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

项目总结 C9orf72基因单等位基因GGGGCC重复扩增次数最多 额颞部痴呆(FTD)和肌萎缩侧索硬化症(ALS)常见的已知遗传原因，两个 致命和不可逆转的神经退行性疾病。鉴于没有有效的治疗FTD(AND)的方法 阿尔茨海默氏症相关的痴呆)，迫切需要新的治疗策略。靶向C9orf72基因 CRISPR/Cas9基因组编辑本身可能提供一种治疗干预。 我们建立了一种新的双重sgRNA策略，该策略可以在保留C9orf72重复区域的同时切除C9orf72重复区域 患者来源的诱导多能干细胞(IPSCs)中的所有外显子。切除消除了病理性 在这些IPSCs来源的神经元中，C9-FTD的特征和维持正常的C9orf72蛋白水平。vt.给出 该区域的序列保守，这种编辑策略适用于所有患者，而不考虑基因 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，它可以跨越 静脉注射后血脑屏障(BBB)，并将CRISPR基因组编辑送到整个 BBBS完好无损的健康动物的大脑。SNC具有许多理想的特性，包括能够 提供多种生物制剂、高输送效率、用于配基偶联的多种表面化学、小颗粒 尺寸、出色的体内稳定性、良好的生物相容性和可扩展生产。结合我们独特的C9orf72 编辑方法通过我们创新的SNC，能够系统地提供全脑基因组编辑，我们的目标是 开发一种安全有效的基因组编辑疗法来治疗C9-FTD。在目标1中，我们将最大化 我们的gRNA策略是通过高效地识别切除C9orf72重复区域的sgRNA对来实现的 有丝分裂后患者来源的神经元在体外的副作用。在目标2中，我们将优化SNC配方和 Ai14 Report和C9-FTD小鼠模型脑内最大给药方案 投放功效。在目标3中，我们将确定切除卵巢的长期生物安全性和治疗效果。 C9-FTD小鼠模型中静脉注射SNC的C9ORF72重复区域 随着拟议的支持IND的研究的成功完成，我们将优化一个候选人 针对C9orf72突变的治疗，并在人类神经元中达到最高的治疗效果 体外和体内C9-FTD小鼠模型。然后，最佳的治疗方法将准备好扩大规模， 非人灵长类动物和大鼠的制造/CMC开发和启用IND的安全性研究。我们会有 此外，还回答了外地的一些悬而未决的问题，包括是否提高了编辑效率 有丝分裂神经元不同于有丝分裂细胞，如何在整个过程中广泛传递多个sgRNA的CRISPR/Cas9 以及是否有可能逆转或阻止有症状的小鼠的临床表型。我们的 目标是加快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.