CRISPR editing therapy for Duchenne muscular dystrophy

杜氏肌营养不良症的 CRISPR 编辑疗法

• 批准号: 10638041
• 负责人: Dongsheng Duan
• 金额: $ 53.02万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638041
• 关键词: Abbreviations; Affect; Alanine; Animal Model; Antigen-Presenting Cells; Attenuated; Bacterial Proteins; Biological Models; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR/Cas technology; Canis familiaris; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cytokeratin-8 Staining Method; Cytotoxic T-Lymphocytes; DNA cassette; Data; Dependovirus; Development; Digestion; Disease; Duchenne muscular dystrophy; Dystrophin; Engineering; Epitopes; Epstein-Barr Virus Nuclear Antigens; Equilibrium; Frameshift Mutation; Future; Generations; Genes; Genetic; Genetic Transcription; Genome engineering; Glycine; Granzyme; Guide RNA; Histologic; Human; Immune; Immune response; Immunity; Immunologics; Immunosuppression; Innate Immune Response; Maps; Mediating; MicroRNAs; Modeling; Monitor; Mus; Muscle; Muscle Cells; Myopathy; Oligonucleotides; Outcome; Pathology; Patients; Peptides; Pharmaceutical Preparations; Physiological; Proteins; Protocols documentation; RNA; Site; System; T cell infiltration; T cell response; T-Lymphocyte Epitopes; TLR9 gene; Testing; Therapeutic; Translating; Translations; Work; adeno-associated viral vector; canine model; cellular transduction; clinically relevant; cytokine; cytotoxic; effective therapy; effector T cell; gene therapy; genome editing; human disease; improved; mdx mouse; mouse model; multicatalytic endopeptidase complex; novel strategies; null mutation; prednisolone; prevent; promoter; protein degradation; public health relevance; response; restoration; therapeutic genome editing; transgene expression; vector; vector genome; vector induced

Project Description Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene. CRISPR/Cas9 editing holds promise to treat DMD at its genetic root. Since DMD affects all muscles in the body, effective therapy for DMD would require bodywide muscle delivery. Adeno-associated virus (AAV) vector is the only delivery system that can efficiently reach all body muscles. For this reason, AAV has been the vector of choice for CRISPR-mediated gene repair therapy for DMD. The AAV vector leads to persistent transgene expression. Continuous Cas9 expression creates two problems. First, it increases the odds of off-target editing. Second, the cytotoxic T lymphocyte (CTL) response to the bacterial-derived Cas9 protein can eliminate the treated cells and abolish the therapy. Many approaches have been developed to monitor off-target editing and improve gene editing fidelity. However, it has been elusive to model the Cas9-specific CTL response. Mouse studies revealed a limited cellular response that failed to eliminate Cas9 transduced cells. In fact, we and others have observed nearly lifelong Cas9 expression, muscle pathology amelioration, and function improvement in mdx mice, the most used mouse DMD model. In contrast to the mouse model, dystrophic canines are considered better models for informing human trials. To determine whether Cas9 immunity is a hurdle for AAV-mediated DMD CRISPR therapy, we performed a comprehensive study in four independent canine models (normal canines and three different canine DMD models) using both Cas9 and non-Cas9 AAV vectors via local and systemic delivery. We found compelling evidence suggesting that the Cas9, but not non-Cas9, AAV vector induced a robust CTL response and eliminated gene-edited dystrophin-positive myofibers. Our studies established a reliable model system to study Cas9 immunity. Importantly, it opens the door to developing and validating strategies that may mitigate the Cas9-specific CTL response in a clinically relevant large animal model. In this proposal, we will leverage our findings to explore novel strategies that may support persistent therapeutic editing without inducing the CTL response in the canine DMD model. Our studies will pave the way for translating AAV CRISPR therapy to DMD patients in the future. Our findings will also inform the translation of AAV CRISPR therapy for other human diseases.

项目描述 Duchenne肌肉营养不良（DMD）是由肌营养不良蛋白基因中的无效突变引起的。 CRISPR/CAS9 编辑有望在其遗传根部处理DMD。由于DMD会影响体内的所有肌肉，因此有效 DMD的治疗将需要全身肌肉递送。腺相关病毒（AAV）载体是唯一的 可以有效地达到所有身体肌肉的输送系统。因此，AAV一直是首选的向量 用于DMD的CRISPR介导的基因修复疗法。 AAV载体导致持续的转基因表达。 连续的CAS9表达会产生两个问题。首先，它增加了脱靶编辑的几率。第二， 细胞毒性T淋巴细胞（CTL）对细菌衍生的CAS9蛋白的反应可以消除处理的细胞 并废除这种疗法。已经开发了许多方法来监视脱靶编辑和改进 基因编辑保真度。但是，对于CAS9特异性CTL响应进行建模是难以捉摸的。小鼠研究 揭示了有限的细胞反应，未能消除CAS9转导的细胞。实际上，我们和其他人有 观察到几乎终生的Cas9表达，肌肉病理学改善和MDX的功能改善 小鼠，最常用的鼠标DMD模型。与小鼠模型相反，营养不良犬被视为 更好的模型，用于为人类试验提供信息。确定CAS9免疫是否是AAV介导的障碍 DMD CRISPR疗法，我们在四种独立的犬模型（正常 通过局部和 系统性交付。我们发现了令人信服的证据，表明CAS9而不是非Cas9，AAV矢量 诱导了强大的CTL响应，并消除了基因编辑的肌营养不良蛋白阳性肌纤维。我们的研究 建立了一个可靠的模型系统来研究CAS9免疫力。重要的是，它为发展和 验证可能减轻临床相关的大动物中CAS9特异性CTL反应的策略 模型。在此提案中，我们将利用我们的发现来探索可能支持持久性的新型策略 治疗性编辑而无需诱导犬DMD模型中的CTL响应。我们的研究将为 将来将AAV CRISPR疗法转化为DMD患者的方法。我们的发现还将告知 对其他人类疾病的AAV CRISPR疗法的翻译。