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

神经退行性疾病中完整突变等位基因特异性 CRISPR 的遗传基础

• 批准号: 10447597
• 负责人: Jong-Min Lee
• 金额: $ 43.1万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447597
• 关键词: Affect; Alleles; Alzheimer' s Disease; Animal Model; Behavioral; Biochemical; Biological; Biological Assay; Biological Models; Brain; CAG repeat; Cell model; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Sequence Alteration; Development; Disease; Drug Targeting; Equilibrium; Evolution; Family member; Fibroblasts; Foundations; Future; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genetic Variation; Guide RNA; Haplotypes; Human Genetics; Huntington Disease; Individual; Injections; Investigation; Knock-in Mouse; Knock-out; Knowledge; Location; Measures; Mediating; Medicine; Messenger RNA; Methods; Modeling; Molecular; Mouse Strains; Mus; Mutation; Nervous system structure; Neurodegenerative Disorders; Neuroglia; Neuronal Differentiation; Neurons; Nonsense-Mediated Decay; Nuclear; Observational Study; Outcome; Participant; Pathway interactions; Patients; Prevention; Production; Proteins; RNA; Research; Research Project Grants; Resources; Risk; Route; Safety; Site; Site-Directed Mutagenesis; Specificity; Stains; Tail; Technology; Testing; Testis; Therapeutic; Therapeutic Intervention; Transcript; Transcription Initiation Site; Transgenes; Transgenic Mice; Treatment Efficacy; Variant; Veins; Vertebral column; base; behavioral outcome; behavioral phenotyping; disease-causing mutation; dosage; effective therapy; experimental study; flexibility; frontotemporal lobar dementia-amyotrophic lateral sclerosis; in vivo; induced pluripotent stem cell; innovation; insertion/deletion mutation; knowledge base; molecular phenotype; mutant; nerve stem cell; novel; pre-clinical; precision medicine; preclinical study; prevent; success; targeted treatment; therapeutic target; transcriptome; transcriptome sequencing

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.

摘要/摘要 神经退行性疾病中完全突变型等位基因特异性CRISPR的遗传学基础 许多显性神经退行性疾病的病例无视有效治疗的发展，尽管 它们长期已知的遗传原因和许多隐含的潜在途径，反映了定义的困难 通过侧重于机制的调查，合理地确定药物靶点。或者，发展和 基因靶向/降低方法的成熟凸显了致病突变本身的价值。 作为治疗的靶点。尽管有希望，但降低信使核糖核酸的方法受到以下固有限制 重复治疗的要求、等位基因特异性和剂量控制。我们最近开发了一种完整的 基于CRISPR基因编辑技术的等位基因特异性DNA打靶策略 克服传统的信使核糖核酸靶向方法的关键限制。我们的沉默策略达到了完美 通过使用SNP变异选择性地在突变体上创建CRISPR PAM位点来实现等位基因特异性 等位基因/单倍型。重要的是，我们新的CRISPR基因沉默策略针对的是单倍型骨架 携带致病突变，因此不依赖于 疾病等位基因，提供广泛适用的治疗平台。在这里，我们建议确定治疗方法 基于PAM改变SNP(PAS)的CRISPR策略1)阻止转录或2)诱导 在亨廷顿病(HD)的细胞和动物模型中，无意义介导的突变等位基因的衰退。 简而言之，我们将：1)确定突变的等位基因特异性转录是否通过CRISPR(TP-CRISPR)防止 和无义介导的CRISPR衰变(NMD-CRISPR)有效地在 从HD患者和动物模型的脑来源的神经细胞，2)检测选择性沉默 发育重要基因(即hTt)的突变等位基因影响神经分化能力 诱导多能干细胞(IPSC)，3)比较神经元和神经胶质细胞的靶向效率，4)确定 突变型等位基因特异性的分子/细胞、行为后果和临床前治疗效果 体内CRISPR。我们预计这项研究将1)提名最佳CRISPR目标站点和策略 HD，2)生成关于突变特定CRISPR效率的知识库，以及3)评估 以PAS为基础的等位基因特异性CRISPR作为HD的治疗干预，为新的 HD的创新治疗途径和2)对其他显性神经退行性变的概念验证 疾病。

项目成果

Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR/Cas9.

• DOI: 10.1172/jci.insight.141042
• 发表时间: 2022-10-10
• 期刊: JCI INSIGHT
• 影响因子: 8
• 作者: Shin, Jun Wan;Hong, Eun Pyo;Choi, Doo Eun;Seong, Ihn Sik;Park, Seri S.;Whittaker, Madelynn N.;Kleinstiver, Benjamin P.;Chen, Richard Z.;Lee, Jong-Min
• 通讯作者: Lee, Jong-Min

Haplotype-specific insertion-deletion variations for allele-specific targeting in Huntington's disease.

• DOI: 10.1016/j.omtm.2022.03.001
• 发表时间: 2022-06-09
• 期刊: Molecular therapy. Methods & clinical development
• 作者: Shin JW;Shin A;Park SS;Lee JM
• 通讯作者: Lee JM

PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease.

• DOI: 10.1016/j.omtm.2022.08.005
• 发表时间: 2022-09-08
• 期刊: MOLECULAR THERAPY METHODS & CLINICAL DEVELOPMENT
• 作者: Shin, Jun Wan;Hong, Eun Pyo;Park, Seri S.;Choi, Doo Eun;Zeng, Sophia;Chen, Richard Z.;Lee, Jong-Min
• 通讯作者: Lee, Jong-Min