Xue-Jun Zhao1, Keaton Solo1, Huifan Shi1, Summar Siddiqui2, Christine DeAntonis2, Lisa Rice2, Paloma H Giangrande2, AI-Walid Mohsen 1,3* Paolo Martini2, Jerry Vockley 1,3
1Division of Medical Genetics, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA, 2Moderna Therapeutics, Rare Diseases, 200 Technology Square, Cambridge, MA, USA, 3Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA
*Corresponding author: AI-Walid Mohsen, PhD; Department of Pediatrics, University of Pittsburgh, 4401 Penn Ave, Rangos Research Center, Room 5157, Pittsburgh, PA 15224; firstname.lastname@example.org, +1 412-692-6498
BACKGROUND: Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive fatty acid β-oxidation disorder that results from a block in the first intramitochondrial step of long chain fatty acid β-oxidation. This block results in the accumulation of long chain fatty acid CoA esters in cells. The current treatment includes avoiding fasting and maintaining a low-fat, high-carbohydrate diet that replaces long chain triglycerides with medium chain triglycerides (MCT). With the limited treatment options for VLCAD deficiency patients, VLCAD deficiency is a good candidate for mRNA therapy. Novel messenger RNA (mRNA) organ-targeted therapies enable cells to produce replacement proteins, which affords the possibility to reverse the enzyme defect. In this study, we investigated the effect of therapeutic mRNA transfection to rescue VLCAD activity in VLCAD knockout mouse embryonic fibroblasts (MEFs).
METHODS: VLCAD knockout MEFs were transfected with synthetic VLCAD mRNAs formulated as the therapeutic agent. After 48 hours transfection, cell lysates were assessed for VLCAD protein presence using western blots and enzyme activity using the ETF fluorescence reduction assay. Long chain fatty acid oxidation (FAO) flux analysis was performed on transfected live cells. ATP production (ATPLite assay) and ROS levels (MitoSox Red assay) were also measured. RESULTS: Transfection of VLCAD mRNA into deficient MEFs resulted in increased presence of VLCAD protein, increased enzyme activity in cell extracts, enhanced ATP production, and decreased levels of superoxide in mitochondria. In addition, VLCAD mRNA transfected MEFs showed improvement in VLCAD activity as indicated by mitochondrial fatty acid utilization using whole cell oleate oxidation assay. DISCUSSION: Our findings demonstrate the potential therapeutic value of this novel mRNA treatment approach in restoring mitochondrial fatty acid oxidation in VLCAD deficient cells, and in animal models of VLCAD deficiency.