Correction of Neurological Disease via Allele Specific Excision of Pathogenic Repeats

项目总结/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。尽管早期临床 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 扩张障碍尚未成为该领域中任何生物技术公司的焦点。我们的提议带来了 与一个学术研究团队一起开发一套基于CRISPR-Cas的协同套件， 用于治疗亨廷顿病（HD）和C9 ORF 72肌萎缩侧索硬化（ALS）的药物。我们将工程师 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- 分别引起来自人HTT和C9 ORF 72基因座的扩增重复。我们的策略是 与致病重复序列位于同一单倍型上的常见杂合SNP的等位基因 扩增，然后工程化CRISPR-Cas9以用于高选择性的切割，在扩增的一侧或两侧进行。 突变等位基因重复，以驱动其切除，具有两层递送创新。我们的开拓者项目（研究 项目1，RP 1）将通过将突变的HTT特异性CRISPR-Cas9包装到新的 腺相关病毒（AAV）的血清型在脑中具有稳健和广泛的生物分布 非人灵长类动物的薄壁组织（NHP）。我们将实施一项创新战略， 盒子暂时限制了它自己的表达。我们将确定和推进临床前铅成分 通过利用3个专用资源核心的IND使能研究，（i）评估 遗传水平，（ii）向动物施用试剂并观察它们的行为，以及（iii）评估分子和 从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 基因组编辑为基础的疗法，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RP 2中，我们将应用基于AAV的切除， 方法来建立C9 ORF 72驱动的ALS的同源实验治疗。与RP 1的协同作用包括 CMC创新生产新型AAV，重新使用自调节CRISPR-Cas盒和病毒 以及对IND药理学、毒理学和生物分布研究的监管反馈。我们 我们将通过pre-IND推进RP 2。对于RP 3，我们将建立一个一流的，变革性的体内模型， 通过将RP 1中使用的临床前先导CRISPR-Cas9组合重新配制成 高度创新的“Cas9 RNP单颗粒”，其中两亲肽将基因编辑器递送到神经元 在注射时。我们将开发单颗粒制造方法，以实现体外和体内功效 HD模型的研究。与RP 1项目的广泛协同作用和驻地协调员的全面支持将使 我们希望在项目结束时将这种方法推进到IND前。这一努力的总和将从根本上建立一个 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式，以及进展 用于一种疾病（HD）的临床前先导制剂发展为IND，而用于另一种疾病（C9 ORF ALS）的临床前先导制剂发展为pre-IND。

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.