Purpose

To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO).

Design

Experimental study for a combination gene therapy that uses both gene ablation and gene replacement.

Participants

This study included 2 kinds of human RHO mutation knock-in mouse models: Rho^P23H and Rho^D190N. In total, 23 Rho^P23H/P23H, 43 Rho^P23H/+, and 31 Rho^D190N/+ mice were used for analysis.

Methods

This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA.

Main Outcome Measures

Electroretinographic and histologic analysis.

Results

The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both Rho^P23H and Rho^D190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy.

Conclusions

As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.

中文翻译：

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基于聚类规则间隔短回文重复的基因组手术治疗常染色体显性色素性视网膜炎

 目的

开发一种通用基因疗法，以克服因视紫红质 ( RHO ) 突变导致的色素性视网膜炎 (RP) 的遗传异质性。

 设计

使用基因消融和基因替代的组合基因疗法的实验研究。

 参加者

本研究包括2种人类RHO突变敲入小鼠模型： Rho ^P23H和Rho ^D190N 。总共使用 23 只Rho ^P23H/P23H 、43 只Rho ^P23H/+和 31 只Rho ^D190N/+小鼠进行分析。

 方法

这项研究涉及使用双腺相关病毒 (AAV) 的基因治疗，该病毒 (1) 通过改进的基于聚类规则间隔短回文重复的基因删除，以与突变无关的方式破坏内源性Rho基因的表达，以及 (2) 启用表达通过外源 cDNA 野生型蛋白质。

 主要观察指标

视网膜电图和组织学分析。

 结果

注射 AAV 后 3 个月，视网膜下注射联合消融和替换基因疗法后的外核层 (ONL) 厚度比仅基因替换疗法产生的 ONL 厚度大约增加 17% 至 36%。此外，视网膜电图检查结果表明，使用消融和替换基因疗法（ P < 0.001）， Rho ^P23H和Rho ^D190N疾病模型的 a 波和 b 波得到更显着的保留，但基因替换单一疗法则不然。

 结论

作为概念证明，我们的结果表明，根据两种人类突变敲入模型的光感受器结构和功能测量，消融和替换策略可以改善疾病进展。这些结果证明了消融和替换策略治疗由不同Rho突变引起的 RP 的效力。此外，由于消融和替换治疗与突变无关，因此该策略可用于治疗眼科和其他领域的多种显性疾病。使用消融和替换基因疗法的临床试验将使研究人员能够确定这种策略是否为患有感兴趣疾病的患者带来任何益处。