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

CRISPR/Cas9 基因编辑可挽救常染色体显性色素性视网膜炎啮齿动物模型的视力

基本信息

批准号：
9388267
负责人：
Shaomei Wang
金额：
$ 26.25万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9388267
关键词：
Ablation Address Affect Alleles Animal Model Blindness CRISPR/Cas technology Catalytic RNA Cells Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Cone Contralateral Custom DNA Binding Dependovirus Development Disease Essential Genes Event Eye Eyedrops Genes Genetic Genomics Guide RNA Histology Human In Vitro Inheritance Patterns Inherited Intervention Investigation Leber&apos s amaurosis Link Mediating Modeling Mus Mutation Paper Pathway interactions Patients Photoreceptors Plasmids Point Mutation Process RPE65 protein Rat-1 Rattus Replacement Therapy Reporting Retina Retinal Retinal Degeneration Retinal Diseases Retinal Dystrophy Retinitis Pigmentosa Rhodopsin Rodent Model Safety Site Staphylococcus aureus Testing Time Transgenes Vision clinical practice clinically relevant comparative efficacy cytotoxicity gain of function gene therapy improved in vivo inherited retinal degeneration loss of function mutant nanoparticle neuronal cell body new technology novel personalized medicine photoreceptor degeneration postnatal prevent promoter protein complex repaired sex subretinal injection tool trafficking vector visual cycle

项目摘要

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.

摘要视网膜色素变性（RP）是遗传性视网膜变性中视力丧失的主要原因，影响超过15万人仅在美国。RP的遗传原因多种多样，包括显性、隐性和性别的不同突变。连锁遗传模式致病基因的发现为基因工程的发展铺平了道路治疗成功的例子是RPE 65基因治疗Leber先天性黑蒙2型（LCA 2）。LCA 2，一个常染色体隐性遗传（功能丧失）RP是由RPE 65突变引起的，RPE 65是视网膜视觉周期中的一个必需基因通路然而，最近一项临床试验的报告显示，载体治疗的视网膜变性率眼睛与对侧未治疗的眼睛相似，这引起了人们的担忧，即一旦开始，变性过程可能不能仅仅通过提供一个正确的基因拷贝就完全停止。此外，大多数遗传性视网膜疾病患者受到显性疾病（功能获得）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。没有治疗方法。定制DNA结合的出现包括TALE和CRISPR/cas9在内的模块提供了特异性消除突变基因的新工具。作为原则的证明，我们证明了通过选择性地切除显性视紫红质， S334 ter转基因（rhoS 334）在大鼠模型严重常染色体显性视网膜色素变性（adRP）。视网膜下注射的指导RNA（gRNA）/Cas9质粒产生rhoS 334的等位基因特异性破坏，预防视网膜营养不良，改善的视觉功能（Mol Ther. 2016年3月; 24（3）：556-63）。使用CRISPR/Cas9作为体内基因的可行性治疗显性遗传疾病的患者还不清楚，但个性化药物的开发，以前无法治愈的疾病可以通过研究新技术来实现，其中CRISPR是最重要的技术。到目前为止，这是一种新颖且有前途的方法。我们建议研究CRISPR/Cas9介导的特异性免疫抑制剂的长期安全性和有效性。通过翻译传递消除S334 ter-3大鼠中的显性突变。此外，我们还整合了第二个adRP 啮齿类动物模型（P23 H-1），重现了最常见的adRP人类突变，并对比了 S334 ter-3大鼠。这两种adRP模型将使我们能够研究治疗后的疗效和长期安全性。 CRISPR/CAS9在视网膜变性的早期、中期和晚期阶段被递送到光感受器中，并表征在视网膜变性的早期、中期和晚期阶段，疗效与干预时间的关系。