| The mitochondrion is a unique organelle that predominantly functions to produce useful cellular energy in the form of adenosine triphosphate (ATP). Unlike non-nuclear eukaryotic organelles (except for chloroplasts), mitochondria have their own DNA (mitochondrial DNA, mtDNA), as well as transcriptional and translational machinery that utilizes the mitochondrial code. mtDNA is subject to mutation, which is the proximate cause of several mitochondrial-related human illnesses. Most mitochondrial diseases have been linked to single transfer RNA (tRNA) point mutations located in mtDNA or mutations in structural proteins. Such examples of mtDNA-related mitochondrial diseases include: Myoclonic Epilepsy with Ragged Red Fibers (MERRF) and Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS), or Leber's Hereditary Optical Neuropathy. Patients experiencing either of these disorders will have a decrease in mitochondrial protein synthesis leading to impaired mitochondrial function and ultimately affecting a host of metabolic functions. The technology for introducing exogenous DNA to the nucleus of cells for therapeutic purposes is well established, but delivery of exogenous DNA to the matrix of mitochondria where mtDNA resides is a new field. A popular viral vector for nuclear gene delivery, adeno-associated virus (AAV), possesses a low immunogenicity profile, and has a distinct ability to transduce a variety of cell types. For correction of mitochondrial diseases, AAV requires a modification to the intracellular trafficking pathway to target its payload to mitochondria. Previously, Yu et al. introduced a version of AAV bearing a mitochondrial targeting sequence (MTS) on its capsid, which showed that it can target and successfully deliver exogenous genes for expression in mitochondria for the purpose of correcting a mutation in a structural gene.;The central hypothesis of this dissertation is mitochondrial-targeted AAV gene delivery may benefit from an optimal combination of AAV genome structure (single stranded vs. self-complementary), promoter (mitochondrial heavy strand promoter vs. cytomegalovirus promoter), transcription terminator (mitochondrial terminator vs. poly A cleavage site), mitochondrial-targeting sequencing, and a transgene optimized to utilize the mitochondrial genetic code. Successful gene delivery to mitochondria from vectors containing the optimized transgene served as the single most important parameter, setting the stage for the development of further vectors to address human diseases. |
