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

大约10%-30%的常染色体显性遗传性视网膜色素变性(ADRP)病例是由视紫红质(Rho)基因突变引起的，Rho基因编码视杆感光细胞中的视觉色素。已有150多个不同的Rho显性突变的功能特征被证明是通过显性负效应或作用机制的获得而起作用的。因此，沉默或选择性破坏突变等位基因是Rho连锁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性修饰的强大方法，从而避免了一些团体宣传的治疗Rho连锁ADRP的“击倒和替换”策略引起的几个担忧(例如，过度表达或异位错误表达导致的视黄醇毒性)。其他研究工作(包括基于反义寡核苷酸和基于CRISPR/Cas9的技术)集中在对某些突变等位基因的选择性破坏上，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-SEDIMS-X(通过Cas9-exo靶向共同的SNPs选择性破坏突变等位基因)-有望克服这些障碍和限制。SEDIMS-X应用CRISPR/Cas9技术，通过针对少数常见的SNP而不是单个致病突变来选择性地破坏突变等位基因。SEDIMS-X采用了新型的高保真Cas9-exoexo融合变体，能够从单个gRNA定向的双链断裂中增强破坏性Indels。在令人鼓舞的初步结果的基础上，本项目进一步探索了SEDIMS-X在ADRP连锁Rho突变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，通过建立高通量的基于细胞的报告系统来设计和筛选有效的Cas9-exo/gRNA化合物，验证它们在人皮肤成纤维细胞中的等位基因特异性，并从转录物和蛋白质水平分析患者来源的视杆细胞光感受器前体细胞的有效性和功能结果。此外，我们将仔细评估这一新方法的潜在非目标活动。我们预计，SEDIMS-X将通过克服目前的一些限制，扩展和补充现有的针对Rho相关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.