Gunewardene Niliksha, Lam Patrick, Song Jiwei, Nguyen Trung, Ruiz Shannon Mendez, Wong Raymond C B, Wise Andrew K, Richardson Rachael T
Bionics Institute, East Melbourne, Victoria 3002, Australia; Department of Medical Bionics, The University of Melbourne, Fitzroy, Victoria 3065, Australia.
Bionics Institute, East Melbourne, Victoria 3002, Australia.
Hear Res. 2025 Mar;457:109170. doi: 10.1016/j.heares.2024.109170. Epub 2024 Dec 20.
In the adult mammalian cochlea, hair cell loss is irreversible and causes deafness. The basic helix-loop transcription factor Atoh1 is essential for normal hair cell development in the embryonic ear. Over-expression of Atoh1 in the adult cochlea by gene therapy can convert supporting cells (cells that underlie hair cells) into a hair cell lineage. However, the regeneration outcomes can be inconsistent. Given that hair cell development is regulated by multiple signalling and transcriptional factors in a temporal and spatial manner, a more complex combinatorial approach targeting additional transcription factors may be required for efficient hair cell regeneration. There is evidence that epigenetic factors are responsible for the lack in regenerative capacity of the deaf adult cochlea. This study aimed to develop a combined gene therapy approach to reprogram both the genome and epigenome of supporting cells to improve the efficiency of hair cell regeneration. Adult Pou4f3-DTR mice were used in which the administration of diphtheria toxin was used to ablate hair cells whilst leaving supporting cells relatively intact. A single adeno-associated viral construct was used to express human Atoh1, Pou4f3 and short hairpin RNA against Kdm1a (regeneration gene therapy) at two weeks following partial or severe hair cell ablation. The average transduction of the inner supporting cells, as measured by the control AAV2.7m8-GFP vector in the deaf cochlea, was only 8 % while transduction in the outer sensory region was <1 %. At 4- and 6-weeks post-treatment the number of Myo+ hair cells in the control and regeneration gene therapy-treated mice were not significantly different. Of note, although both control and regeneration gene therapy treated cochleae contained supporting cells that co-expressed the hair cell marker Myo7a and the supporting cell marker Sox2, the regeneration gene therapy treated cochleae had significantly higher numbers of these cells (p < 0.05). Furthermore, among these treated cochleae, those that had more hair cell loss had a higher number of Myo7a positive supporting cells (R2=0.33, Pearson correlation analysis, p < 0.001). Overall, our results indicate that the adult cochlea possesses limited intrinsic spontaneous regenerative capacity, that can be further enhanced by genetic and epigenetic reprogramming.
在成年哺乳动物的耳蜗中，毛细胞损失是不可逆的，并会导致耳聋。碱性螺旋-环转录因子Atoh1对胚胎期耳朵中正常毛细胞的发育至关重要。通过基因疗法在成年耳蜗中过表达Atoh1可将支持细胞（位于毛细胞下方的细胞）转化为毛细胞谱系。然而，再生结果可能并不一致。鉴于毛细胞的发育在时间和空间上受到多种信号传导和转录因子的调控，可能需要一种更复杂的针对其他转录因子的组合方法来实现高效的毛细胞再生。有证据表明，表观遗传因素是成年失聪耳蜗再生能力缺失的原因。本研究旨在开发一种联合基因疗法，对支持细胞的基因组和表观基因组进行重编程，以提高毛细胞再生的效率。使用成年Pou4f3-DTR小鼠，通过给予白喉毒素来消除毛细胞，同时使支持细胞相对保持完整。在部分或严重毛细胞消融两周后，使用单一腺相关病毒构建体来表达人Atoh1、Pou4f3和针对Kdm1a的短发夹RNA（再生基因疗法）。在失聪耳蜗中，通过对照AAV2.7m8-GFP载体测量，内侧支持细胞的平均转导率仅为8%，而外侧感觉区域的转导率<1%。在治疗后4周和6周，对照小鼠和接受再生基因疗法治疗的小鼠中Myo+毛细胞的数量没有显著差异。值得注意的是，尽管对照和接受再生基因疗法治疗的耳蜗中都含有同时表达毛细胞标记物Myo7a和支持细胞标记物Sox2的支持细胞，但接受再生基因疗法治疗的耳蜗中这些细胞的数量明显更多（p<0.05）。此外，在这些接受治疗的耳蜗中，毛细胞损失更多的耳蜗中Myo7a阳性支持细胞的数量更高（R2=0.33，Pearson相关性分析，p<0.001）。总体而言，我们的结果表明，成年耳蜗具有有限的内在自发再生能力，通过基因和表观遗传重编程可以进一步增强这种能力。
