Developing intrabody therapeutics for mitochondrial DNA heteroplasmy

项目总结 许多疾病都是由线粒体DNA的正常和致病突变形式引起的。 (MtDNA)。适度减少这种异质性，有利于正常的mtDNA，可能会导致重大患者 利益。我们打算识别识别某些特定结构的单链抗体(ScFv)。 致病线粒体DNA序列。因为单链抗体可以抑制聚合酶，所以我们试图鉴定新的抗体。 阻止导致原发线粒体疾病的特定病理性mtDNA变体的复制。单链抗体 已发现识别靶向DNA结构的一般家族，称为G-四链体 (G4S)利用阳性和阴性噬菌体筛选，我们将分离出具有结合特定G4的能力的单链抗体 结构。我们将在生物物理和生化实验中表征单链抗体，以确定领先的候选者 区分致病线粒体DNA结构和正常线粒体DNA结构。然后，销售线索scFv将通过以下功能实现 线粒体靶向序列在线粒体中的靶向表达以测试有益的异质性 换档。表现出最高亲和力和最有效地阻断靶标聚合酶活性的单链抗体 然后将在细胞培养中研究序列，细胞摄取、定位和表型对 将测定异质性和线粒体耗氧量。这种mt-scFv的优点 表达方式是，它是选择性的，很容易靶向线粒体，并且可以重复维持 实现了健康效益。

PROJECT SUMMARY Numerous diseases arise from the presence of both normal and pathogenic mutant forms of mitochondrial DNA (mtDNA). A modest reduction of this heteroplasmy in favor of the normal mtDNA can lead to significant patient benefit. We intend to identify single-chain antibodies (scFvs) that recognize structures unique to some pathogenic mtDNA sequences. Because scFvs can inhibit polymerases, we seek to identify novel antibodies that block replication of particular pathological mtDNA variants that cause primary mitochondrial disorders. ScFvs have been identified that recognize the general family of DNA structures being targeted, called G-quadruplexes (G4s). Using positive and negative phage screening, we will isolate scFvs with the ability to bind a specific G4 structure. We will characterize scFvs in biophysical and biochemical experiments to identify lead candidates that discriminate between pathogenic and normal mtDNA structures. Lead scFvs will then be functionalized with mitochondrial-targeting sequences for targeted expression in mitochondria to test for beneficial heteroplasmy shift. ScFvs that exhibit the highest affinity and most potent blockage of polymerase activity at the target sequence will then be studied in cell culture, where cell uptake, localization, and phenotypic effects on heteroplasmy and mitochondrial oxygen consumption will be determined. The advantages of this mt-scFv expression approach are that it is selective, easily targeted to mitochondria, and can be repeated to maintain the achieved health benefit.