Optimization of CRISPR genome editor and its delivery strategy for C9orf72 frontotemporal dementia

项目摘要 在C9 orf 72基因的单个等位基因中杂合六核苷酸（GGGGCC）重复扩增是最常见的。 额颞叶痴呆（FTD）和肌萎缩侧索硬化症（ALS）的常见已知遗传原因， 致命和不可逆的神经退行性疾病。鉴于FTD没有有效的治疗方法（ 阿尔茨海默氏症相关的痴呆症），迫切需要新的治疗策略。靶向C9 orf 72基因 通过CRISPR/Cas9基因组编辑本身可以提供治疗性干预。 我们已经建立了一种新的双重sgRNA策略，该策略可以切除C9 orf 72重复区，同时保留C9 orf 72重复区。 患者来源的诱导多能干细胞（iPSC）中的所有外显子。切除消除了病理性 C9-FTD的标志，并在这些iPSC衍生的神经元中保持正常的C9 orf 72蛋白水平。给定 由于该区域的序列保守性，这种编辑策略适用于所有患者，无论其遗传背景如何。 背景或突变大小。我们还设计了一种新型的二氧化硅纳米胶囊（SNC）平台， CRISPR基因组编辑器在静脉内（IV）注射后通过血脑屏障（BBB），并将CRISPR基因组编辑器递送至整个 具有完整BBB的健康动物的脑。SNC具有许多期望的性质，包括能够 提供多种生物制剂，高递送效率，用于配体缀合的多功能表面化学，小颗粒 尺寸、优异的体内稳定性、良好的生物相容性和可规模化生产。结合我们独特的C9 orf 72 编辑方法与我们的创新SNC能够全脑系统交付的基因组编辑器，我们的目标是 开发安全有效的基因组编辑疗法来治疗C9-FTD。在目标1中，我们将最大限度地提高 我们的gRNA策略是通过鉴定高效切除C9 orf 72重复区的sgRNA对， 体外有丝分裂后患者衍生神经元的副作用。在目标2中，我们将优化SNC配方， Ai 14报告基因和C9-FTD小鼠模型中最大脑递送的注射方案， 递送功效。在目标3中，我们将确定切除肿瘤的长期生物安全性和治疗效果。 C9-FTD小鼠模型中通过SNC IV给药的C9 orf 72重复区。 随着拟议的IND使能研究的成功完成，我们将优化候选人 靶向C9 orf 72突变的治疗剂，并在人类神经元中达到最高的治疗功效， 体外和体内C9-FTD小鼠模型。最佳的治疗方法将准备好进行规模放大， 在非人灵长类动物和大鼠中进行的生产/CMC开发和IND使能安全性研究。我们会一起 此外，还讨论了该领域的一些未决问题，包括是否有编辑效率， 有丝分裂神经元与有丝分裂细胞不同，如何在整个细胞中广泛递送具有多个sgRNA的CRISPR/Cas9 小鼠大脑以及是否有可能逆转或阻止有症状小鼠的临床表型。我们 我们的目标是加速神经退行性疾病的基因组编辑走向临床。

PROJECT SUMMARY A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases. Given that there are no effective treatments for FTD (an Alzheimer’s-related dementia), novel therapeutic strategies are urgently needed. Targeting the C9orf72 gene itself by CRISPR/Cas9 genome editing may provide a curative intervention. We have established a novel dual sgRNA strategy, which can excise the C9orf72 repeat region while preserving all exons in patient-derived induced pluripotent stem cells (iPSCs). The excision eliminated pathological hallmarks of C9-FTD and preserved normal C9orf72 protein levels in neurons derived from these iPSCs. Given sequence conservation in this region, this editing strategy applies to all patients, regardless of genetic background or mutation size. We have also engineered a novel silica nanocapsule (SNC) platform that can cross the blood-brain barrier (BBB) after intravenous (IV) injection and deliver CRISPR genome editors to the whole brain of healthy animals with intact BBBs. The SNCs possess many desirable properties including the ability to deliver diverse biologics, high delivery efficiency, versatile surface chemistry for ligand conjugation, small particle sizes, excellent in vivo stability, good biocompatibility, and scalable production. Combining our unique C9orf72 editing approach with our innovative SNC capable of brain-wide systemic delivery of genome editors, we aim to develop a safe and efficiency genome editing therapy to treat C9-FTD. In Aim 1, we will maximize efficiency of our gRNA strategy by identifying sgRNA pairs that excise the C9orf72 repeat region with high efficiency and no side effects in post-mitotic patient-derived neurons in vitro. In Aim 2, we will optimize the SNC formulation and the injection regimen for maximal delivery to the brain in Ai14 reporter and C9-FTD mouse models, maximizing delivery efficacy. In Aim 3, we will determine the long-term biosafety and therapeutic efficacy of excising the C9orf72 repeat region via IV delivery of SNC in the C9-FTD mouse model. With the successful completion of the proposed IND-enabling studies, we will have optimized a candidate therapeutic that targets the C9orf72 mutation, and reaches the highest therapeutic efficacy in human neurons in vitro and in a C9-FTD mouse model in vivo. The optimal therapeutic will then be ready for scale-up, manufacturing/CMC development and IND-enabling safety studies in nonhuman primate and rat. We will have additionally addressed a number of open questions in the field, including whether editing efficiencies in post- mitotic neurons differ from mitotic cells, how to deliver CRISPR/Cas9 with multiple sgRNAs widely throughout the mouse brain and whether it is possible to reverse or arrest clinical phenotypes in symptomatic mice. Our goal is to accelerate genome editing for neurodegenerative diseases toward the clinic.