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US-12618070-B2 - Increasing Atoh1 life to drive sensorineural hair cell differentiation

US12618070B2US 12618070 B2US12618070 B2US 12618070B2US-12618070-B2

Abstract

The present disclosure provides compositions and methods for treating subjects at risk for or with sensorineural hearing loss by modulating the rate of Atoh1 protein degradation to increase levels of Atoh1 protein.

Inventors

  • Albert Edge
  • Yen-Fu Cheng
  • Judith Kempfle
  • Dunia Abdul-Aziz

Assignees

  • MASSACHUSETTS EYE AND EAR INFIRMARY

Dates

Publication Date
20260505
Application Date
20220304

Claims (13)

  1. 1 . An expression vector comprising a polynucleotide encoding a human Atoh1 variant polypeptide, wherein the human Atoh1 variant polypeptide is a long-lived human Atoh1 variant polypeptide comprising a mutation at the amino acid position corresponding to amino acid 334 of SEQ ID NO: 1, and wherein the long-lived human Atoh1 variant polypeptide is at least 95% identical to SEQ ID NO: 1.
  2. 2 . The expression vector of claim 1 , wherein the amino acid at the position corresponding to amino acid 334 of SEQ ID NO: 1 is not serine.
  3. 3 . The expression vector of claim 1 , wherein the long-lived human Atoh1 variant polypeptide comprises SEQ ID NO: 1 with a mutation of S334A.
  4. 4 . The expression vector of claim 1 , wherein the long-lived human Atoh1 variant polypeptide further comprises a mutation at the amino acid position corresponding to amino acid 331 of SEQ ID NO: 1.
  5. 5 . The expression vector of claim 4 , wherein the amino acid at the position corresponding to amino acid 331 of SEQ ID NO: 1 is not serine.
  6. 6 . The expression vector of claim 1 , wherein the long-lived human Atoh1 variant polypeptide comprises SEQ ID NO: 1 with mutations S331A/S334A.
  7. 7 . The expression vector of claim 1 , which is a viral vector.
  8. 8 . The expression vector of claim 7 , wherein the viral vector is selected from the group consisting of recombinant retrovirus, adenovirus, adeno-associated virus, and lentivirus.
  9. 9 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a human Atoh1 variant polypeptide, wherein the human Atoh 1 variant polypeptide is a long-lived human Atoh1 variant polypeptide comprising a mutation at the amino acid position corresponding to amino acid position 334 of SEQ ID NO: 1, and wherein the long-lived human Atoh1 variant polypeptide is at least 95% identical to SEQ ID NO: 1.
  10. 10 . The isolated nucleic acid molecule of claim 9 , wherein the long-lived human Atoh1 variant polypeptide comprises SEQ ID NO: 1 with a mutation of S334A.
  11. 11 . The isolated nucleic acid molecule of claim 9 , wherein the long-lived human Atoh1 variant polypeptide comprises SEQ ID NO: 1 with mutations S331A/S334A.
  12. 12 . The isolated nucleic acid molecule of claim 9 , wherein the nucleotide sequence further encodes a 5′ untranslated region.
  13. 13 . The isolated nucleic acid molecule of claim 9 , which is at least 95% identical to SEQ ID NO: 2.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional of U.S. patent application Ser. No. 16/358,579, filed Mar. 19, 2019, which is a continuation of U.S. patent application Ser. No. 15/502,113, filed Feb. 6, 2017, now abandoned, which is a 371 U.S. National of PCT Application No. PCT/US2015/043976, filed on Aug. 6, 2015, which claims priority to U.S. Patent Application Ser. No. 62/034,040, filed on Aug. 6, 2014; and U.S. Patent Application Ser. No. 62/034,459, filed on Aug. 7, 2014, the entire contents of which applications are incorporated herein by reference. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named “Sequence_Listing.txt.” The ASCII text file, created on Mar. 2, 2022, is 10 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety. TECHNICAL FIELD This invention relates to the generation of sensorineural hair cells, and more particularly to the use of modulation of Atoh1 expression to generate sensorineural hair cells. BACKGROUND There are six distinct sensory organs in the mammalian inner ear: the three cristae of the semicircular canals, the two maculae of the saccule and utricle, and the organ of Corti of the cochlea. The organ of Corti is the organ of hearing. The receptor cell for hearing is the hair cell of the cochlea (referred to herein as a hair cell, a sensory hair cell, or a sensorineural hair cell). Hair cells are limited in number and do not regenerate in mammals; damage or death of these cells leads to hearing loss (Edge and Chen, Curr. Opin. Neurobiol., 18:377-382 (2008)). Therapeutic compositions and methods to increase sensorineural hair cell number and/or function in the cochlea are required to address hearing loss. SUMMARY The present disclosure provides compositions and methods for generating hair cells by modulating expression of Atoh1 by inhibiting proteasome degradation of the Atoh1 protein. The data presented herein shows a role of the ubiquitin proteasome pathway in post-translational regulation of Atoh1, identifies several amino acids in the N terminus as important in determining lifespan of the Atoh1 protein, and identifies the HECT, UBA and WWE domain containing 1 (Huwe1) protein as an E3 ubiquitin ligase for Atoh1. Thus, in a first aspect the invention provides methods for treating a disorder, e.g., sensorineural hearing loss or vestibular dysfunction, associated with loss of auditory hair cells in a subject. The methods include administering a therapeutically effective amount of proteasome inhibitor to the subject, e.g., to the inner ear of the subject. Also provided herein is the use of a proteasome inhibitor for the treatment of sensorineural hearing loss associated with loss of auditory hair cells in a subject. In some embodiments, the proteasome inhibitor is selected from the group consisting of Bortezomib, Carfilzomib, NPI-0052, MLN9708, CEP-18770, and ONX0912. In some embodiments, the methods include comprising administering an HDAC inhibitor, an EZH2/HMT inhibitor, or a DNMT inhibitor, in combination with a proteasome inhibitor, to the subject, e.g., to the inner ear of the subject. Also provided herein is the use of one or more of an HDAC inhibitor, an EZH2/HMT inhibitor, or a DNMT inhibitor, in combination with a proteasome inhibitor, for the treatment of sensorineural hearing loss associated with loss of auditory hair cells in a subject. In some embodiments, the HDAC inhibitor is selected from the group consisting of: Sodium Butyrate, Trichostatin A, hydroxamic acids, cyclic tetrapeptides, trapoxin B, depsipeptides, benzamides, electrophilic ketones, aliphatic acid compounds, phenylbutyrate, valproic acid, hydroxamic acids, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589), entinostat (MS275), romidepsin, C1994, and mocetinostat (MGCD0103). In some embodiments, the EZH2/HMT inhibitor is selected from the group consisting of Deazaneplanocin A; GSK J1; GSK126; EPZ005687; E7438; EI1 (Qi et al., 2012, supra); EPZ-6438; GSK343; BIX-01294, UNC0638, BRD4770, EPZ004777, AZ505 and PDB 4e47, and those described in Garapaty-Rao et al., Chem. Biol. 20(11):1329-1339 (2013); 20130303555; and WO2012/005805; see, e.g., Wagner and Jung, Nature Biotechnology 30:622-623(2012). In some embodiments, the DNMT inhibitor is selected from the group consisting of azacytidine, decitabine, Zebularine (1-((3-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one), procainamide, procaine, (−)-epigallocatechin-3-gallate, MG98, hydralazine, RG108, and Chlorogenic acid. In some embodiments, the proteasome inhibitor is selected from the group consisting of Bortezomib, Carfilzomib, NPI-0052, MLN9708, CEP-18770, and ONX0912. In some embodiments, the methods uses include application of the HDAC inhibitor, DNMT inhibitor, the EZH2/HMT inhibitor, or the proteasome inhibitor, to the round window membrane, e.g., intra-tympanic injection, intra