Correction of Neurological Disease via Allele Specific Excision of Pathogenic Repeats

项目摘要/摘要 基因组编辑的一个核心承诺是它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、单基因神经退变条件和核苷酸三联体的研究进展 扩张障碍并不是这一领域任何一家生物技术公司关注的焦点。我们的建议带来了 联合一组学术研究人员开发一流的CRISPR-CAS协同套件 治疗亨廷顿病(HD)和C9ORF72肌萎缩侧索硬化症(ALS)。我们将进行工程设计 并部署一种编辑方法，以Ind级的效力和突变的等位基因选择性切除疾病- 分别导致人HTT和C9ORF72基因座的扩展重复。我们的战略是基于确定 与致病重复序列位于同一单倍型上的常见杂合性SNPs的等位基因 扩张，然后在一侧或两侧设计CRISPR-Cas9以实现高选择性切割 突变的等位基因重复，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，RP1)将通过将突变的HTT特异性CRISPR-Cas9包装到一个新的 已发展的腺相关病毒(AAV)血清型，在脑内具有广泛的生物分布 非人灵长类的薄壁组织(NHP)。我们将实施CRISPR-CAS9创新战略 卡带在时间上限制了它自己的表达。我们将确定并推进临床前铅的成分 通过启用IND的研究，利用3个专用资源核心来(I)评估分子结果 遗传水平，(Ii)给动物注射试剂并观察它们的行为，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 以基因组编辑为基础的治疗学，将提供项目管理支持并领导准备 监管意见书，目标是在计划结束前提交HD Ind。在RP2中，我们将应用基于AAV的切除 建立C9ORF72驱动的ALS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 拥有它，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Pre-Ind推进RP2。对于RP3，我们将为体内建立一流的、变革性的范式 基因组编辑治疗通过将RP1中使用的临床前领先CRISPR-Cas9组合重新配制为 高度创新的Cas9 RNP单粒子，其中两亲性多肽将基因编辑器递送到神经元 在注射时。我们将开发单粒制品的制造方法，以实现体外和体内的效果 高清模型的研究。与RP1项目的广泛协同作用和RCS的全面支持将使 我们将把这一方法推进到项目结束前的IND。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张性疾病的体内基因组编辑的新范式 一种疾病的临床前铅制剂，HD，到IND，以及另一种这样的疾病，C9ORF ALS，到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.