CRISPR-Cas9 based treatment of dominant retinal degeneration

摘要 遗传性视网膜变性是一组遗传异质性的疾病，其特征在于视网膜变性患者的死亡。 外层神经视网膜的感光细胞。几十年来，医生和科学家 我梦想着通过某种形式的基因来预防遗传性视网膜疾病患者的视力丧失 替代疗法因此，最近AAV介导的基因转染的临床成功并不令人惊讶。 用于治疗早期RPE 65相关疾病的增强已经非常令人兴奋， 提供了一个新的信心水平，这种方法也将是有用的治疗其他隐性 和X染色体连锁疾病然而，AAV介导的基因扩增不太可能有效地作为一种替代疗法。 单独治疗大量患有显性遗传疾病的患者， 毒性蛋白产物的突变依赖性形成（即功能变体的获得）。 治疗显性功能获得性疾病，纠正突变等位基因或抑制异常 将需要成绩单。基于CRISPR的基因组编辑领域的最新进展显示出巨大的潜力。 对此类应用的承诺。例如，在最近的一项研究中，我们证明了视网膜下注射 CRISPR/Cas9构建体可用于破坏DNA中致病的Pro23 His视紫红质等位基因， 通过非同源末端连接（NEHJ）在猪视网膜中的水平。不幸的是，疾病等位基因的效率 缺失显著低于具有临床显著效果所需的量。针对这一贫困 效率（以及脱靶效应是体内基因组编辑的一个真实的问题），我们决定 将这一更新申请集中在最近开发的基于CRISPR的方法上， 转录和切割特定的mRNA，而不是永久的DNA修饰。这些新开发 与传统方法相比， 基于CRISPR的策略。同样，由于这些方法不需要DNA切割和修复，因此它们具有 潜在的是显着更有效，反过来使它们更适合于体内应用。 本申请中概述的基于CRISPR的方法将在患者特异性iPSC衍生的细胞中进行测试。 体外视网膜类器官和体内Pro23 His视紫红质突变体猪。本提案中的研究将 评估使用这种新的CRISPR生物学维度治疗常染色体 显性视网膜疾病

Abstract Inherited retinal degenerations are a genetically heterogeneous group of disorders characterized by death of the light-sensing photoreceptor cells of the outer neural retina. For decades, physicians and scientists have dreamed of preventing vision loss in patients affected with an inherited retinal disease via some form of gene replacement therapy. Thus, it is not surprising that the recent clinical success of AAV-mediated gene augmentation for the treatment of early stage RPE65-associated disease has been tremendously exciting, providing a new level of confidence that this approach will also be useful for the treatment of other recessive and X-linked disorders. However, AAV-mediated gene augmentation is not likely to be effective as a stand- alone treatment for the large population of patients with dominantly inherited diseases that result from mutation dependent formation of a toxic protein product (i.e. gain of function variants). To treat dominant gain of function diseases, correction of the mutant allele or suppression of the abnormal transcript will be required. Recent advances in the field of CRISPR-based genome editing have shown great promise for such applications. For instance, in a recent study, we demonstrated how the subretinal injection of a CRISPR/Cas9 construct could be used to disrupt the disease-causing Pro23His rhodopsin allele at the DNA level in pig retina via nonhomologous end joining (NEHJ). Unfortunately, the efficiency of disease allele deletion was significantly lower than that required to have a clinically significant effect. In response to this poor efficiency (and the fact that off-target effects are a real concern with in vivo genome editing), we have decided to focus this renewal application on very recently developed CRISPR-based approaches to suppress transcription and cleave specific mRNAs rather than permanent DNA modification. These newly developed approaches are significantly less likely to induce deleterious off-target genomic modifications than traditional CRISPR-based strategies. Likewise, as these approaches do not require DNA cleavage and repair they have the potential to be significantly more efficient, in turn making them more suitable for in vivo applications. The CRISPR-based approaches outlined in this application will be tested in patient-specific iPSC-derived retinal organoids in vitro and the Pro23His rhodopsin mutant pig in vivo. The studies in this proposal will evaluate the feasibility of using this new dimension of CRISPR biology for the treatment of autosomal dominant retinal diseases.