Correction of Neurological Disease via Allele Specific Excision of Pathogenic Repeats

通过等位基因特异性切除致病重复序列来纠正神经系统疾病

• 批准号: 10668665
• 负责人: JENNIFER A DOUDNA
• 金额: $ 468.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668665
• 关键词: Alleles; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Behavior; Biodistribution; Biological Assay; Biotechnology; C9ALS; C9ORF72; CRISPR/Cas technology; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Convection; Dedications; Dependovirus; Development; Disease; Disease model; Dose; Engineering; Excision; Feedback; Fibroblasts; Formulation; Genes; Genetic; Guide RNA; Haplotypes; Heterozygote; Histologic; Human; Huntington Disease; In Vitro; Induced pluripotent stem cell derived neurons; Injections; Investigational Therapies; Lead; Mendelian disorder; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nucleotides; Outcome; Pathogenicity; Patients; Pharmacology; Pharmacology and Toxicology; Phenotype; Preparation; Process; Production; Property; Rattus; Reagent; Research; Research Personnel; Research Project Grants; Resources; Ribonucleoproteins; Rodent Model; Route; Serotyping; Side; Sum; System; Technology; Testing; Therapeutic; Therapeutic Index; Toxic effect; Toxicology; Transfection; Triplet Multiple Birth; Viral; Virus; Work; animal tissue; brain parenchyma; brain tissue; brain volume; cell transformation; design; efficacy evaluation; efficacy study; experience; genome editing; improved; in vivo; innovation; knock-down; lead candidate; manufacture; mortality; mouse model; mutant; nervous system disorder; nonhuman primate; novel; peptide amphiphiles; postmitotic; pre-clinical; prevent; programs; response; screening; synergism; therapeutic genome editing; tool

PROJECT SUMMARY / ABSTRACT A central promise of genome editing is its potential to treat monogenic disease. Despite early-stage clinical progress for CRISPR-Cas based approaches, monogenic neurodegenerative conditions and nucleotide triplet expansion disorders have not been a focus of any biotechnology company in this space. Our proposal brings together a team of academic investigators to develop a synergistic suite of first-in-class CRISPR-Cas based therapeutics for Huntington's disease (HD) and C9ORF72 amyotrophic lateral sclerosis (ALS). We will engineer and deploy an editing approach that excises, with IND-grade potency and mutant allele-selectivity, the disease- causing expansion repeat from human HTT and C9ORF72 loci, respectively. Our strategy is based on identifying alleles of commonly heterozygous SNPs that reside on the same haplotype as the disease-causing repeat expansion, and then engineering CRISPR-Cas9 for high selectivity of cleavage, on one or both sides of the mutant allele repeat, to drive its excision, with two tiers of delivery innovation. Our trailblazer project (Research Project 1, RP1) will develop an HD therapeutic by packaging mutant HTT-specific CRISPR-Cas9 into a newly developed serotype of adeno-associated virus (AAV) with robust and broad biodistribution in the brain parenchyma of nonhuman primates (NHP). We will implement an innovative strategy in which the CRISPR-Cas9 cassette temporally limits its own expression. We will identify and advance the preclinical lead composition through IND-enabling studies leveraging 3 dedicated Resource Cores to (i) assess molecular outcomes at the genetic level, (ii) administer reagents to animals and observe their behavior, and (iii) assess molecular and histological outcomes from cells and animal tissues. An Administrative Core led by experienced developers of genome editing-based therapeutics, will provide project-management support and lead on preparation of regulatory submissions, aiming to file an HD IND by program end. In RP2, we will apply the AAV-based excision approach to build a cognate experimental therapeutic for C9ORF72-driven ALS. Synergies with RP1 include CMC innovation to manufacture novel AAV, re-use of the self-regulating CRISPR-Cas cassette and virus harboring it, and regulatory feedback on IND-enabling pharmacology, toxicology, and biodistribution studies. We will advance RP2 through pre-IND. For RP3, we will establish a first-in-class, transformative paradigm for in vivo genome editing therapy by reformulating the preclinical lead CRISPR-Cas9 combination used in RP1 into a highly innovative “Cas9 RNP monoparticle” in which amphiphilic peptides deliver the gene editor to neurons upon injection. We will develop approaches for monoparticle manufacture to enable ex vivo and in vivo efficacy studies in HD models. Extensive synergies with RP1 project and comprehensive support by the RCs will enable us to advance this approach to pre-IND by program end. The sum total of this effort will establish a fundamentally new paradigm for in vivo genome editing applicable to all nucleotide repeat expansion disorders, and advance preclinical lead formulations for one disease, HD, to IND, and another such disease, C9ORF ALS, to pre-IND.

项目概要/摘要 基因组编辑的一个核心承诺是其治疗单基因疾病的潜力。尽管处于早期临床阶段 基于 CRISPR-Cas 的方法、单基因神经退行性疾病和核苷酸三联体的进展 扩张障碍并不是该领域任何生物技术公司关注的焦点。我们的建议带来 与学术研究人员团队一起开发基于 CRISPR-Cas 的一流协同套件 亨廷顿病 (HD) 和 C9ORF72 肌萎缩侧索硬化症 (ALS) 的治疗方法。我们将设计 并部署一种编辑方法，以 IND 级效力和突变等位基因选择性切除疾病- 分别引起人类 HTT 和 C9ORF72 基因座的扩增重复。我们的战略基于识别 与致病重复位于同一单倍型上的常见杂合 SNP 的等位基因 扩增，然后对 CRISPR-Cas9 进行改造，以在一侧或两侧进行高选择性切割 突变等位基因重复，以驱动其切除，通过两层递送创新。我们的开拓者项目（研究 项目 1，RP1）将通过将突变型 HTT 特异性 CRISPR-Cas9 包装到新的 开发出腺相关病毒（AAV）血清型，在大脑中具有强大且广泛的生物分布 非人类灵长类动物（NHP）的实质。我们将实施一项创新战略，其中 CRISPR-Cas9 盒暂时限制其自身的表达。我们将确定并推进临床前先导成分 通过利用 3 个专用资源核心的 IND 支持研究来 (i) 评估分子结果 遗传水平，(ii) 对动物施用试剂并观察其行为，以及 (iii) 评估分子和 细胞和动物组织的组织学结果。由经验丰富的开发人员领导的管理核心 基于基因组编辑的疗法，将提供项目管理支持并领导准备 监管提交，旨在在项目结束前提交 HD IND。在 RP2 中，我们将应用基于 AAV 的切除 为 C9ORF72 驱动的 ALS 构建同源实验疗法的方法。与 RP1 的协同效应包括 CMC创新制造新型AAV，重复使用自我调节CRISPR-Cas盒和病毒 包含它，以及对支持 IND 的药理学、毒理学和生物分布研究的监管反馈。我们 将通过预 IND 推进 RP2。对于 RP3，我们将为体内建立一流的变革性范例 通过将 RP1 中使用的临床前先导 CRISPR-Cas9 组合重新配制为基因组编辑疗法 高度创新的“Cas9 RNP 单粒子”，其中两亲性肽将基因编辑器传递给神经元 注射后。我们将开发单粒子制造方法，以实现离体和体内功效 HD 模型的研究。与 RP1 项目的广泛协同作用以及区域协调员的全面支持将使 我们将在计划结束时将这种方法推进到预 IND 阶段。这项努力的总和将从根本上建立 适用于所有核苷酸重复扩增疾病的体内基因组编辑的新范例，并推进 针对一种疾病 HD 的临床前先导制剂，用于 IND，以及另一种此类疾病 C9ORF ALS 的临床前先导制剂。