Developing intrabody therapeutics for mitochondrial DNA heteroplasmy

项目摘要 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）。这种异质性的适度减少有利于正常的mtDNA，可导致显著的患者死亡。 效益我们打算鉴定单链抗体（scFvs），其识别一些特定的结构。 致病线粒体DNA序列。由于单链抗体可以抑制聚合酶，我们试图鉴定新的抗体， 阻断导致原发性线粒体疾病的特定病理性线粒体DNA变异体的复制。ScFvs 已经确定了识别被靶向的DNA结构的一般家族，称为G-四链体 （G4）。使用阳性和阴性噬菌体筛选，我们将分离具有结合特异性G4 结构我们将在生物物理和生物化学实验中表征scFv，以鉴定 区分致病和正常的mtDNA结构。然后将先导scFv用以下物质官能化： 用于在线粒体中靶向表达以测试有益异质性的线粒体靶向序列 班在靶点处表现出最高亲和力和最有效的聚合酶活性阻断的ScFv 然后将在细胞培养中研究序列，其中细胞摄取，定位和表型效应， 异质性和线粒体耗氧量将被确定。这种mt-scFv的优点是 表达方法的优点是它是选择性的，容易靶向线粒体，并且可以重复以维持线粒体的功能。 取得了健康效益。

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.