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.

抽象的视网膜炎色素（RP），遗传性视网膜变性的视力丧失的主要原因影响超过15万人仅在美国。 RP有多种遗传原因，包括不同的突变，具有显性，隐性和性别链接的继承模式。引起疾病基因的鉴定为发展基因的发展铺平了道路疗法。成功的例子是RPE 65基因疗法，用于LEBER先天性瘤2型（LCA2）。 lca2，an 常染色体隐性（功能丧失）RP是由RPE65突变引起的，RPE65是视网膜视觉周期中的必要基因路径。但是，一项临床试验的最新报告表明，载体处理的视网膜变性率眼睛与对侧未经处理的眼睛相似，引起了人们的关注，一旦开始，变性过程可能会不仅可以通过提供正确的基因副本来完全停止。此外，大多数继承的视网膜疾病患者受到主要形式疾病的影响（功能增长），在这些情况下，需要减少有毒产物而不是提供基因的正确副本。没有可用的治疗方法。定制的DNA结合的出现包括Tale和CRISPR/CAS9在内的模块为特别消融突变基因提供了新的工具。作为原则的证明，我们证明了通过选择性消除主要的视紫红质，在体内使用CRISPR/CAS9作为基因疗法的可行性 S334Ter转基因（Rhos334）在大鼠中模拟严重的常染色体显性视网膜炎色素炎（ADRP）。视网膜下注射引导RNA（GRNA）/cas9质粒产生的等位基因特异性破坏Rhos334，防止视网膜营养不良，并且改进的视觉功能（MolTher。20163月； 24（3）：556-63）。将CRISPR/CAS9用作体内基因的可行性为患者修复占主导地位疾病的治疗尚不清楚，但开发了个性化药物以前可以通过调查新技术来实现以前不可治疗的疾病，其中CRISPR是最多的到目前为止，新颖而有前途。我们建议调查CRIS/CAS9介导的特定的长期安全性和功效通过翻译递送在S334Ter-3大鼠中的主要突变消融。此外，我们合并了第二个ADRP 啮齿动物模型（P23H-1）概括了最频繁的ADRP人类突变，并将其对比 S334Ter-3大鼠。 ADRP的这两个模型将使我们能够调查之后的功效和长期安全性 CRISPR/CAS9在视网膜变性的早期，中期和晚期输送到感光器中功效与干预时间的关系。