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，其特征是死亡 视网膜外神经的感光细胞。几十年来，医生和科学家一直 梦想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介导的基因在临床上的成功也就不足为奇了 加强对早期RPE65相关疾病的治疗是非常令人兴奋的， 提供了一个新的信心水平，这种方法也将有助于治疗其他隐性 和X连锁障碍。然而，AAV介导的基因扩增不太可能作为一种标准有效。 对大量以遗传性疾病为主的患者的单独治疗 依赖突变的有毒蛋白质产物的形成(即功能变体的获得)。 治疗显性获得功能疾病，纠正突变等位基因或抑制异常 需要成绩单。基于CRISPR的基因组编辑领域的最新进展显示出很大的进步 对这类应用的承诺。例如，在最近的一项研究中，我们演示了视网膜下注射 CRISPR/Cas9构建可用于在DNA上干扰致病的Pro23His视紫红质等位基因 通过非同源末端连接(NEHJ)在猪视网膜中的表达。不幸的是，疾病等位基因的效率 缺失显著低于达到临床显著效果所需的水平。为了回应这个可怜的人 效率(以及脱靶效应是体内基因组编辑的一个真正令人担忧的问题)，我们决定 将此续订申请集中在最近开发的基于CRISPR的方法上，以抑制 转录和切割特定的mRNAs，而不是永久性的DNA修饰。这些新开发的 与传统的方法相比，这种方法引起有害的非目标基因组修饰的可能性要小得多 基于CRISPR的战略。同样，由于这些方法不需要DNA切割和修复，它们具有 可能会显著提高效率，进而使它们更适合体内应用。 本申请中概述的基于CRISPR的方法将在患者特定的IPSC派生的情况下进行测试 视网膜体外有机体和体内Pro23His视紫红质突变猪。这项建议中的研究将 评价CRISPR生物学的这一新维度治疗常染色体的可行性 主要的视网膜疾病。

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.