Testing Optimal Gene Editor for an Alzheimer's CRISPR therapeutic.

测试阿尔茨海默病 CRISPR 疗法的最佳基因编辑器。

• 批准号: 10746716
• 负责人: Subhojit Roy
• 金额: $ 211.28万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10746716
• 关键词: Advisory Committees; Alzheimer' s Disease; Alzheimer' s disease therapeutic; Alzheimer' s disease therapy; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Attenuated; Biological Assay; Brain; CRISPR therapeutics; CRISPR/Cas technology; Cell surface; Cells; Clinic; Clinical; Clinical Paths; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Consensus; Consultations; Deposition; Disease; Dose; Down Syndrome; Early Onset Alzheimer Disease; Endosomes; Equilibrium; Etiology; Event; Exons; Eye diseases; FDA approved; Failure; Future; Gene Proteins; Genes; Genetic; Genome; Goals; Guide RNA; Hand; Human; Human Amyloid Precursor Protein; Human Genetics; Injections; Kinetics; Knock-in; Knock-in Mouse; Lead; Letters; Measures; Mediating; Mission; Modality; Mouse Protein; Mus; Nerve Regeneration; Neurofibrillary Tangles; Neurons; Organoids; Outcome; Pathologic; Pathway interactions; Persons; Pharmaceutical Preparations; Phase I/II Trial; Physiological; Physiology; Positioning Attribute; Protein C; Protein Fragment; Protein Truncation; Proteins; Proteolysis; Public Health; Quantitative Reverse Transcriptase PCR; RNA; Recommendation; Research; Research Personnel; Retina; Role; Safety; Surface; Testing; Therapeutic; Time; Transcript; Transmembrane Domain; United States National Institutes of Health; Variant; adeno-associated viral vector; base editing; beta-site APP cleaving enzyme 1; cost; efficacy testing; experimental study; extracellular; genome-wide; in vivo; induced pluripotent stem cell; meetings; mouse model; neuroprotection; nonhuman primate; novel; novel therapeutics; prevent; response; secretase; tau Proteins; tau aggregation; therapeutic genome editing; translation to humans

Summary/Abstract: The overall goal of this U01 proposal is to advance a unique CRISPR-based therapeutic for Alzheimer’s disease (AD) towards the pre-IND stage, by determining the best genome-editor/gRNA combination that can be ultimately used in clinical trials. Though there is an enormous unmet need for developing therapeutics for AD, we do not have drugs that can unequivocally slow down the relentless course of AD. We have developed a gene-editing based strategy targeting APP – a gene with a central and indisputable role in AD; strongly supported by human genetics. We do not eliminate the APP gene, but edit out a pentapeptide YENPTY endocytic domain at the C- terminus of APP, which blocks the entire pathologic APP β-cleavage pathway (including β-amyloid). The transmembrane domain and the N-terminus remains intact, and the ∆C APP is retained on the cell surface, leading to an increase in APP α-cleavage, which in turn upregulates neuroprotective and neuroregenerative APP fragments. Thus, our strategy shifts the balance of APP cleavage from pathologic to physiologic, without eliminating the gene. Since the targeted events are upstream, our strategy should be applicable to forms of AD – sporadic and familial – and also to early-onset AD in Down syndrome, where APP triplication on Ch.21 invariably causes AD. So far, we have used the classical SaCas9 to demonstrate safety and efficacy of our approach in vivo, but SaCas9 is too big to fit into a single AAV, which will be necessary for ultimate delivery in humans. Moreover, since there is no FDA-approved gene-editing clinical trial in the brain yet, and the best genome-editor is unknown. Here, we propose to first test two promising small genome-editors (and corresponding gRNAs) that can fit into a single AAV – SaCas9 and NmCas9 – using genome-scale on- and off- target assays (Aim 1). The best Lead-Editor/gRNA combinations emerging from these experiments will be tested for efficacy, safety, predictability, and durability in human brain organoids and a novel APP mouse model where the entire mouse APP was replaced with human APP (Aim 2). Collectively, these studies will not only clarify the best genome-editor/RNA combination for editing the APP gene, but also help future trials in the brain gene- editing space by determining the best editor for brain-relevant applications. Towards the end of our projects, we propose to hold an INTERACT meeting with the FDA – in consultation with our Clinical/Translational Advisory Team – to get regulatory input and consensus on pre-IND NHP studies. Upon completion, our studies will not only offer a novel therapeutic in an arena of enormous unmet need and litany of failures, but also provide a clinical path for future gene-editing efforts in this therapeutic space.

摘要/摘要： U01 提案的总体目标是推进一种独特的基于 CRISPR 的阿尔茨海默病治疗方法 (AD) 进入 IND 前阶段，通过确定最终可实现的最佳基因组编辑器/gRNA 组合 用于临床试验。尽管开发 AD 疗法的需求尚未得到满足，但我们并不 拥有可以明确减缓 AD 持续进程的药物。我们开发了基因编辑 基于 APP 的策略——该基因在 AD 中具有无可争议的核心作用；得到人类的大力支持 遗传学。我们没有消除 APP 基因，而是编辑了 C- 处的五肽 YENPTY 内吞结构域 APP 的末端，阻断整个病理性 APP β-裂解途径（包括 β-淀粉样蛋白）。这 跨膜结构域和 N 末端保持完整，ΔC APP 保留在细胞表面， 导致 APP α-裂解增加，进而上调神经保护和神经再生 APP 碎片。因此，我们的策略将 APP 裂解的平衡从病理性转变为生理性，而无需 消除基因。由于目标事件位于上游，因此我们的策略应该适用于 AD 的形式 – 散发性和家族性 – 以及唐氏综合症中的早发性 AD，其中 APP 在 Ch.21 上三倍 总是会导致AD。到目前为止，我们已经使用经典的 SaCas9 来证明我们的安全性和有效性 体内方法，但 SaCas9 太大，无法放入单个 AAV 中，这对于最终在体内的递送是必要的 人类。此外，由于目前还没有 FDA 批准的大脑基因编辑临床试验，最好的 基因组编辑器未知。在这里，我们建议首先测试两个有前途的小型基因组编辑器（和 相应的 gRNA），可以适应单个 AAV – SaCas9 和 NmCas9 – 使用基因组规模的on-和off- 目标测定（目标 1）。这些实验中出现的最佳 Lead-Editor/gRNA 组合将接受测试 人脑类器官的有效性、安全性、可预测性和耐久性，以及一种新型 APP 小鼠模型，其中 整个小鼠 APP 被人类 APP 取代（目标 2）。总的来说，这些研究不仅将阐明 用于编辑APP基因的最佳基因组编辑器/RNA组合，同时也有助于未来大脑基因的试验- 通过确定与大脑相关的应用程序的最佳编辑器来编辑空间。在我们的项目即将结束时，我们 建议与 FDA 举行 INTERACT 会议 – 与我们的临床/转化咨询顾问协商 团队 – 就 IND 前 NHP 研究获得监管意见和共识。完成后，我们的学习不会 不仅在巨大的未满足需求和一连串失败的领域提供了一种新颖的治疗方法，而且还提供了一种 该治疗领域未来基因编辑工作的临床路径。