CRISPR therapy in the canine DMD model

犬 DMD 模型中的 CRISPR 疗法

基本信息

批准号：
10700268
负责人：
Dongsheng Duan
金额：
$ 9.96万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10700268
关键词：
Abbreviations Adult Affect Alanine Animal Model Antigen-Presenting Cells Attenuated Bacterial Proteins Biological Models CD8B1 gene CRISPR/Cas technology Canis familiaris Cells Clustered Regularly Interspaced Short Palindromic Repeats 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 response Immune system Immunity Immunologics Immunosuppression Injections Innate Immune Response Light Maps Mediating MicroRNAs Modeling Monitor Mus Muscle Muscle Cells Mutate Mutation Myopathy Oligonucleotides Outcome Pathology Patients Peptides Pharmaceutical Preparations Physiological Plant Roots Proteins Protocols documentation RNA Reporting Site System T cell response T-Lymphocyte Epitopes TLR9 gene Testing Translating Translations Tumor-infiltrating immune cells Work adeno-associated viral vector canine model cellular transduction clinically relevant combinatorial cytokine cytotoxic effective therapy effector T cell endonuclease functional improvement 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 therapeutic genome editing transgene expression vector vector genome vector-induced

项目摘要

Project Summary/Abstract 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. We have observed nearly lifelong (18 months) Cas9 expression, muscle pathology amelioration, and function improvement in adult mdx mice, the most commonly 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 develop and validate strategies that may mitigate the Cas9-specific CTL response in a clinically relevant large animal model. In this proposal, we will leverage our new findings to explore various 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.

项目概要/摘要杜氏肌营养不良症 (DMD) 是由肌营养不良蛋白基因无效突变引起的。 CRISPR/Cas9 编辑有望从基因根源上治疗 DMD。由于 DMD 影响身体的所有肌肉，因此有效 DMD 的治疗需要全身肌肉输送。腺相关病毒（AAV）载体是唯一的可以有效到达全身肌肉的输送系统。因此，AAV 已成为首选载体用于 CRISPR 介导的 DMD 基因修复疗法。 AAV 载体导致持久的转基因表达。连续的 Cas9 表达会产生两个问题。首先，它增加了脱靶编辑的可能性。第二，细胞毒性 T 淋巴细胞 (CTL) 对细菌来源的 Cas9 蛋白的反应可以消除被处理的细胞并取消治疗。已经开发了许多方法来监控脱靶编辑并改进基因编辑保真度。然而，对 Cas9 特异性 CTL 反应进行建模一直难以实现。小鼠研究揭示了有限的细胞反应，未能消除 Cas9 转导的细胞。我们观察到近成人 mdx 的终生（18 个月）Cas9 表达、肌肉病理改善和功能改善小鼠，最常用的小鼠 DMD 模型。与小鼠模型相反，营养不良的犬齿考虑为人体试验提供更好的模型。确定 Cas9 免疫是否是一个障碍 AAV 介导的 DMD CRISPR 疗法，我们对四只独立的犬进行了全面的研究使用 Cas9 和非 Cas9 AAV 载体的模型（正常犬科动物和三种不同的犬科 DMD 模型）通过局部和全身递送。我们发现令人信服的证据表明 Cas9，而非非 Cas9， AAV 载体诱导了强大的 CTL 反应并消除了基因编辑的肌营养不良蛋白阳性肌纤维。我们的研究建立了可靠的模型系统来研究Cas9免疫。重要的是，它打开了发展之门并验证可减轻临床相关大型动物中 Cas9 特异性 CTL 反应的策略模型。在本提案中，我们将利用我们的新发现来探索可能支持的各种策略在犬 DMD 模型中，持续的治疗编辑不会诱导 CTL 反应。我们的研究将为未来将 AAV CRISPR 疗法应用于 DMD 患者铺平道路。我们的研究结果还将告知 AAV CRISPR 疗法对其他人类疾病的转化。