Evaluation of adeno-associated viral (AAV) mediated gene replacement therapy as a therapeutic option for SLC25A4 deficiency

项目总结/摘要 线粒体氧化磷酸化（OXPHOS）的破坏与以下疾病的发生有关： 通常影响高能量需求组织的生化改变，特别是骨骼和心脏 肌肉.一种遗传性常染色体隐性骨骼肌病和肥厚型心肌病有关联 核DNA编码的线粒体蛋白功能丧失，由于溶质中的移码突变， 携带者家族25，成员4（SLC 25 A4; c.523delC，p.Q175RfxX38）。SLC 25 A4编码心肌 腺嘌呤核苷酸转运子-1（ANT 1，SLC 25 A4）的同种型，其在野生型状态下是一个关键的 线粒体代谢的组成部分。具有SLC 25 A4缺乏的患者显示乳酸酸中毒，持续性 肾上腺素能激活和继发于全身骨骼肌肌病的劳力性不耐受 是肥厚型心肌病最终，心肌增厚和心功能障碍进展至结束- 需要心脏移植的阶段性心力衰竭。 目前没有任何疾病缓解疗法可用于该患者队列。然而，腺- 相关病毒（AAV）介导的基因替代疗法已经成为治疗疾病的有力策略 遗传性单基因疾病的修饰。我们研究的长期目标是开发一种治疗 基因替代策略治疗SLC 25 A4缺陷。本提案的目的是进一步说明 疾病表型以及合成和评估重组AAV（rAAV）载体的功效 在患者来源的细胞系和类器官模型的体外模型中。这个问题的核心假设是 我们认为，AAV介导的基因置换可以改善生物化学和功能效应， SLC 25 A4缺乏症，可以更果断地预防疾病进展。具体目标是 建议如下： 1.表征患者来源细胞系中的SLC 25 A4缺陷表型。 2.合成用于递送密码子优化的SLC 25 A4 cDNA的重组AAV载体， 骨骼肌和心肌细胞。 3.评估AAV-SLC 25 A4病毒转导在患者来源的细胞系中的功效。 这些实验将提高我们对SLC 25 A4的分子机制的理解 这一发现证实了AAV平台的缺陷，并且允许我们评估AAV平台在相关临床前模型中的功效。 此外，我将获得的技能，在这个奖学金将有助于建立我作为一个独立的调查员 一位外科医生兼科学家专注于翻译基因替代疗法的发展。

Project Summary/Abstract Disruption of mitochondrial oxidative phosphorylation (OXPHOS) is associated with the development of biochemical alterations that typically affect tissues with a high energy demand, particularly skeletal and cardiac muscle. An inherited autosomal recessive skeletal myopathy and hypertrophic cardiomyopathy has been linked to loss of function of a nuclear DNA-encoded mitochondrial protein, due to a frameshift mutation in solute carrier family 25, member 4 (SLC25A4; c.523delC, p.Q175RfxX38). SLC25A4 encodes the heart-muscle isoform of the adenine nucleotide translocator-1 (ANT1, SLC25A4), which in the wild-type state is a critical component of mitochondrial metabolism. Patients with SLC25A4 deficiency display lactic acidosis, persistent adrenergic activation, and exertional intolerance secondary to both a general skeletal muscle myopathy as well as a hypertrophic cardiomyopathy. Ultimately, myocardial thickening and cardiac dysfunction progress to end- stage heart failure necessitating cardiac transplantation. There are not currently any disease-modifying therapies available for this patient cohort. However, adeno- associated viral (AAV) mediated gene replacement therapies have emerged as a powerful strategy for disease modification of inherited monogenic disorders. The long-term goal of our research is to develop a therapeutic gene replacement strategy to treat SLC25A4 deficiency. The objective of this proposal is to further characterize the disease phenotype as well as to synthesize and evaluate the efficacy of a recombinant AAV (rAAV) vector in an in vitro model of patient-derived cell lines and organoid models. The central hypothesis of this proposal is that AAV-mediated gene replacement can ameliorate the biochemical and functional effects of SLC25A4 deficiency and can more decisively prevent disease progression. The specific aims of this proposal are: 1. Characterize the SLC25A4 deficiency phenotype in patient-derived cell lines. 2. Synthesize a recombinant AAV vector for delivery of codon-optimized SLC25A4 cDNA to skeletal and cardiac myocytes. 3. Evaluate the efficacy of AAV-SLC25A4 viral transduction in patient-derived cell lines. These experiments will improve our understanding of the molecular mechanisms underlying SLC25A4 deficiency as well as allow us to evaluate the efficacy of an AAV platform in a relevant preclinical model. Moreover, the skills I will acquire during this fellowship will help to establish me as an independent investigator and a surgeon-scientist focused on the development of translational gene replacement therapies.