Abstract: Functional assay development for tafazzin enzyme replacement candidate selection In the proposed research project, we will perform key biochemical, cellular and physiological studies to evaluate the potential of various modified recombinant tafazzin enzyme therapeutics to become the first effective treatment for Barth Syndrome, a rare, life-threatening disorder with no efficacious therapy. Barth Syndrome is a rare X-linked disorder affecting 1:300,000 live births, resulting from defects in the gene encoding Tafazzin, an acyltransferase that modifies cardiolipin to the tetralinoleoyl form and is essential for mitochondrial respiration. Patients with Barth Syndrome develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists for these patients. We are developing potential enzyme replacement therapeutics in which recombinant tafazzin is modified to contain a cellular penetrating peptide that promotes uptake into tafazzin-deficient cells, sometimes in conjunction with an endosomal escape peptide to facilitate escape from lysosomal degradation. We have found that these recombinant tafazzin enzyme replacement therapeutics (rTERTs) can enter tafazzin-deficient cells, localize to mitochondria and correct both cardiolipin remodeling and mitochondrial respiration defects, in vitro and in vivo to varying degrees. To facilitate the commercialization of these reagents, we will develop standardized biochemical, cellular and physiological assays to directly compare enzymatic function, cellular uptake, rescue of cardiolipin remodeling defects, and rescue of defective mitochondrial respiration in cultured cells. Based on these studies, we have developed a selection algorithm to identify a cohort of potential lead candidates that demonstrate the greatest biochemical and biological activity in vitro. The top candidates identified by this algorithm will undergo production process optimization and evaluation for activity in vivo. The in vivo evaluation will consist of pharmacokinetics, tissue distribution, toxicology and efficacy studies in tafazzin knockout mice using an in house tafazzin LC-MS peptide detection assay, an in house ELISA assay to measure antibody formation, in conjunction with measurements of tafazzin function and left ventricular function. We believe that our studies will establish standardized assays to characterize potential enzyme replacement therapies for Barth Syndrome in the R61 phase, thereby facilitating evaluation of potential lead candidates in a mouse model of Barth Syndrome in the R33 phase, ultimately leading to the identification of a lead candidate for further clinical development.

Project Narrative Barth Syndrome is a genetic disorder characterized by muscle dysfunction that results from deficiency of an enzyme called tafazzin. No disease-specific treatments are available and patients uniformly die prematurely. Developing a commercially viable enzyme replacement reagent will have important implications for treating this disorder in human populations.