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: 10684251
• 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/10684251
• 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; Central Nervous System; Clinical Trials; Code; Codon Nucleotides; Complement; Cre driver; Cre-LoxP; Cytoplasm; DNA; DNA Sequence; Dependovirus; Development; Disease; Dose; Exhibits; Faculty; Fibroblasts; Foundations; Functional disorder; Future; Genes; Heart; Hereditary Disease; Human; Hybrids; 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; 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; Training and Education; Transduction Gene; Transgenic Mice; Treatment Efficacy; Variant; Wild Type Mouse; adeno-associated viral vector; 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 这项提案旨在为申请者开发基因疗法的长期职业计划提供关键培训 神经线粒体疾病患者的治疗方法，目前还没有得到批准的治疗方法。 基于腺相关病毒(AAV)的基因治疗是治疗遗传性疾病的一种很有前途的选择 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一种基于AAV的基因 SURF1相关Leigh综合征的替代疗法，SURF1相关Leigh综合征是一种早期发病的神经退行性疾病，以及 在体内证明了其有效性和安全性。线粒体DNA编码的ATPase 6(MTATP6)突变 基因代表了神经线粒体疾病的另一组常见原因。与SURF1不同的是， 对于核DNA编码基因，MTATP6的基因替换提出了更多的挑战。首先，没有合适的载体 将基因直接送入线粒体。一种可能的方法是异位表达式，在该表达式中，一个通配类型副本 在细胞核中表达，ATP6蛋白通过线粒体被重新定位到线粒体 目标序列。另一个障碍是没有可用的MTATP6小鼠模型，而且同种异体 仅在细胞培养中检测到MTATP6的表达。 为了解决这些问题，申请人寻求开发第一个MTATP6小鼠模型，该模型使用一种新颖的 线粒体靶向碱基编辑技术，在小鼠MTATP6基因中产生截断突变。 然后，她将使用这个小鼠模型来评估同种异体表达的体内有效性和安全性 MTATP6通过AAV递送。在K99阶段，申请者将开发和优化鼠标的基础编辑器 MTATP6基因，建立条件截短的MTATP6小鼠模型。她还将开发一种AAV 载体传递异位MTATP6并在同时携带MTATP6和MTATP6的患者来源的细胞系中测试载体 截断突变和m.8993T&gt；G错义突变，MTATP6中最常见的疾病变异。在 R00阶段，申请人将鉴定截短的MTATP6小鼠模型，并评估其安全性和 甲型肝炎病毒基因治疗的体内疗效。这项研究将为MTATP6的翻译研究铺平道路 为MTATP6的进一步研究奠定了基础 变种。 申请人将获得线粒体生物学、碱基编辑和实验室的关键知识和技能 在她的K99期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因以下方面的专长而得到加强 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 在世界级研究机构德克萨斯大学西南分校的培训机会。杰出的导师， 无与伦比的资源和她所在部门的坚定承诺将加强申请人的候选人资格， 并过渡到一个独立的终身教职教师职位。