Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11.
Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11.
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DOI：
10.3390/biom12070914
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发表时间：
2022-06-29
期刊：
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
--
作者：
遗传性耳聋的基因治疗是一种新兴的听力恢复治疗方法。然而，该方法尚未转化为临床应用。为了进一步发展内耳基因治疗，我们设计了一种新的小鼠模型，该模型携带跨膜通道1基因(TMC1)的人类突变，并对小鼠的听觉表型进行了表征。TMC1在小鼠和人体内形成机械感觉转导通道，是听觉功能所必需的。我们发现，携带等同于人类p.N199I突变(p.N193I)的小鼠由于毛细胞感觉转导的丧失而导致严重的先天性听力损失。接下来，我们对包装成AAV9-PHP.B衣壳的病毒有效载荷进行了优化和筛选。将载体注射到Δ/Δ小鼠内耳和人源化TMC 1-p.N193I小鼠模型中。在注射的小鼠中评估听性脑干反应(ABR)、失真产物耳声发射(DPOAEs)、细胞存活和生物分布。在注射AAV9-PHP.B-CB6-hTMC1-WPRE的TMC1-p.N193I小鼠中，我们发现了广谱、持久的听觉功能恢复。ABR和DPOAE阈值在整个频率范围内与野生型小鼠相同。生物分布分析显示，病毒DNA/RNA在对侧耳、脑和肝脏中存在，但没有明显的毒性。结论：构建的AAV9-PHP.B-CB6-hTMC1-WPRE有望成为治疗隐性TMC1突变所致遗传性耳聋的基因治疗试剂。
Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene (Tmc1) and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of Tmc1Δ/Δ mice and the new humanized Tmc1-p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in Tmc1-p.N193I mice injected with AAV9-PHP.B-CB6-hTMC1-WPRE. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B-CB6-hTMC1-WPRE construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive TMC1 mutations.
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假定的毛细胞机械转导通道 TMC1 和 TMEM16 蛋白之间的结构关系。
影响因子：
7.7
作者：
Ballesteros A;Fenollar-Ferrer C;Swartz KJ
通讯作者：
Swartz KJ
影响因子：
16.2
作者：
Pan B;Akyuz N;Liu XP;Asai Y;Nist-Lund C;Kurima K;Derfler BH;György B;Limapichat W;Walujkar S;Wimalasena LN;Sotomayor M;Corey DP;Holt JR
通讯作者：
Holt JR
影响因子：
30.8
作者：
Kurima, K;Peters, LM;Griffith, AJ
通讯作者：
Griffith, AJ
影响因子：
25
作者：
Géléoc, GSG;Holt, JR
通讯作者：
Holt, JR
影响因子：
15.9
作者：
Kawashima, Yoshiyuki;Geleoc, Gwenaelle S. G.;Griffith, Andrew J.
通讯作者：
Griffith, Andrew J.