.g., loss of hair cells related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), and subjects with abnormal hair cells (e.g., hair cells that do not function properly when compared to normal hair cells), damaged hair cells (e.g., hair cell damage related to trauma (e.g., acoustic trauma or head trauma), disease or infection, ototoxic drugs, or aging), or reduced hair cell numbers due to genetic mutations or congenital abnormalities. The compositions and methods described herein can also be used to promote or increase hair cell survival (e.g., increase survival of damaged hair cells, promote repair of damaged hair cells, or preserve hair cells in a subject at risk of loss of hair cells (e.g., loss of hair cells due to age, exposure to loud noise, disease or infection, head trauma or ototoxic drugs)).

[0167] The compositions and methods described herein can also be used to prevent or reduce ototoxic drug-induced hair cell damage or death (e.g., cochlear and/or vestibular hair cell damage or death) in subjects who have been treated with ototoxic drugs, or who are currently undergoing or soon to begin treatment with ototoxic drugs. Ototoxic drugs are toxic to the cells of the inner ear, and can cause sensorineural hearing loss, vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), tinnitus, or a combination of these symptoms. Drugs that have been found to be ototoxic include aminoglycoside antibiotics (e.g., gentamycin, neomycin, streptomycin, tobramycin, kanamycin, vancomycin, and amikacin), viomycin, antineoplastic drugs (e.g., platinum-containing chemotherapeutic agents, such as cisplatin, carboplatin, and oxaliplatin), loop diuretics (e.g., ethacrynic acid and furosemide), salicylates (e.g., aspirin, particularly at high doses), and quinine. In some embodiments, the methods described herein prevent or reduce hair cell damage or death related to acoustic trauma, disease or infection, head trauma, or aging.

[0168] The transgene operably linked to a Myo15 promoter (e.g., a polynucleotide that contains a first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof) for treatment of a subject as described herein can be a transgene that encodes a protein expressed in healthy hair cells (e.g., cochlear and/or vestibular hair cells, e.g., a protein that plays a role in hair cell development, function, cell fate specification, regeneration, survival, or maintenance, or a protein that is deficient in a subject with sensorineural hearing loss and/or vestibular dysfunction) or another therapeutic protein of interest. The transgene may be selected based on the cause of the subject's hearing loss or vestibular dysfunction (e.g., if the subject's hearing loss or vestibular dysfunction is associated with a particular genetic mutation, the transgene can be a wild-type form of the gene that is mutated in the subject, or if the subject has hearing loss associated with loss of hair cells, the transgene can encode a protein that promotes hair cell regeneration), the severity of the subject's hearing loss or vestibular dysfunction, the health of the subject's hair cells, the subject's age, the subject's family history of hearing loss or vestibular dysfunction, or other factors. The proteins that may be expressed by a transgene operably linked to a Myo15 promoter for treatment of a subject as described herein include ACTG1, FSCN2, RDX, POU4F3, TRIOBP, TPRN, XIRP2, ATOH1, GFI1, CHRNA9, CIB3, CDH23, PCDH15, KNCN, DFNB59, OTOF, MKRN2OS, LHX3, TMC1, MYO15, MYO7A, MYO6, MYO3A, MYO3B, GRXCR1, PTPRQ, LCE6A, LOXHD1, ART1, ATP2B2, CIB2, CACNA2D4, CABP2, EPS8, EPS8L2, ESPN, ESPNL, PRPH2, STRC, SLC8A2, ZCCHC12, LRTOMT2, LRTOMT1, USH1C, ELFN1, TTC24, DYTN, KCP, CCER2, LRTM2, KCNA10, NTF3, CLRN1, CLRN2, SKOR1, TCTEX1 D1, FCRLB, SLC17A8, GRXCR2, BDNF, SERPINE3, NHLH1, HSP70, HSP90, ATF6, PERK, IRE1, and BIP.

[0169] Treatment may include administration of a composition containing the nucleic acid vectors (e.g., AAV viral vectors) containing a Myo15 promoter described herein in various unit doses. Each unit dose will ordinarily contain a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route of administration and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Dosing may be performed using a syringe pump to control infusion rate in order to minimize damage to the inner ear (e.g., the cochlea). In cases in which the nucleic acid vectors are AAV vectors (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, or PHP.S vectors), the viral vectors may be administered to the patient at a dose of, for example, from about 1×10.sup.10 vector genomes (VG) to 1×10.sup.15 VG (e.g., 1×10.sup.10VG, 2×10.sup.10VG, 3×10.sup.10VG, 4×10.sup.10VG, 5×10.sup.10 VG, 6×10.sup.10VG, 7×10.sup.10VG, 8×10.sup.10VG, 9×10.sup.10VG, 1×10.sup.11 VG, 2×10.sup.11 VG, 3×10.sup.11 VG, 4×10.sup.11 VG, 5×10.sup.11 VG, 6×10.sup.11 VG, 7×10.sup.11 VG, 8×10.sup.11 VG, 9×10.sup.11 VG, 1×10.sup.12VG, 2×10.sup.12VG, 3×10.sup.12 VG, 4×10.sup.12 VG, 5×10.sup.12 VG, 6×10.sup.12 VG, 7×10.sup.12 VG, 8×10.sup.12 VG, 9×10.sup.12 VG, 1×10.sup.13 VG, 2×10.sup.13 VG, 3×10.sup.13 VG, 4×10.sup.13 VG, 5×10.sup.13 VG, 6×10.sup.13 VG, 7×10.sup.13 VG, 8×10.sup.13 VG, 9×10.sup.13 VG, 1×10.sup.14 VG, 2×10.sup.14 VG, 3×10.sup.14 VG, 4×10.sup.14 VG, 5×10.sup.14 VG, 6×10.sup.14 VG, 7×10.sup.14 VG, 8×10.sup.14 VG, 9×10.sup.14 VG, 1×10.sup.15 VG) in a volume of 1 μL to 200 μL (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL).

[0170] The compositions described herein are administered in an amount sufficient to improve hearing, improve vestibular function (e.g., improve balance or reduce dizziness or vertigo), reduce tinnitus, increase expression of a therapeutic protein encoded by a transgene, increase function of a therapeutic protein encoded by a transgene, prevent or reduce hair cell damage, prevent or reduce hair cell death (e.g., ototoxic drug-induced hair cell death, age-related hair cell death, or noise (e.g., acoustic trauma)-related hair cell death), promote or increase hair cell development, increase hair cell numbers (e.g., promote or induce hair cell regeneration), increase or promote hair cell survival, or improve hair cell function. Hearing may be evaluated using standard hearing tests (e.g., audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hearing measurements obtained prior to treatment. Vestibular function may be evaluated using standard tests for balance and vertigo (e.g., eye movement testing (e.g., ENG or VNG), posturography, rotary-chair testing, ECOG, VEMP, and specialized clinical balance tests) and may be improved by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to measurements obtained prior to treatment. In some embodiments, the compositions are administered in an amount sufficient to improve the subject's ability to understand speech. The compositions described herein may also be administered in an amount sufficient to slow or prevent the development or progression of sensorineural hearing loss and/or vestibular dysfunction (e.g., in subjects who carry a genetic mutation associated with hearing loss or vestibular dysfunction, who have a family history of hearing loss or vestibular dysfunction (e.g., hereditary hearing loss or vestibular dysfunction), or who have been exposed to risk factors associated with hearing loss or vestibular dysfunction (e.g., ototoxic drugs, head trauma, acoustic trauma, or infection) but do not exhibit hearing impairment or vestibular dysfunction (e.g., vertigo, dizziness, or imbalance), or in subjects exhibiting mild to moderate hearing loss or vestibular dysfunction). Expression of the therapeutic protein encoded by the transgene operably linked to a Myo15 promoter in the nucleic acid vector administered to the subject may be evaluated using immunohistochemistry, Western blot analysis, quantitative real-time PCR, or other methods known in the art for detection protein or mRNA, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to expression prior to administration of the compositions described herein. Hair cell numbers, hair cell function, or function of the therapeutic protein encoded by the nucleic acid vector administered to the subject may be evaluated indirectly based on hearing tests or tests of vestibular function, and may be increased by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hair cell numbers, hair cell function, or function of the therapeutic protein prior to administration of the compositions described herein. Hair cell damage or death may be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more) compared to hair cell damage and death typically observed in untreated subjects. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more, following administration of the compositions described herein. The patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the composition depending on the dose and route of administration used for treatment. Depending on the outcome of the evaluation, the patient may receive additional treatments.

Kits

[0171] The compositions described herein can be provided in a kit for use in treating sensorineural hearing loss or vestibular dysfunction. Compositions may include a polynucleotide described herein (e.g., a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof, that optionally contains a linker between the first region and the second region), nucleic acid vectors containing such polynucleotides, and nucleic acid vectors containing a polynucleotide described herein operably linked to a transgene encoding a protein of interest (e.g., a protein that can be expressed in hair cells to treat hearing loss and/or vestibular dysfunction). The nucleic acid vectors may be packaged in an AAV virus capsid (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV9, Anc80, Anc80L65, DJ/9, 7m8, or PHP.B). The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the composition.

EXAMPLES

[0172] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: Generation of the Murine Myo15 Promoter

[0173] Regions of evolutionary conservation in the vertebrate Myo15 promoter were first identified using the UCSC Genome Browser (genome.ucsc.edu). The region immediately upstream of the Myo15 translation start site (−1 to −1157, SEQ ID NO: 1) and an upstream region containing non-coding exon1 of the Myo15 gene (−6755 to −7209, SEQ ID NO: 2) were synthesized and joined together as a single DNA fragment by de novo gene synthesis (SEQ ID NO: 13). The total size of the truncated Myo15 promoter is 1611 bp versus more than 7000 bp for the entire genomic region.

[0174] Experiments evaluating tropism (cell type targeting) and the extent and duration of transgene expression by the Myo15 promoter relative to the cytomegalovirus (CMV) promoter in mouse cochlea resulted in the Myo15 promoter, but not the CMV promoter, yielding selective expression in cochlear hair cells. An AAV construct was created with Myo15 driving expression of Aequorea coerulescens green fluorescent protein (AcGFP) to analyze the progression of gene expression relative to the matched standard AAV construct with CMV. Transgene expression was evaluated in experiments in which virus was delivered to the mouse cochlea in neonatal mice, in adult mice, and ex vivo in cochlear explants.

[0175] To evaluate transgene expression, AAV-Myo15-GFP virus was infused via posterior semi-circular canal to 6-8-week-old C57Bl/6J male mice. Mice recovered from surgery and were euthanized and perfused with 10% normal buffered formalin 10 days later. The inner ear temporal bone was harvested and decalcified in 8% EDTA for 3 days. The cochlea or vestibular system were dissected from the de-calcified temporal bone and mounted on a slide for imaging. Using a ubiquitous promoter, AAV-CMV-GFP induced GFP expression in many cell types within the cochlea including inner hair cells, outer hair cells, spiral ganglion neurons, mesenchymal cells, and glia (FIG. 1A). Using the hair cell-specific promoter, AAV-Myo15-GFP induced expression exclusively in the inner and outer hair cells (FIG. 1B). In the vestibular system, AAV-Myo15-GFP induced expression exclusively in vestibular hair cells (FIG. 2).

Example 2: Generation of a Minimal Myo15 Promoter

[0176] A series of promoters are generated and placed upstream of a fluorescent reporter (e.g., GFP, AcGFP, or luciferase). Promoters that are generated include:

1) SEQ ID NO: 3 fused to SEQ ID NO: 2;
2) SEQ ID NO: 4 fused to SEQ ID NO: 2;
3) A fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6, or 7) fused to SEQ ID NO: 2;
4) SEQ ID NO: 8 fused to SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);
5) SEQ ID NO: 1 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11 or 12);
6) SEQ ID NO: 3 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);
7) SEQ ID NO: 4 fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);
8) A fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6, or, 7) fused to a fusion of SEQ ID NO: 8 and SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12);
9) A fusion of SEQ ID NO: 3 and SEQ ID NO: 4 (e.g., SEQ ID NO: 5, 6, or 7);

11) SEQ ID NO: 1;

12) SEQ ID NO: 2;

13) SEQ ID NO: 17;

14) SEQ ID NO: 18;

[0177] 15) SEQ ID NO: 17 fused to SEQ ID NO: 18;
16) SEQ ID NO: 18 fused to SEQ ID NO: 17;
17) SEQ ID NO: 19 fused to SEQ ID NO: 18;
18) SEQ ID NO: 20 fused to SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24);
19) SEQ ID NO: 17 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24); and
20) SEQ ID NO: 19 fused to a fusion of SEQ ID NO: 20 and SEQ ID NO: 21 (e.g., SEQ ID NO: 22, 23, or 24).The promoter constructs are packaged into an AAV serotype capable of transducing hair cells (e.g., AAV1, AAV2, AAV6, AAV9, Anc80, or Anc80L65).

[0178] Viral promoter constructs are used to infect organotypic cochlear explants. After 48 hours of incubation with virus, explants are imaged and analyzed using fluorescence intensity of the reporter to gauge hair cell-specific expression.

Example 3: Administration of a Composition Containing a Nucleic Acid Vector Containing a Myo15 Promoter to a Subject with Sensorineural Hearing Loss

[0179] According to the methods disclosed herein, a physician of skill in the art can treat a patient, such as a human patient, with sensorineural hearing loss so as to improve or restore hearing. To this end, a physician of skill in the art can administer to the human patient a composition containing an AAV vector (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV9, Anc80, Anc80L65, DJ/9, 7m8, or PHP.B) containing a polynucleotide that contains first region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 1 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 3-7, e.g., SEQ ID NOs 3 and 4) and/or a second region having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity) to SEQ ID NO: 2 or a functional portion or derivative thereof (e.g., any one or more of SEQ ID NOs: 8-12, e.g., SEQ ID NOs: 8 and 9), that optionally contains a linker between the first region and the second region, operably linked to a transgene that encodes a therapeutic protein. For example, the polynucleotide operably linked to the transgene that encodes a therapeutic protein may be SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 36, or SEQ ID NO: 37. The composition containing the AAV vector may be administered to the patient, for example, by local administration to the inner ear (e.g., injection into the perilymph), to treat sensorineural hearing loss.

[0180] Following administration of the composition to a patient, a practitioner of skill in the art can monitor the expression of the therapeutic protein encoded by the transgene, and the patient's improvement in response to the therapy, by a variety of methods. For example, a physician can monitor the patient's hearing by performing standard tests, such as audiometry, ABR, electrocochleography (ECOG), and otoacoustic emissions following administration of the composition. A finding that the patient exhibits improved hearing in one or more of the tests following administration of the composition compared to hearing test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

Example 4: Murine Myo15 Promoter Sequences Containing Portions of SEQ ID NO: 1 and SEQ ID NO: 2 Induce Transgene Expression in Hair Cells

[0181] Neonatal cochlear explants were established from P0 wildtype CD1 mice. Briefly, neonatal mice were euthanized according to IACUC approved protocols. Inner ears were removed from the head and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil and the roof of the cochlear duct was removed exposing the sensory epithelium. Cochlear explants were plated, lumenal side of the cochlear duct facing up, on Mat-tek dishes coated in 15% Matrigel. Cochlear explant culture media consisting of DMEM with 10% FBS and 10 ug/mL ciproflaxin was added directly to the culture. Virus containing the Myo15 1 kb (SEQ ID NO: 15) promoter or the Myo15 mini intron (SEQ ID NO: 16) promoter (AAV 2/9-Myo15 1 kb-GFP or AAV2/9-Myo15 mini intron-GFP) was added to the culture media at a concentration of 1E+10 genome copies/mL. Cultures were incubated in media containing virus for 48 hours in standard culture conditions of 5% CO.sub.2 at 37° C. Cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before antibody staining. Antibodies used: Rabbit anti-Myo6 (Proteus), Donkey anti-Rabbit Alexa 568 (Thermo Fisher). As shown in FIGS. 3A-3B, both the Myo15 1 kb (SEQ ID NO: 15) and Myo15 mini intron (SEQ ID NO: 16) promoters induced expression of GFP specifically in hair cells.

Example 5: Myo15 Promoter Sequences Induce Transgene Expression in Hair Cells in Murine Cochlea

[0182] Neonatal cochlear explants were established from P0 wildtype B6.CAST-Cdh23Al+/Kjn mice. Briefly, neonatal mice were euthanized according to IACUC approved protocols. Inner ears were removed from the head and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil and the roof of the cochlear duct was removed exposing the sensory epithelium. Cochlear explants were plated, lumenal side of the cochlear duct facing up, on prepared 3D rat tail collagen matrices. Cochlear explant culture media consisting of DMEM with 7% FBS and 1U/μL penicillin G was added directly to the culture. Virus containing the murine Myo15 1 kb (SEQ ID NO: 15) promoter, human Myo15 0.6 kb (SEQ ID NO: 26), or human Myo15 1.2 kb (SEQ ID NO: 25) promoter (AAV1-Myo15 1 kb-GFP, AAV1-Myo15 0.6 kb-GFP, or AAV1-Myo15 1.2 kb-GFP) was added to the culture media at a concentration of 1E+11 vg/culture. Cultures were incubated in media containing virus for 72 hours in standard culture conditions of 5% CO.sub.2 at 37° C. Cultures were then fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before anti-GFP antibody staining. GFP signal, as detected with anti-GFP antibody, is shown (FIGS. 4A-4C). In a separate experiment, AAV1-1.2 kb human Myo15-GFP or AAV1-1 kb murine Myo15-GFP was delivered to adult mouse cochlea via round window injection at a dose of 2.65E+12 vg/ml. Whole mount images (FIGS. 4D-4E) or sections (FIGS. 4F-4G) show hair cell-specific signal in the apex, middle, and base of the cochlea, as detected with anti-GFP antibody. Arrows in FIG. 4F point to GFP-labeled cochlear hair cells. As shown in FIGS. 4F and 4G, expression with both the 1.2 kb human Myo15 promoter and 1 kb murine Myo15 promoter was restricted primarily to inner hair cells.

Example 6: Murine Myo15 Promoter Sequences Induce Transgene Expression in Cochlea of Non-Human Primates In Vivo

[0183] Murine Myo15 promoter specificity was tested in non-human primates. Thirty microliters of AAV1-CMV-GFP or AAV1-Myo15 (SEQ ID NO: 13)-GFP were injected into the cochleas through the round window membrane at 15 μI/min. The animals were sacrificed four weeks after AAV injection, and the cochleas were harvested and processed as a surface preparation to examine transgene expression without antibody augmentation. AAV1 had high infectivity. Under the ubiquitous CMV promoter, GFP was expressed in hair cells, supporting cells, and fibrocytes in the lateral wall in the rhesus cochlea (FIG. 5A). In contrast, the 1.6 kb murine Myo15 promoter (SEQ ID NO: 13) restricted GFP transgene expression to hair cells in a cynomolgus cochlea (FIGS. 5B-5C).

Example 7: The Murine Myo15 Promoter Enhances Biological Efficacy of AAV-Mouse Tmc1 in Tmc1 Knockout Mice Compared to a Ubiquitous Promoter

[0184] Tmc1 knockout (KO) mice were anesthetized with isoflurane, hair clipped, and providone iodine was applied to the skin. An incision was made under the left ear from above the cheek muscle to behind the ear. Skin was separated and muscle teased and cleared to expose the posterior canal. A drill bit was used to make a small hole in the canal and bone was kept dry with fine cotton tips. A polyamide/polyethylene tubing was inserted into the hole and sealed with bone glue. A micropump with a Hamilton syringe was used to deliver 1 μl of vector (AAV-CMV1-mouse TMC1 or AAV-Myo15 (SEQ ID NO: 13)-mouse TMC1) with 0.05 μl of trypan blue into the IL space at 100 nl/min rate. After delivering 1 μl, 5 minutes were allowed to elapse for the fluid to reach the apex of the cochlea and to prevent back flow and leakage. The tube was bent and cut near the bone. Muscle was pulled back into place and skin was glued together. Mice were given 0.01 cc of Meloxicam before being allowed to recover under a heating lamp. Animals were checked for 5 days post-op for signs of pain or infection. Twenty-one to twenty-eight days post-op animals were anesthetized with ketamine and xylazine and the auditory brainstem response (ABR) was measured. As shown in FIG. 6, restoration of ABR thresholds was greatly improved in homozygous Tmc1 KO mice injected with AAV-Myo15-mouse TMC1 compared to AAV-CMV-mouse TMC1.

Example 8: Human Myo15 Promoter Sequences Induce Transgene Expression in Cochlea of Non-Human Primates In Vivo

[0185] A composition containing an AAV1 vector carrying an EGFP transgene under the control of the 1.2 kb human myosin 15 promoter (SEQ ID NO: 25) or the 0.6 kb human myosin 15 promoter (SEQ ID NO: 26) was delivered bilaterally into the inner ear of cynomolgus monkeys by way of injection through the round window membrane. Injections (30 μL) of AAV1-1.2 kb Myosin 15-GFP (viral titer at 1.06×10.sup.13 genome copies/mL) or AAV1-0.6 kb Myosin 15-GFP (viral titer at 1.22×10.sup.13 genome copies/mL) were performed at an injection rate of 15 μL/min. Four weeks post-injection, inner ears were removed, and a surface preparation of the basilar membranes was made. GFP expression was used to monitor Myosin 15 promoter activity (FIGS. 7A and 8A, one of the representative ears is shown). AAV1-1.2 kb Myosin 15-GFP transduction of the inner ear of cynomolgus monkeys resulted in GFP expression across the entire baso-apical axis of the cochlea with high frequency at base and low frequency at apex (FIG. 7A, frequencies at kHz). High magnification images at 2.8 kHz showed GFP expression was observed within inner hair cells (IHCs) and outer hair cells (OHC) (FIG. 7B upper panel). Similarly, AAV1-0.6 kb Myosin 15-GFP transduction of the inner ear of cynomolgus monkeys resulted in GFP expression across the entire baso-apical axis of the cochlea with high frequency at base and low frequency at apex (FIG. 8A, frequencies at kHz). High magnification images at 2.8 kHz showed GFP expression was observed within IHCs and OHCs (FIG. 8B upper panel). Immunohistochemistry for myosin 7A (Myo7A) was used to visualize hair cells and nuclei were stained with DAPI (FIG. 7B and FIG. 8B, middle and lower panels).

Example 9: Myo15 Promoter Variants Induce Transgene Expression in Murine Cochlear Explants

[0186] Neonatal Cochlear Explant Culture and Viral Transduction

[0187] Neonatal cochlear explants were established from P0 wildtype B6.CAST-Cdh23Ahl+/Kjn mice. Briefly, neonatal mice were euthanized according to Institutional Animal Care and Use Committee approved protocols. Inner ears were removed from the head and the cochlea was carefully removed using microdissection. The modiolus was removed from the center of the coil and the roof of the cochlear duct was removed exposing the sensory epithelium. Cochlear explants were plated, lumenal side of the cochlear duct facing up, on prepared 3D rat tail collagen matrices. Cochlear explant culture media consisting of DMEM with 7% FBS and 1 U/μL penicillin G was added directly to the culture. Virus containing the murine Myo15 1 kb (SEQ ID NO: 15) promoter, human Myo15 0.6 kb (SEQ ID NO: 26), human Myo15 1.2 kb (SEQ ID NO: 25) promoter, murine Myo15 0.5 kb promoter (SEQ ID NO: 16), murine Myo15 A promoter (SEQ ID NO: 27), murine Myo15 B promoter (SEQ ID NO: 28), murine Myo15 C promoter (SEQ ID NO: 29), murine Myo15 A-B promoter (SEQ ID NO: 30), murine Myo15 A-C promoter (SEQ ID NO: 31), murine Myo15 B-C promoter (SEQ ID NO: 32), murine Myo15 B-A-C promoter (SEQ ID NO: 33), murine Myo15 C-A-B promoter (SEQ ID NO: 34), or murine Myo15 B-C-A promoter (SEQ ID NO: 35; AAV1-Myo15 1 kb-GFP, AAV1-Myo15 0.6 kb-GFP, AAV1-Myo15 1.2 kb-GFP, AAV1-Myo15 0.5 kb-GFP, AAV1-Myo15 A-GFP, AAV1-Myo15 B-GFP) was added to the culture media at a concentration of 1×10.sup.11 vg/culture. Cultures were incubated in media containing virus for 72 hours in standard culture conditions of 5% CO2 at 37° C.

[0188] GFP Quantification

[0189] After 72 hours in culture with virus-containing media, cultures were fixed in 4% PFA, permeabilized with 0.01% TritonX100, and blocked with 10% normal donkey serum before anti-Myo7a and/or anti-GFP antibodies. Samples were imaged using confocal microscopy (Zeiss LSM 810) using uniform settings (laser intensity and gain) between samples. ImageJ was used to quantify GFP intensity values per cell.

[0190] mRNA Quantification

[0191] After 72 hours in culture with virus-containing media, cultures were snap frozen on dry ice and RNA was extracted using PicoPure RNA Isolation Kit (Arcturus cat #0204). GFP and Myo7A mRNA were quantified by qPCR using the Quantinova Probe RT-PCR Kit (Qiagen cat #208354) for all cDNA synthesis and RT-PCR reactions. GFP expression levels were normalized to Myo7a expression.

[0192] Results

[0193] Quantification of GFP expression from murine Myo15 promoters with hair cell-specific expression using quantitative PCR (qPCR) showed that expression levels of the GFP transgene from the mMyo15 1.6 kb, 1 kb, and 0.5 kb promoters were robust and comparable to each other in degree of GFP expression (FIG. 9). Comparison of GFP expression driven by a variety of murine or human Myo15 promoters showed differences in GFP expression in Myo7a-positive hair cells, with the mMyo15 1 kb promoter yielding the highest levels of GFP expression per cell (FIG. 10).

[0194] Analysis of whole mount images of neonatal cochlear explant cultures infected with a subset of the Myo15 promoter variants described in Table 2 demonstrated that the promoter sequence of SEQ ID NO: 27 (Myo15 A) was neither sufficient nor necessary for general promoter activity or hair cell specificity as shown in FIGS. 11B (SEQ ID NO: 27), 11E (SEQ ID NO: 30), 11F (SEQ ID NO: 31), and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 27 did provide a splice donor site that increased expression levels when paired with a splice acceptor site as shown in FIGS. 11A (SEQ ID NO: 16) and 11H (SEQ ID NO: 33), thus introducing an intron into the promoter sequence. The promoter sequence of SEQ ID NO: 28 (Myo15 B) alone was found to be a core promoter that conferred general promoter activity as shown in FIG. 11C (SEQ ID NO: 28), but did not drive hair cell-specific GFP expression in the absence of the promoter sequence of SEQ ID NO: 29 (Myo15 C) as shown in FIGS. 11C (SEQ ID NO: 29) and 11G (SEQ ID NO: 32). The promoter sequence of SEQ ID NO: 29 (Myo15 C) was found to be an enhancer that restricted activity of the core promoter (SEQ ID NO: 28) to inner and outer hair cells as shown in FIG. 11G (SEQ ID NO: 32), but was not sufficient for general promoter activity in the absence of the core promoter as shown in FIG. 11D (SEQ ID NO: 29). The promoter sequences of SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29 were reordered into various combinations and still resulted in hair cell specific promoter activity as shown in FIGS. 11H (SEQ ID NO: 33) and 11I (SEQ ID NO: 34), although not all rearrangements were tolerated, as shown in FIG. 11J (SEQ ID NO: 35). These data are summarized in Table 3, below.

TABLE-US-00003 TABLE 3 Promoter activity and hair cell specificity of murine Myo15 promoters Promoter Size (bp) Promoter activity Hair cell specificity A-B-C 520 Yes Yes (SEQ ID NO: 16) A 240 No n/a (SEQ ID NO: 27) B  95 Yes No (SEQ ID NO: 28) C 185 No n/a (SEQ ID NO: 29) A-B 335 No n/a (SEQ ID NO: 30) A-C 425 No n/a (SEQ ID NO: 31) B-C 280 Yes Yes (SEQ ID NO: 32) B-A-C 520 Yes Yes (SEQ ID NO: 33) C-A-B 520 Yes Yes (SEQ ID NO: 34) B-C-A 520 No n/a (SEQ ID NO: 35)

[0195] Myo15 promoter activity was further assessed to determine the strength of expression of Myo15 promoter variants that exhibited hair cell specificity, including the Myo15 0.5 kb (SEQ ID NO: 16), Myo15 B-C(SEQ ID NO: 32), and Myo15 B-A-C(SEQ ID NO: 33). Expression levels of the GFP reporter from the Myo15 0.5 kb, Myo15 B-C, and Myo15 B-A-C promoters were robust and comparable to each other. The Myo15 C-A-B promoter was generated by rearranging positions of the splice donor and acceptor sites and resulted in cryptic splicing into the middle of the GFP cDNA, as evidenced by higher expression levels detected with GFP probes located in the 3′ end of the transcript versus the 5′ end of the transcript (FIG. 12).

[0196] Quantitative image analysis of GFP expression levels was performed in Myo7a-positive hair cells to assess expression levels of GFP per hair cell for various Myo15 promoters, including the 0.5 kb mMyo15 promoter (also referred to as a Myo15 A-B-C promoter; SEQ ID NO: 16), Myo15 A-B promoter (SEQ ID NO: 30), Myo15 A-C promoter (SEQ ID NO: 31), Myo15 B-C promoter (SEQ ID NO: 32), Myo15 A promoter (SEQ ID NO: 27), Myo15 B promoter (SEQ ID NO: 28), Myo15 C promoter (SEQ ID NO: 29), Myo15 B-A-C promoter (SEQ ID NO: 33), Myo15 C-A-B promoter (SEQ ID NO: 34), and the Myo15 B-C-A promoter (SEQ ID NO: 35). The Myo15 A-B-C promoter (SEQ ID NO: 16) yielded the highest expression of GFP per cell (FIG. 13A). Background levels of fluorescence were determined per dataset based on autofluorescence of tissues (<2000 units for FIG. 13A and <6000 units for FIG. 13B).

Other Embodiments

[0197] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. Other embodiments are in the claims.