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.

项目摘要/摘要 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。我们已经观察到终身（18个月）CAS9表达，肌肉病理学改善和成人MDX的功能改善小鼠，最常用的鼠标DMD模型。与小鼠模型相反，营养不良犬是被认为是为人类试验提供的更好模型。确定CAS9免疫是否是障碍 AAV介导的DMD CRISPR疗法，我们在四个独立的犬类中进行了全面研究使用Cas9和non-Cas9 AAV向量的型号（正常犬和三种不同的犬DMD模型）通过本地和系统交付。我们发现了令人信服的证据，表明CAS9而不是非Cas9， AAV载体诱导了强大的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。重要的是，它打开了开发的大门并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，我们将利用我们的新发现来探索各种可能支持的策略持续的治疗编辑，而无需在犬DMD模型中诱导CTL响应。我们的研究将将来将AAV CRISPR疗法转化为DMD患者的道路。我们的发现也将告知对其他人类疾病的AAV CRISPR疗法的翻译。