Genetic foundation for complete mutant allele-specific CRISPR in neurodegenerative diseases

总结/摘要 标题：神经退行性疾病中完全突变等位基因特异性CRISPR的遗传基础 许多显性神经退行性疾病的病例无视有效治疗的发展， 他们的长期已知的遗传原因和许多牵连的潜在途径，反映了难以界定 通过机制研究，合理确定药物靶点。另一方面，发展和 基因靶向/降低方法的成熟突出了致病突变本身的价值 作为治疗的目标。虽然有希望，但降低mRNA的方法受到生物学特性的固有限制。 需要重复治疗、等位基因特异性和剂量控制。我们最近开发了一套完整的 基于CRISPR基因编辑技术的等位基因特异性DNA靶向策略， 克服了常规mRNA靶向方法的关键限制。我们的消音策略达到了完美的效果 等位基因特异性，通过使用SNP变异，在突变体上选择性地产生CRISPR PAM位点 等位基因/单倍型。重要的是，我们的新型CRISPR基因沉默策略靶向了 携带致病突变，因此不依赖于类型，大小和位置的 疾病等位基因，提供广泛适用的治疗平台。在这里，我们建议确定治疗 基于PAM改变SNP（PAS）的CRISPR策略的潜力1）防止转录或2）诱导 在细胞和亨廷顿病（HD）动物模型中突变等位基因的无义介导的衰减。 简言之，我们将：1）确定突变等位基因特异性CRISPR转录预防（TP-CRISPR） 和无义介导的CRISPR衰变（NMD-CRISPR）有效地将基因敲除突变引入细胞中。 来源于HD患者和动物模型脑的神经元细胞，2）测试是否选择性沉默 发育重要基因的突变等位基因（即，HTT）影响神经元分化能力， 诱导多能干细胞（iPSC），3）比较神经元和神经胶质中的靶向效率，以及4）确定 突变等位基因特异性的分子/细胞、行为后果和临床前治疗功效 CRISPR in vivo.我们预计这项研究将1）提名最佳的CRISPR靶向位点和策略， HD，2）生成关于多克隆特异性CRISPR的效率的知识库，以及3）评估多克隆特异性CRISPR的潜力。 基于PAS的等位基因特异性CRISPR作为HD的治疗性干预，为1）新的 HD的创新治疗途径和2）其他显性神经退行性疾病的概念验证 疾病

SUMMARY / ABSTRACT Title: Genetic foundation for complete mutant allele-specific CRISPR in neurodegenerative diseases Many cases of dominant neurodegenerative disorders defy the development of effective treatments despite their long-known genetic causes and numerous implicated underlying pathways, reflecting difficulty in defining rational drug targets through investigations focusing on mechanisms. Alternatively, development and maturation of gene targeting/lowering approaches have highlighted the value of disease-causing mutation itself as the target of treatments. Although promising, mRNA-lowering approaches suffer from inherent limitations of requirement of repeated treatments, allele-specificity, and dosage controls. We recently developed a complete allele-specific DNA targeting strategy based on CRISPR gene editing technology using PAM-altering SNP to overcome key limitations of conventional mRNA targeting approaches. Our silencing strategy achieves perfect allele specificity by using SNP variations that create CRISPR PAM sites selectively on the mutant allele/haplotype. Importantly, our novel CRISPR gene silencing strategy targets the haplotype backbone that carries the disease-causing mutation, and therefore does not depend on the type, size, and location of the disease allele, providing broadly applicable therapeutic platforms. Here, we propose to determine therapeutic potential of PAM-Altering SNP (PAS)-based CRISPR strategies 1) to prevent the transcription or 2) to induce nonsense-mediated decay of the mutant allele in cells and animal models of Huntington's disease (HD). Briefly, we will: 1) determine whether mutant allele-specific Transcription Prevention by CRISPR (TP-CRISPR) and Nonsense-Mediated Decay by CRISPR (NMD-CRISPR) efficiently introduce knockout mutations in neuronal cells derived from patients and brains of animal models of HD, 2) test whether selective silencing of mutant allele of a developmentally important gene (i.e., HTT) influences neuronal differentiation capability of induced pluripotent stem cells (iPSC), 3) compare targeting efficiency in neurons and glia, and 4) determine molecular/cellular, behavioral consequences, and pre-clinical therapeutic efficacy of mutant allele-specific CRISPR in vivo. We anticipate this research will 1) nominate optimal CRISPR targeting sites and strategies for HD, 2) generate knowledge base regarding efficiency of mutant-specific CRISPR, and 3) evaluate potential of PAS-based allele-specific CRISPR as therapeutic intervention for HD, providing 1) genetic foundation for novel and innovative therapeutic routes for HD and 2) proof-of-concept for other dominant neurodegenerative diseases.