Allele-specific suppression of pathogenic Bestrophin-1 transcripts by targeted CRISPR/Cas9-mediated genome editing

近年来，我们对孤儿疾病分子基础的理解取得了重大进展，这使得建议精确的治疗方法成为可能，这些治疗方法通常针对特定类型的遗传缺陷进行个性化定制。最值得注意的是，基因替代疗法已被广泛关注的常染色体隐性遗传疾病的蛋白质表达的完全或部分损失是由安全和有效的腺相关病毒基因转移拯救。相反，常染色体显性突变，特别是那些导致功能获得或以显性负性方式起作用的突变，仍然具有挑战性的解决，因为突变的基因产物可能与正常的基因拷贝相互作用，因此需要在考虑替代治疗之前首先去除。通过CRISPR/Cas9基因组编辑技术特异性消除突变转录物来治疗阴性疾病。为了证明这种方法的可行性，我们专注于显性负性bestrophin-1（BEST 1）突变（Y227 N）已知导致Best疾病，一种成年早期威胁视力的黄斑病变。为此，我们已经建立了患者来源的细胞，并随后将这些细胞分化为视网膜色素上皮（RPE），这是一种有丝分裂后组织，已知具有BD的主要病理学。在这种体内细胞培养系统中，我们希望证明在消除显性负性基因表达后恢复（增加）正常BEST 1通道活性的可行性。在第二系列实验中，我们专注于翻译方面，以探索所需基因组编辑工具向RPE的递送途径以及在活体小鼠眼睛中的治疗效果。总之，我们的体外和体内工作可以作为概念验证，以进一步开发CRISPR/Cas驱动的精准医学方法，并为治疗显性阴性基因突变患者铺平道路。

In recent years, major advances in our understanding of the molecular underpinnings of orphan diseases have made it feasible to advise precise therapies, often individually tailored to a specific type of genetic defect. Most notably, gene replacement therapy has found wide attention for the autosomal recessive disorders where complete or partial loss of protein expression is rescued by safe and efficient adeno-associated viral gene transfer. In contrast, autosomal dominant mutations, specifically those leading to gain of function or acting in a dominant negative fashion, are still challenging to address as the mutated gene product may interact with the normal gene copy and thus needs to be removed first before a replacement treatment can be considered.With the present proposal we want to explore a novel therapeutic option to treat dominant-negative disease by specifically eliminating mutant transcripts via the CRISPR/Cas9 genome editing technology. To demonstrate feasibility of such an approach, we focus on a dominant-negative bestrophin-1 (BEST1) mutation (Y227N) known to cause Best disease, a sight-threatening maculopathy of early adulthood. To this end, we have established patient-derived cells and have subsequently differentiated these cells into retinal pigment epithelium (RPE), a post-mitotic tissue known to harbor the primary pathology of BD. In this in vivo cell culture system, we want to demonstrate feasibility of restoring (increasing) normal BEST1 channel activity after ablating dominant-negative gene expression. In a second line of experiments, we focus on translational aspects to explore delivery routes of the required genome editing tools to the RPE and efficacy of treatment in a living mouse eye. Together, our in vitro and in vivo work may serve as proof-of-concept to further develop CRISPR/Cas-driven approaches in precision medicine and to pave the way for treatment of patients with dominant-negative gene mutations.