CRISPR/Cas9 Gene Editing to Rescue Vision in Rodent Model for Autosomal Dominant Retinitis Pigmentosa

摘要 视网膜色素变性是遗传性视网膜变性导致视力丧失的主要原因，影响超过15万人 仅在美国。RP的遗传原因是多样的，包括显性突变、隐性突变和性别突变。 相互关联的继承模式。致病基因的鉴定为基因的发展铺平了道路 治疗。成功的例子是RPE 65基因治疗Leber先天性2型黑色素(LCA2)。LCA2，AN 常染色体隐性(功能丧失)RP是由视网膜视觉周期中的一个重要基因RPE65的突变引起的 路径。然而，最近一项临床试验的报告显示，经载体治疗的视网膜退行性变的比率 与未经治疗的对侧眼睛相似，这引发了人们的担忧，即一旦开始，变性过程可能 而不是仅仅通过提供正确的基因拷贝就能完全阻止。此外，大多数遗传性视网膜疾病 患者受到主型疾病(功能增强)的影响，在这些情况下，需要减少有毒产品 而不是提供正确的基因拷贝。没有可用的治疗方法。定制DNA绑定的出现 包括TALE和CRISPR/Cas9在内的模块提供了专门去除突变基因的新工具。作为原则的证明， 我们证明了使用CRISPR/Cas9作为体内基因治疗的可行性，通过选择性地消融优势视紫红质 S334ter转基因(RhoS334)在严重常染色体显性遗传性视网膜色素变性(ADRP)模型大鼠中的表达。视网膜下注射 引导RNA(GRNA)/Cas9质粒产生RhoS334的等位基因特异性中断，防止视网膜营养不良，以及 改善的视觉功能(摩尔·希尔2016年3月；24(3)：556-63)。CRISPR/Cas9作为体内基因的可行性研究 修复显性遗传性疾病的治疗方法尚不清楚，但为患者开发个性化药物 以前无法治愈的疾病可以通过研究新技术来实现，其中CRISPR是最多的 到目前为止，它很新奇，也很有前途。我们建议研究CRISPR/Cas9介导的特异性药物的长期安全性和有效性 翻译递送消融S334ter-3大鼠的显性突变。此外，我们还加入了第二个adrp。 啮齿动物模型(P23H-1)，概括了最常见的adrp人类突变，并对比了 S334ter-3大鼠。这两个模型的ADRP将使我们能够调查的有效性和长期安全性 CRISPR/Cas9在视网膜变性的早期、中期和晚期被传递到光感受器，并表征 疗效与干预时间的关系。

Abstract Retinitis pigmentosa (RP), a major cause of vision loss in inherited retinal degenerations affects more than 150,000 people in the USA alone. There are diverse genetic causes of RP including different mutations with dominant, recessive and sex- linked inheritance patterns. The identification of disease-causing genes has paved the way for development of gene therapies. The successful example is RPE 65 gene therapy for Leber congenital amaurosis type 2 (LCA2). LCA2, an autosomal recessive (loss of function) RP, is caused by mutations in RPE65, an essential gene in the retinal visual cycle pathway. However, a recent report from one clinical trial showed that the rate of retinal degeneration in the vector-treated eye was similar to that in the contralateral untreated eye, raising concerns that once started, the degeneration processes may not be entirely stopped just by supplying a correct copy of the gene. Moreover, majority of the inherited retinal disease patients are affected by dominant form diseases (gain of function), in these cases, reducing the toxic product is required rather than supplying a correct copy of the gene. There is no treatment available. The advent of customized DNA-binding modules, including TALE and CRISPR/cas9 provides new tools to specifically ablate mutant genes. As proof of principle, we demonstrate the feasibility of using CRISPR/Cas9 in vivo as gene therapy by selectively ablating the dominant rhodopsin S334ter transgene (rhoS334) in rats that model severe autosomal dominant retinitis pigmentosa (adRP). Subretinal injection of guide RNA (gRNA)/Cas9 plasmid generated allele-specific disruption of rhoS334, prevented retinal dystrophy, and improved visual function (Mol Ther. 2016 Mar; 24 (3): 556-63). The feasibility of using CRISPR/Cas9 as in vivo gene therapy to repair dominant-inherited diseases for patients is not yet clear, but development of personalized medicines for previously untreatable diseases can be realized through investigation into new technologies, of which CRISPR is the most novel and promising so far. We propose to investigate the long-term safety and efficacy of CRISPR/Cas9 mediated specific ablation on the dominant mutation in S334ter-3 rats by translational delivery. In addition, we incorporated a second adRP rodent model (P23H-1) that recapitulates the most frequent adRP human mutation and contrasts the fast degeneration in S334ter-3 rats. These two models for adRP will allow us to investigate the efficacy and long-term safety after CRISPR/CAS9 delivered into the photoreceptors at early, mid and late stages of retinal degeneration and to characterize the relationship of efficacy to time-of-intervention.