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

项目概要/摘要 该提案旨在为申请人开发基因疗法的长期职业计划提供重要的培训 针对患有神经线粒体疾病的患者的方法，目前尚无批准的治疗方法。 基于腺相关病毒（AAV）的基因疗法是遗传性疾病的一种有前景的治疗选择 最近的大量临床试验证明了这一点。申请人之前的研究开发了基于AAV的基因 SURF1 相关 Leigh 综合征（一种早发性神经退行性疾病）的替代疗法，以及 证明了其在体内的有效性和安全性。线粒体 DNA 编码的 ATP 酶 6 (MTATP6) 突变 基因代表神经线粒体疾病的另一组常见原因。与 SURF1 不同的是， 核DNA编码的基因，对MTATP6的基因替换提出了更多的挑战。首先，没有合适的载体 将基因直接传递到线粒体中。一种潜在的方法是同位素表达，其中野生型拷贝 MTATP6 在细胞核中表达，并且使用线粒体将 ATP6 蛋白重新定位到线粒体 靶向序列。另一个障碍是没有可用的 MTATP6 小鼠模型，并且同位素 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，申请人寻求使用一种新颖的方法开发第一个 MTATP6 小鼠模型 线粒体靶向碱基编辑技术，可在小鼠 MTATP6 基因中产生截短突变。 然后，她将使用该小鼠模型来评估同位素表达的体内功效和安全性 MTATP6 通过 AAV 传递。 K99阶段，申请人将开发并优化小鼠碱基编辑器 MTATP6基因，并生成条件截短的MTATP6小鼠模型。她还将开发 AAV 载体递送同位素 MTATP6 并用 MTATP6 在患者来源的细胞系中测试载体 截短突变和 m.8993T>G 错义突变，这是 MTATP6 中最常见的疾病变异。在 R00阶段，申请人将表征截短的MTATP6小鼠模型，并评估安全性和 AAV基因治疗的体内功效。这项研究将为 MTATP6 的转化研究奠定明确的道路 截短突变患者，并为其他MTATP6的未来发展提供基础 变体。 申请人将获得线粒体生物学、碱基编辑和 在 K99 阶段进行转基因小鼠建模，以补充她之前的 AAV 基因治疗培训 和神经药理学。此外，申请人的职业发展将通过以下专业知识得到加强： 一个杰出的咨询/指导委员会，以及无与伦比的资源和充足的教育和 世界一流研究机构 UT Southwestern 的培训机会。出色的辅导， 无与伦比的资源以及她所在部门的坚定承诺将增强申请人的候选资格， 并过渡到独立的终身教授职位。

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.