Engineered Isoforms of Tafazzin Demonstrate Differential Cardiolipin Remodeling Activity and Protein Stability and Inform Potential Gene and Enzyme replacement therapies

Junya Awata, Rahul Raghav, Xilin Wang, Michael T. Chin
Molecular Cardiology Research Institute Tufts Medical Center Boston, MA

Barth Syndrome is a rare X-linked disorder affecting 1:140,000 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 and sometimes in conjunction with a polyhistidine tag to facilitate protein purification. To assess the effects of these protein modifications, we have generated recombinant lentiviruses to express these modified proteins in mouse embryo fibroblasts isolated from tafazzin knockout mice. We have found that the majority of these recombinant tafazzin constructs can correct cardiolipin remodeling defects in these cells but those that do not seem to be limited by protein stability. Selected lentiviruses are being tested for rescue of CL remodeling in tafazzin KO mice. To facilitate the development of recombinant tafazzin enzyme replacement therapeutics (rTERTs), proteins shown to be active in lentiviruses are being expressed in a mammalian floating HEK293 expression system. Crude lysates containing these recombinant proteins are being tested for cardiolipin remodeling enzymatic activity in vitro and some have been shown to be active. Candidates shown to be active are being partially purified, with iterative optimization of the extraction and purification procedures with repeated monitoring of enzyme activity. Soluble proteins will undergo crystallization for structural determination, if possible. To facilitate the commercialization of these reagents, candidates are being subjected to 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 will identify a cohort of potential lead candidates that demonstrate the greatest biochemical and biological activity in vitro for further efficacy testing in tafazzin KO mice.