RESTORATION OF VLCAD IN KNOCKOUT MICE AND DEFICIENT HUMAN FIBROBLASTS USING NOVEL mRNA TECHNOLOGY: A MODEL TO TREAT FATTY ACID β-OXIDATION DISORDERS

Ermal Aliu1, Stephanie Mihalik1, Shawn Hillier3, Huifang Shi1, Catherine Kochersperger1, Anuradha Karunanidhi1, Xuling Zhu3, Christine DeAntonis3, Summar Siddiqui3, Kristine Burke3, Al-Walid Mohsen1, Patrick Finn3, Paolo Martini3, Jerry Vockley1,2

1Division of Medical Genetics, Department of Pediatrics, University of Pittsburgh, PA, USA 2Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA 3Moderna Therapeutics, Rare Diseases, 200 Technology Square, Cambridge, MA, USA

Introduction

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disorder identifiable by newborn screening. VLCAD patients present with severe cardiomyopathy, hypoketotic hypoglycemia, or intermittent recurrent rhabdomyolysis. VLCAD catalyzes the initial dehydrogenation step of fatty acid β-oxidation in mitochondria. The current treatment circumvents the metabolic block by avoiding fasting and maintaining a low-fat diet that includes medium-chain fats. To eliminate the hypoglycemia and reduce pathologic metabolites, we have restored liver VLCAD protein expression through targeted mRNA treatment in mouse models.

Materials and Methods

Initially, mRNA coding for human VLCAD was transfected into cultured human cells using Lipofectamine 2000 or Mirus Trans-It. Cells were evaluated for protein expression and activity, restitution of β-oxidation flux, and metabolite reduction by acylcarnitine quantitation. Mitochondrial localization was assessed using immunofluorescence microscopy. Mice deficient in VLCAD were then injected with mRNA encapsulated in a liver-targeting lipid nanoparticle. Livers were isolated and examined for protein expression and enzyme activity.

Results

VLCAD mRNA transfected well and expressed enzymatically active protein in HeLa cells and VLCAD mutant fibroblasts. The translated VLCAD trafficked to mitochondria where it was clipped to the correct size product. VLCAD transfection reduced accumulation of C16-carnitine and improved β-oxidation flux. Expressed protein was stable for at least 144 hours. Finally, when VLCAD liver-targeting constructs were injected into tail veins of VLCAD knockout mice, VLCAD was expressed and persisted for at least 116 hours.

Conclusions

VLCAD liver-targeted mRNA treatment results in the expression of protein in vitro and in vivo. The lead human mRNA VLCAD constructs produce abundant active protein in mutant human cells. Future mouse studies will determine whether VLCAD mRNA transfected into liver rescues the phenotype, especially in response to stress.

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