VLCAD deficient patient fibroblasts show improvement with peroxisomeproliferator activated receptor drug treatment


Presented By:

Olivia D’Annibale, MPH1, Anuradha Karunanidhi, MS2, Colin O’Carroll, PhD, MBA3, Jerry Vockley, MD, PhD1,2, and Al-Walid Mohsen, PhD1,2,*

1Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA, 2Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, 3Reneo Pharmaceuticals, San Diego, CA, USA

*Correspondence email: aam27@pitt.edu and phone: (412) 378-6666

Background: Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disease that prevents the body from utilizing long chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a PPARδ agonist that modulates expression of the genes for fatty acid -oxidation enzymes and many involved in oxidative phosphorylation. The purpose of this project was to assess the effect of REN001 on VLCAD activity and cellular bioenergetics.

Methods: VLCAD deficient cells due to missense mutations were used: FB671 (p.L540P plus an RNA splicing mutation) and FB833 (p.V174M/p.E609K). Patient and control cells were grown until 85% confluency, and treated with REN001 at 0, 15, 30, 60, and 120 nM for 48 hr. Cells were harvested for western blot, enzyme assay, ATP Production, and Seahorse Mito Stress test and ATP Rate assay.

Results: Western blot showed an increase in VLCAD signal in deficient cells at all concentrations compared to untreated. VLCAD activity increased at 30 nM and 60 nM REN001, in FB671 and FB833 by 8% and 46%, respectively. ATPlite assay showed increased ATP production at 15 nM and 30 nM, in FB671 and FB833 respectively, compared to the 0 nM. Seahorse assay of whole cell oxygen consumption showed increases in both cell lines in basal respiration, maximal respiration, and spare capacity at 30 nM REN001 when compared to untreated (p < 0.0001). Seahorse ATP Rate assay showed an increase in glycolytic and mitochondrial ATP production at 60 nM REN001 in both cells lines (p < 0.0001).

Discussion: Treating VLCAD deficient patient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein, enzyme activity, and a decrease in cellular stress. These results identify REN001 as a potential therapy for VLCAD, and a clinical trial in patients will start soon.

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