Developing intrabody therapeutics for mitochondrial DNA heteroplasmy

点击翻译按钮获取中文摘要

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.