Abstract 816 Mechanisms in Hereditary Disease Platform, Tuesday, 5/4

Mitochondrial proteins are either encoded from nuclear genomic DNA (nucDNA), translated in the cytoplasm, and imported into the mitochondria in a process signaled by a specific presequence, or they are transcribed from mitochondrial DNA (mtDNA) and translated inside the mitochondria on mitochondrial ribosomes. Defects in any of these mitochondrial proteins may lead to disorders of energy metabolism resulting in lactic acidosis, retinopathies, myopathies, or encephalopathies. Here, we have constructed several adeno-associated virus (AAV) vectors as potential therapies for disorders caused by mutations in either mtDNA or nucDNA affecting oxidative metabolism. Using green florescence protein (GFP) as a reporter gene, we have created fusion proteins to examine the expression and localization of these proteins within mitochondria. The first vectors tested contained only a presequence (from either cytochrome oxidase subunit 8 or ATP synthase subunit 9) fused in-frame with GFP. In vitro infection with these rAAV vectors in human embryonic kidney 293 cells resulted in expression of a GFP fusion that was localized within mitochondria as shown by both florescence microscopy and immunogold electron microscopy. Vectors were then constructed containing hydrophobic portions of mtDNA-encoded genes (ATP synthase subunit 6 or NADH dehydrogenase subunit 6) converted to the nuclear genetic code for optimal expression. These hydrophobic domains were cloned in frame between the presequence and GFP. Our initial data indicated a block in mitochondrial import of these proteins that correlated with their hydrophobicity. In contrast, when pyruvate dehydrogenase (PDH) E1α, a protein that is normally nuclear-encoded, was fused to GFP and expressed in a rAAV vector, it was efficiently imported into mitochondria both in vitro in 293 cells and in vivo in rat spinal neurons. Deficiency of the PDH complex is the most common disorder leading to congenital lactic acidosis and the most common mutations of PDH are in the E1α component. Future experiments with a similar vector (without the GFP fusion) will test the feasibility of this approach as a therapy for patients with PDH E1α defects.