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.