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

C9orf72额颞叶痴呆的CRISPR基因组编辑器优化及其递送策略

基本信息

批准号：
10746565
负责人：
Claire Clelland
金额：
$ 87万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-18 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10746565
关键词：
5&apos Untranslated Regions Acceleration Address Adult Alleles Alzheimer&apos s Disease Alzheimer&apos s disease related dementia Animals Biodistribution Biological Assay Biological Markers Biological Products Blood - brain barrier anatomy Brain C9FTD C9ORF72 CRISPR/Cas technology Cells Cessation of life Chemistry Clinic Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Conserved Sequence Dementia Development Disease Engineering Excision Exons Formulation Fragile X Syndrome Frontotemporal Dementia Future Genes Genetic Genome Goals Guide RNA Health Expenditures Heterozygote Human Huntington Disease In Vitro Induced pluripotent stem cell derived neurons Injections Intravenous Ligands Link Mitotic Mus Mutation Neurodegenerative Disorders Neurons Particle Size Pathologic Pathology Patients Production Property Proteins Rattus Reagent Regimen Reporter Silicon Dioxide Spinocerebellar Ataxias Surface System Therapeutic Therapeutic Effect Therapeutic Index Therapeutic Intervention Transgenic Mice Transgenic Organisms Treatment Efficacy Wild Type Mouse Work behavioral outcome biomaterial compatibility blood-brain barrier crossing c9FTD/ALS clinical phenotype clinical translation clinically relevant curative treatments delivery vehicle disability dosage effective therapy frontotemporal lobar dementia amyotrophic lateral sclerosis gene function genome editing in vivo in vivo Model induced pluripotent stem cell innovation intravenous injection manufacture mouse model mutant nanocapsule nonhuman primate novel novel therapeutic intervention postmitotic preservation safety study scale up side effect therapeutic candidate therapeutic genome editing therapeutic target tool

项目摘要

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.

项目摘要在C9ORF72基因的单个等位基因中，杂合六核苷酸（GGGGCC）重复膨胀是最多的额颞痴呆（FTD）和肌萎缩性侧面硬化症（ALS）的经常已知的遗传原因，两个致命的和不可逆的神经退行性疾病。鉴于没有有效的FTD治疗迫切需要与阿尔茨海默氏症相关的痴呆症）。靶向C9ORF72基因 CRISPR/CAS9基因组编辑本身可以提供治疗性干预措施。我们已经建立了一种新颖的双SGRNA策略，可以在保留时行使C9orf72重复区域患者衍生的诱导多能干细胞（IPSC）中的所有外显子。切除消除了病理来自这些IPSC的神经元中C9-FTD和保留的正常C9ORF72蛋白水平的标志。给出该区域的序列保守性，该编辑策略适用于所有患者，而不论一般性背景或突变大小。我们还设计了一个新型的二氧化硅纳米胶囊（SNC）平台，该平台可以交叉静脉注射（IV）注射后的血脑屏障（BBB）并将CRISPR基因组编辑提供给整个具有完整BBB的健康动物的大脑。 SNC具有许多理想的特性，包括提供潜水员生物制剂，高递送效率，多功能表面化学，用于配体共轭，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的产生。结合我们独特的C9orf72 通过我们的创新SNC进行编辑方法，能够全身传递基因组编辑者，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AIM 1中，我们将最大化我们的GRNA策略通过识别SGRNA对以高效率行使C9ORF72重复区域在体外有丝分裂后患者衍生的神经元的副作用。在AIM 2中，我们将优化SNC公式和在AI14记者和C9-FTD鼠标模型中，注射方案最大的注射方案，最大交付效率。在AIM 3中，我们将确定切除的长期生物安全和治疗效率 C9ORF72通过C9-FTD小鼠模型中的SNC递送重复区域。随着拟议的指定研究的成功完成，我们将优化候选人针对C9ORF72突变的治疗性，并达到人类神经元中最高的治疗效率体内的C9-FTD小鼠模型。然后，最佳疗法将准备好扩大，非人类灵长类动物和大鼠的制造/CMC开发和辅助安全研究。我们将有此外，还解决了该领域的许多公开问题，包括是否在后期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CRISPR/CAS9 小鼠大脑以及是否有可能在有症状的小鼠中逆转或阻止临床表型。我们的目的是加速基因组编辑，以使神经退行性疾病向诊所加速。