First murine animal model and adeno-associated virus (AAV)-based gene therapy for MTATP6 mitochondrial diseases

首个针对 MTATP6 线粒体疾病的小鼠动物模型和基于腺相关病毒 (AAV) 的基因治疗

• 批准号: 10506768
• 负责人: Qinglan Ling
• 金额: $ 9.11万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10506768
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Address; Animal Model; Behavioral; Biochemical; Biology; Brain; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell physiology; Clinical Trials; Code; Codon Nucleotides; Complement; Cre driver; Cre-LoxP; DNA; DNA Sequence; Dependovirus; Development; Disease; Dose; Exhibits; Faculty; Fibroblasts; Foundations; Future; Genes; Heart; Hereditary Disease; Human; Hybrid Cells; Impairment; In Vitro; Inherited; Institution; Knowledge; Laboratories; Lead; Leigh Disease; Liver; Long-Term Effects; Mentors; Missense Mutation; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proton-Translocating ATPases; Mus; Mutation; Neuraxis; Neurodegenerative Disorders; Neurologic; Neuropharmacology; Nuclear; Pathology; Patients; Phase; Phenotype; Positioning Attribute; Proteins; Recombinant adeno-associated virus (rAAV); Research; Resources; Respiratory physiology; Route; Safety; Scheme; Skeletal Muscle; Specificity; Technology; Terminator Codon; Testing; Therapeutic; Tissues; Training; Transgenic Mice; Treatment Efficacy; Variant; Wild Type Mouse; adeno-associated viral vector; base; base editing; base editor; career; career development; cell growth; complex IV; early onset; efficacy evaluation; gene replacement therapy; gene therapy; heteroplasmy; in vitro testing; in vivo; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial gene replacement; mouse genetics; mouse genome; mouse model; mutant; mutant mouse model; nervous system disorder; new technology; novel; oligomycin sensitivity-conferring protein; skills; tenure track; tool; training opportunity; translational study; vector; vector biodistribution

Project Summary/Abstract This proposal aims to provide crucial training for the applicant’s long-term career plan to develop gene therapy approaches for patients with neurological mitochondrial diseases, which currently have no approved treatment. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option for inherited diseases evident by numerous recent clinical trials. The applicant’s previous study developed an AAV-based gene replacement therapy for SURF1-related Leigh syndrome, an early onset neurodegenerative disease, and demonstrated its efficacy and safety in vivo. Mutations in the mitochondrial DNA-encoded ATPase 6 (MTATP6) gene represents another group of common causes for neurological mitochondrial diseases. Unlike SURF1, a nuclear DNA-encoded gene, gene replacement for MTATP6 poses more challenges. Firstly, no suitable vectors deliver gene directly into mitochondria. A potential approach is the allotopic expression, in which a wildtype copy of MTATP6 is expressed in the nucleus and the ATP6 protein is relocated to mitochondria using a mitochondrial targeting sequence. Another hurdle is that there is no MTATP6 mouse model available, and the allotopic expression of MTATP6 has only been tested in the cell cultures. To address these issues, the applicant seeks to develop the first MTATP6 mouse model using a novel mitochondria-targeting base editing technology, which creates a truncating mutation in the mouse MTATP6 gene. She will then use this mouse model to evaluate the in vivo efficacy and safety of the allotopic expression of MTATP6 via AAV delivery. In the K99 phase, the applicant will develop and optimize the base editor for mouse MTATP6 gene, and generate a conditional truncated MTATP6 mouse model. She will also develop an AAV vector to deliver the allotopic MTATP6 and test the vectors in patient-derived cell lines with both MTATP6 truncating mutations and m.8993T>G missense mutation, the most common disease variant in MTATP6. In the R00 phase, the applicant will characterize the truncated MTATP6 mouse model, and evaluate the safety and efficacy of the AAV gene therapy in vivo. This study will set a clear path for a translational study for MTATP6 patients with truncating mutations, and provide a foundation for the future development for other MTATP6 variants. The applicant will acquire crucial knowledge and laboratory skills in mitochondrial biology, base editing, and transgenic mouse modeling during her K99 phase to complement her previous training on AAV gene therapy and neuropharmacology. Additionally, the applicant’s career development will be enhanced by the expertise of an exceptional advisory/mentoring committee, as well as the unparalleled resources and ample educational and training opportunities at UT Southwestern, a world class research institution. The outstanding mentoring, unmatched resources, and strong commitment from her department will strengthen the applicant’s candidacy for, and transition to, an independent tenure-track faculty position.

项目摘要/摘要 该建议旨在为申请人的长期职业计划提供重要的培训，以制定基因疗法 神经学线粒体疾病患者的方法，目前尚无批准治疗。 基于腺体相关的病毒（AAV）基因疗法是遗传疾病的有前途的治疗选择 许多近期临床试验的证据。申请人先前的研究开发了基于AAV的基因 与Surf1相关的Leigh综合征的替代疗法，一种早期发作神经退行性疾病和 在体内证明了其有效性和安全性。线粒体DNA编码的ATPase 6中的突变（MTATP6） 基因代表了神经线粒体疾病的另一组常见原因。与Surf1不同， 核DNA编码的基因，MTATP6的基因替代具有更多的挑战。首先，没有合适的向量 将基因直接输送到线粒体中。潜在的方法是同种型表达，其中野生型复制 MTATP6在细胞核中表达，然后使用线粒体将ATP6蛋白迁移到线粒体 靶向序列。另一个障碍是没有MTATP6鼠标模型，并且有同倍数 MTATP6的表达仅在细胞培养物中进行了测试。 为了解决这些问题，适用的试图使用小说开发第一个MTATP6鼠标模型 线粒体靶向基础编辑技术，该技术在小鼠MTATP6基因中产生截断的突变。 然后，她将使用此鼠标模型评估同种异体表达的体内效率和安全性 MTATP6通过AAV交付。在K99阶段，申请人将开发并优化鼠标的基本编辑器 MTATP6基因，并生成条件截断的MTATP6小鼠模型。她还将发展一个AAV 向量输送同种异体MTATP6并测试具有MTATP6的患者衍生细胞系中的向量 截断突变和M.8993T> g错义突变，这是MTATP6中最常见的疾病变体。在 R00阶段，适用的将表征截短的MTATP6鼠标模型，并评估安全性和 AAV基因疗法在体内的功效。这项研究将为MTATP6翻译研究树立清晰的途径 截断突变的患者，并为其他MTATP6的未来发展奠定了基础 变体。 申请人将获得线粒体生物学，基础编辑和 在其K99阶段的转基因小鼠建模，以补充她先前对AAV基因疗法的培训 和神经药理学。此外，申请人的职业发展将由专家提高 一个卓越的咨询/指导委员会，以及无与伦比的资源和充足的教育和 在世界一流研究机构UT西南部的培训机会。出色的心理， 无与伦比的资源和部门的坚定承诺将加强申请人的候选人的候选人， 并过渡到独立的终身教师职位。