CRISPR/Enhanced-Deletion Cas9-mediated allele-specific targeting of the rhodopsingene: a novel mutation-independent approach to treat RHO-linked autosomal dominantRetinitis pigmentosa

约10%-30%的常染色体显性视网膜色素变性（adRP）病例是由编码视杆光感受器中视觉色素的视紫红质（RHO）基因突变引起的。超过150种不同的RHO显性突变已被功能表征，并显示通过显性负作用或功能机制的增益来起作用。因此，沉默或选择性破坏突变等位基因是rho相关adRP基因治疗的基本原理。基于CRISPR/ cas9的基因组编辑提供了一种在DNA水平上产生永久性治疗修饰的强大方法，从而避免了“敲除和替代”策略在治疗某些群体传播的RHO-linked adRP时引起的一些问题（例如，由于过度表达或异位错误表达而引起的视网膜毒性）。其他研究工作（包括基于反义寡核苷酸和基于CRISPR/ cas9的技术）主要集中在选择性破坏某些突变等位基因上，因此没有充分解决RHO突变的等位基因异质性。该项目寻求一种新的替代方法-沉淀物- x（通过Cas9-exo靶向常见snp选择性破坏突变等位基因）-有望克服这些障碍和局限性。沉淀物- x应用CRISPR/Cas9技术，通过靶向少数常见snp而不是单个致病突变，选择性地破坏突变等位基因。sediment - x采用新颖的高保真Cas9-exoexo融合变体，能够增强单grna定向双链断裂的破坏性索引。基于令人鼓舞的初步结果，本项目进一步深入探讨了沉淀物- x在adrp相关RHO突变的临床翻译中的适用性和应用。为此，我们将采用严格的工作流程，通过建立基于高通量细胞的报告系统来设计和选择有效的Cas9-exo/gRNA化合物，验证其在人真皮成纤维细胞中的等位基因特异性，并分析其在患者源性杆状光感受器前体细胞中的转录物和蛋白水平的功效和功能结果。此外，我们将仔细评估这种新方法的潜在脱靶活动。我们期望通过克服目前的一些局限性，sediment - x将扩展、补充和扩展现有的RHO-linked adRP患者的临床前、实验性治疗策略。

About 10%-30% of autosomal dominant retinitis pigmentosa (adRP) cases are caused by mutations in the rhodopsin (RHO) gene, encoding the visual pigment in rod photoreceptors. More than 150 different RHO dominant mutations have been functionally characterized, and shown to act via dominant negative effect or gain of function mechanisms. Therefore, silencing or selective disruption of the mutant allele is the fundamental rational for genetic therapies in RHO-linked adRP. CRISPR/Cas9-based genome editing offers a powerful approach to generate permanent therapeutic modification at the DNA level, thereby avoiding several concerns (e.g. retinotoxicity due to overexpression or ectopic mis-expression) raised on the ‘knockdown and replacement’ strategy for the treatment of RHO-linked adRP propagated by some groups. Other research efforts (including antisense oligonucleotide-based and CRISPR/Cas9-based technologies) are focused on selective disruption of certain mutant alleles, and thus do not adequately address the allelic heterogeneity of RHO mutations. This project pursues a novel alternatively approach - SEDIMS-X (selective disruption of the mutant allele through targeting common SNPs by Cas9-exo) - which is expected to overcome such hurdles and limitations. SEDIMS-X applies CRISPR/Cas9 technology to selectively disrupt mutant alleles by targeting few common SNPs rather than the individual disease-causing mutation. SEDIMS-X employs novel high-fidelity Cas9-exoexo fusion variants able to enhance disruptive indels from single gRNA-directed double-strand breaks. Based on encouraging preliminary results, this project further explores in-depth the applicability and utilization of SEDIMS-X on adRP-linked RHO mutations towards clinical translation. To this end, we will apply a stringent workflow to design and select effective Cas9-exo/gRNA compounds through the establishment of a high-throughput cell-based reporter system, validate their allele specificity in human dermal fibroblasts, and analyze the efficacy and functional outcome on transcript and protein level in patient-derived rod photoreceptor precursor cells. Moreover, we will carefully assess potential off-target activity of this novel approach. We expect that SEDIMS-X will extend and complement and extend the existing repertoire of preclinical, experimental therapeutic strategies for patients with RHO-linked adRP by overcoming some of the current limitations.