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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.

项目总结/文摘