A non-viral CRISPR-mediated genome editing delivery platform as a potential therapy for neurogenetic diseases

基因组编辑为许多遗传病的治疗带来了巨大的希望；然而，它在 由于缺乏安全的交付工具以及所需的巨大成本和时间投资，诊所进展缓慢 定制开发个性化疗法。在我们的SCGE计划第一阶段研究中，我们开发了一种化学 基于改良核糖核蛋白(CRNP)的特异性靶向神经细胞的基因递送系统 鞘内(IT)给药后的整个大脑。此应用程序的总体目标是将此 用于治疗两种严重神经发育障碍(NDD)的新基因编辑技术： Angelman综合征(AS)和H1-4综合征(HIST1H1E综合征)。就像是毁灭性的神经发育 人类母体和脑特异性印迹UBE3A基因缺失引起的疾病 染色体15q11-q13区域。ALL AS患者父系染色体中UBE3A基因结构完整 但受UBE3A非编码反义RNA(UBE3A-ATS)介导的转录抑制 机制。已经令人信服地表明，通过反义寡核苷酸(ASO)还原UBE3A-ATS， 拓扑异构酶抑制剂和病毒传递的Cas9基因编辑可以下调UBE3A和 纠正AS小鼠模型中异常的神经表型。H1-4综合征是由 C端移码尾巴(CFT)突变蛋白的作用机制。H1-4综合征有 临床表现与强直性脊柱炎相似，但程度较轻。目前还没有针对H1-4综合征的有效干预措施。因此，一个漫长的- AS和H1-4以及其他NDDS的长期分子治疗是迫切需要的。在我们的临床前研究中使用 作为一个经过良好验证的AS小鼠模型，我们演示了单一IT交付的Ube3a反义靶向 RNP/Cas9有效地抑制父亲染色体上Ube3a的表达，导致纠正 神经行为表型。同样，H1-4 CFT基因的敲除拯救了H1-4的异常表型。 4人源化小鼠。我们提出了基于CRNP的AS和H1-4综合征治疗平台 相同的基因组编辑程序(CRISPR)、传递系统(CRNPs)、路线(IT)、靶细胞类型(神经元)、治疗 机制(基因失活)和总体试验设计。我们从耶鲁汇聚了一支优秀的团队 和拉什大学在临床前、IND支持研究领域拥有强大的互补专业知识， 和临床试验。这项研究的成功将导致有史以来第一次基于基因编辑的AS和H1治疗- 4.更重要的是，它将支持基因组编辑的范式转变；迅速扩大 体内基因编辑和加速基因组编辑转型治疗神经遗传病 将技术转化为临床应用。

Genome editing holds great promise for the treatment of many genetic diseases; however its application in the clinic has been slow due to the lack of the safe delivery tools and significant cost and time investment required to custom-develop individual therapies. In our SCGE program phase 1 study, we developed a chemically modified ribonucleoprotein (cRNP)-based gene delivery system that specifically targets neuronal cells throughout the brain after intrathecal (IT) administration. The overarching goal of this application is to apply this novel gene editing technology towards the treatment of two severe neurodevelopmental disorders (NDD): Angelman syndrome (AS) and H1-4 syndrome (HIST1H1E syndrome). AS is a devastating neurodevelopmental disease caused by the deficiency of the maternal and brain specific imprinting UBE3A gene in human chromosome 15q11-q13 region. The structure of UBE3A is intact in the paternal chromosome in all AS cases but transcriptionally repressed by a non-coding and antisense RNA of UBE3A (UBE3A-ATS) mediated mechanism. It has been shown convincingly that reduction of UBE3A-ATS by antisense oligo (ASO), topoisomerase inhibitors, and virus delivered Cas9 gene editing can de-repress the expression of UBE3A and correct the abnormal neurological phenotypes in AS mouse models. H1-4 syndrome is caused by a gain of function mechanism due to a mutated protein with aberrant C-terminal frameshift tail (CFT). H1-4 syndrome has similar but milder clinical features than AS. There is no effective intervention for H1-4 syndrome. Thus, a long- term molecular therapy for AS and H1-4, as well as other NDDs is urgently needed. In our preclinical study using a well validated AS mouse model, we demonstrated that a single IT delivery of Ube3a antisense-targeting RNP/Cas9efficiently de-represses the expression of Ube3a from the paternal chromosome, leading to correction of neurobehavioral phenotypes. Similarly, the knockdown of H1-4 CFT rescue the abnormal phenotypes in H1- 4 humanized mice. We propose our cRNP-based platform for the treatment of AS and H1-4 syndrome utilizing the same genome editor (CRISPR), delivery system (cRNPs), route (IT), target cell type (neurons), therapeutic mechanism (genetic inactivation) and overall trial design. We have assembled an outstanding team from Yale and Rush University with strong and complementary expertise in the areas of preclinical, IND enabling studies, and clinical trials. The success of this study will lead to the first ever gene editing based therapy for AS and H1- 4. More importantly, it will support a paradigm shift for genome editing; rapidly expanding the number of neurogenetic diseases treated by in vivo gene editing and accelerating the transition of genome editing technology into clinical applications.