Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy

Cardiac alterations (hypertrophic/dilated cardiomyopathy, and rhythm alterations) are one of the major causes of mortality and morbidity in propionic acidemia (PA), caused by the deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC), involved in the catabolism of branched-chain amino acids, cholesterol, and odd-chain fatty acids. Impaired mitochondrial oxidative phosphorylation has been documented in heart biopsies of PA patients, as well as in the hypomorphic Pcca−/−(A138T) mouse model, in the latter correlating with increased oxidative damage and elevated expression of cardiac dysfunction biomarkers atrial and brain natriuretic peptides (ANP and BNP) and beta-myosin heavy chain (β-MHC). Here we characterize the cardiac phenotype in the PA mouse model by histological and echocardiography studies and identify a series of upregulated cardiac-enriched microRNAs (miRNAs) in the PA mouse heart, some of them also altered as circulating miRNAs in PA patients’ plasma samples. In PA mice hearts, we show alterations in signaling pathways regulated by the identified miRNAs, which could be contributing to cardiac remodeling and dysfunction; notably, an activation of the mammalian target of rapamycin (mTOR) pathway and a decrease in autophagy, which are reverted by rapamycin treatment. In vitro studies in HL-1 cardiomyocytes indicate that propionate, the major toxic metabolite accumulating in the disease, triggers the increase in expression levels of miRNAs, BNP, and β-MHC, concomitant with an increase in reactive oxygen species. Our results highlight miRNAs and signaling alterations in the PCC-deficient heart which may contribute to the development of PA-associated cardiomyopathy and provide a basis to identify new targets for therapeutic interventionThis work was supported by Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (grant number SAF2016-76004-R) and by Fundación Isabel Gemio and Fundación La Caixa (LCF/PR/PR16/ 11110018). AFG is funded by the FPI-UAM program, EAB and ARB by the Spanish Ministry of Science, Innovation and Universities (predoctoral fellowships FPU15/02923 and BES-2014-069420, respectively

Gene therapy for the treatment of propionic acidemia

Propionic acidemia is an organic acidemia that results from mutations in the PCCA or PCCB genes responsible for the two protein subunits of the propionyl-CoA carboxylase enzyme. Patients with PA have several metabolic abnormalities including elevated levels of glycine, propionylcarnitine, and methyl citrate. They also experience growth delay, developmental delay, and pathologies involving the brain, heart, pancreas, eyes, and muscles. The only viable treatment options for PA are protein restriction via a formula diet or liver transplantation, but neither of these treatments result in cures. To study the possible benefit of gene therapy for the treatment of PA we generated a mouse model of PA by introducing a hypomorphic human transgene with an A138T mutation onto a Pcca null mouse background. The resulting Pcca-/-(A138T) mice recapitulated many characteristics of PA in humans, and showed similar growth delay and biochemical perturbations. These mice were then used to study the utility of adeno-associated virus (AAV) serotype 8 and adenovirus serotype 5 expressing human PCCA. Both vectors mediated significant reductions in PA metabolite levels. Efficacy lasted for approximately 2 months in adenoviral treated mice but persisted for 1.5 years in male mice treated with AAV vector with expression remaining in the liver, heart, and skeletal muscle. Further studies examined the effect of tissue-specific treatments using alternate AAV serotypes and transcriptional regulation. When PCCA expression was restricted to the liver or muscle of treated mice metabolite levels were significantly lower in both indicating that there was likely a significant amount of these metabolites being produced within the muscle. Together these data provide evidence that PA disease is amenable to treatment with gene therapy and AAV vectors are able to mediate a significant degree of correction over long periods of time in mice. Additionally, the optimal treatment for individuals with PA will include correction of PCC activity in liver and muscle at a minimum to decrease the amount of PA metabolites such as methyl citrate being produced in these tissues. These studies also provide previously unknown insight into the molecular basis of the disease

Mucosal Vaccination by Adenoviruses Displaying Reovirus Sigma 1

We previously developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but had 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generated stronger T cell responses than Ad5 when used for mucosal immunization. New Ad5-fiber-sigma vectors were generated here by varying the number of fiber β-spiral shaft repeats (R) fused between fiber tail and the sigma. Ad5 virions encoding R3, R14, and R20 chimeras were rescued. Increasing chimera length led to their decreasing encapsidation of these proteins in the virions. Ad5-R3 and R14 mediated JAM-1- retargeting in vitro. When used to immunize mice by the intranasal route, Ad5-R3-sigma produced similar luciferase activity to Ad5, but higher serum and vaginal antibody responses. These data suggest optimized Ad-Sigma vectors may be useful vectors for mucosal vaccination

Mucosal Vaccination by Adenoviruses Displaying Reovirus Sigma 1

We previously developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but had 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generated stronger T cell responses than Ad5 when used for mucosal immunization. New Ad5- fiber-sigma vectors were generated here by varying the number of fiber β-spiral shaft repeats (R) fused between fiber tail and the sigma. Ad5 virions encoding R3, R14, and R20 chimeras were rescued. Increasing chimera length led to their decreasing encapsidation of these proteins in the virions. Ad5-R3 and R14 mediated JAM-1- retargeting in vitro. When used to immunize mice by the intranasal route, Ad5-R3-sigma produced similar luciferase activity to Ad5, but higher serum and vaginal antibody responses. These data suggest optimized Ad-Sigma vectors may be useful vectors for mucosal vaccination

Generation of a hypomorphic model of propionic acidemia amenable to gene therapy

Propionic acidemia (PA) is a recessive genetic disease that results in an inability to metabolize certain amino acids and odd-chain fatty acids. Current treatment involves restricting consumption of these substrates or liver transplantation. Deletion of the Pcca gene in mice mimics the most severe forms of the human disease. Pcca(−) mice die within 36 hours of birth, making it difficult to test intravenous systemic therapies in them. We generated an adult hypomorphic model of PA in Pcca(−) mice using a transgene bearing an A138T mutant of the human PCCA protein. Pcca(−/−)(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in patients with PA. This adult model allowed gene therapy testing by intravenous injection with adenovirus serotype 5 (Ad5) and adeno-associated virus 2/8 (AAV8) vectors. Ad5-mediated more rapid increases in PCCA protein and propionyl-CoA carboxylase (PCC) activity in the liver than AAV8 and both vectors reduced propionylcarnitine and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8-mediated long-lasting effects. These data suggest that this PA model may be a useful platform for optimizing systemic intravenous therapies for PA

Mucosal Vaccination by Adenoviruses Displaying Reovirus Sigma 1

We previously developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but had 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generated stronger T cell responses than Ad5 when used for mucosal immunization. New Ad5-fiber-sigma vectors were generated here by varying the number of fiber β-spiral shaft repeats (R) fused between fiber tail and the sigma. Ad5 virions encoding R3, R14, and R20 chimeras were rescued. Increasing chimera length led to their decreasing encapsidation of these proteins in the virions. Ad5-R3 and R14 mediated JAM-1- retargeting in vitro. When used to immunize mice by the intranasal route, Ad5-R3-sigma produced similar luciferase activity to Ad5, but higher serum and vaginal antibody responses. These data suggest optimized Ad-Sigma vectors may be useful vectors for mucosal vaccination