360. Comparison of Promiscuous and Tissue-Targeted AAV Gene Therapy for Systemic Propionic Acidemia Treatment

DIABETES, METABOLIC AND GENETIC DISEASES I against enzymatic degradation in circulation. Polyethylenimine (PEI) has been widely applied as an efficient in vitro transfection agent due to its DNA condensation capacity and intrinsic endosomolytic activity. However, translation of PEI to the clinic has been hampered by limited gene transfer efficiency in vivo due to intracellular and extracellular delivery obstacles and toxicity. While the toxicity of PEI can be reduced dramatically by conjugating polyethylene glycol (PEG) along the backbone, the transfection efficiency of the copolymer remains poor. We hypothesized that this hurdle could be overcome by combining US/MB with PEGylated PEI (PEG-PEI). In this study, we explored if US/MB could significantly enhance gene transfer efficiency of pGL4 luciferase reporter plasmids condensed with PEG-PEI in the mouse liver. The particle size and zeta potential of PEG-PEI/pDNA complexes at various N/P ratios were measured to determine appropriate PEG-PEI/pDNA mass ratio. Three groups of mice were injected, respectively, with naked DNA, PEG-PEI or non-PEGylated PEI condensed pDNA, and MBs into liver through portal vein for 30 sec with simultaneous 60-sec pulse-train US treatment (2.5 MPa, 1MHz). Control groups were treated without US exposure. Luciferase expressions of mouse liver and blood transaminase levels on day 1 were evaluated following the treatment. We found that US/MB significantly enhanced luciferase transgene expression levels of PEG-PEI/pDNA injected mice (~250 fold) compared with the control group injected with PEG-PEI/pDNA without US exposure. Furthermore, these levels were comparable or slightly higher than US/MB + pDNA group. The enhancement effects of gene expression at different PEG-PEI/pDNA mass ratios will be further explored in ongoing studies. The transgene expression levels of non-PEGylated PEI group were marginally improved by US/MB treatment compared to the control group (PEI/pDNA only), however, the expression levels were much lower than US/MB + PEG-PEI/ pDNA group. Transaminase levels in all US/MB treated groups on day 1 after treatment were higher than normal mouse and control groups without US exposure. Further long-term evaluation of US/MB + PEG-PEI/pDNA group showed that transaminase levels returned to normal levels on day 7 after treatment, indicating that transient liver damage was induced by PEG-PEI and acoustic cavitation. Taken together, these results indicated that US/MB significantly enhanced gene transfer efficiency of PEG-PEI condensed pDNA. Next step we will incorporate additional moieties onto PEG-PEI such as targeting ligands to further improve US/MB-mediated gene transfer efficiency.

the traditional view of “liver-directed” gene therapy for metabolic disease. Previously we have generated a hypomorphic model of PA by inserting a human PCCA transgene harboring an A138T mutation identified in human patients onto a Pcca-/- null background. These mice are able to survive to adulthood, have approximately 2% of wild type PCC enzyme activity, and elevations in propionyl-carnitine, methylcitrate (mean: 5.1 μM vs 0.3 μM for wild type), glycine, and ammonia, as well as cardiac defects. Results: Pcca-/-(A138T) mice were administered 5x1011 viral genomes (vg) of AAV8-hPCCA intravenously. A single injection of this vector mediated significant decreases in circulating C3 and MeCit when assayed over the course of the 1.5-year study (MeCit in male mice 1.5 years p.i.=1.3 ±0.4 μM compared to 0.9 ±0.3 μM 1 week p.i.). PCCA expression and PCC enzyme activity in the liver were lower at the 1.5 year time point than that measured at 25 days post injection indicating a loss of expression that didn’t correlate with the maintained MeCit and C3 attenuation. We also generated liver- and muscle-targeted vectors using natural tissue bias of AAV serotypes 8 and 1 along with transcriptional restriction of transthyretin (TTR) and muscle creatine kinase (MCK) enhancer/promoters. Intravenous injection of 5x1011 vg of either AAV8-TTR-PCCA or AAV1-MCKPCCA resulted in significant correction of C3 and MeCit (MeCit = 1.9 ±0.7 μM for mice treated with AAV8-TTR-PCCA and 3.2 ±0.4 μM for mice treated with AAV1-MCK-PCCA). Conclusions: This study provides evidence that gene therapy with AAV vectors is a viable alternative to liver transplantation for propionic acidemia and our findings in Pcca-/-(A138T) mice 1.5 years after single dose administration enforces findings in other animal models that AAV can provide significant long-term metabolic disease correction. These data also provide evidence that inborn errors of metabolism such as PA can benefit from treatment of nonhepatic tissues, primarily muscle, to reduce the level of circulating disease-associated metabolites. Additionally, the use of our modified AAV8-TTR-PCCA and AAV1-MCK-PCCA has allowed us to study the effect of tissue-specific treatment on organ-specific pathology, particularly pathology in the heart.

361. In Vivo Interallelic Complementation Assay at the Mut Locus With Adeno-Associated (AAV) Viral Gene Delivery

Julien S. Senac,1 Charles P. Venditti.1 Genetic and Molecular Biology Branch, NHGRI / NIH, Bethesda, MD. 1

Diabetes, Metabolic and Genetic Diseases I 360. Comparison of Promiscuous and TissueTargeted AAV Gene Therapy for Systemic Propionic Acidemia Treatment Adam J. Guenzel,1 Matthew L. Hillestad,1 Michael A. Barry.1 Mayo Clinic, Rochester, MN.

1

Background: Propionic Acidemia (PA) is an inborn error of metabolism resulting from decreased activity of the propionyl CoA carboxylase enzyme (PCC) caused by mutations in the genes encoding for the PCCA or PCCB subunit. Patients with PA are unable to fully catabolize odd-chain fatty acids and the amino acids valine, isoleucine, threonine, and methionine which results in elevated levels of propionyl CoA, propionylcarnitine (C3), and methyl citrate (MeCit). Downstream effects of these elevations include hyperammonemia and organ-specific defects such as cardiomyopathy, pancreatitis, and neurological defects. Current treatment of PA is limited to dietary restriction of protein to decrease the sources of harmful metabolites. Elective liver transplantation is also used as a treatment, but is not curative as circulating metabolite levels such as C3 and MeCit remain elevated after the procedure, indicating that treatment of non-hepatic tissues may offer an advantage over Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

Methylmalonic acidemia (MMA) is most commonly caused by mutations in methylmalonyl-CoA mutase (MUT). Among the wide spectrum of partial activity or mut- mutations, the mechanism of enzymatic impairment has been defined only for selected missense changes, such as p.G717V, which has been documented to be an adenosylcobalamin Km mutant. We generated transgenic mice that ubiquitously express the murine p.G717V homologue (p.G715V) as a stable transgene (Mut-/-;TgINS-CBA-G715V). We demonstrated that this new mouse model mimics the physiologic and phenotypic manifestations observed in mut- MMA patients such as failure to respond to vitamin B12 injection treatment and dietary induction of severe methylmalonic acidemia. Previous publications have demonstrated that cell lines harboring some MUT mutations, such as p.G717V mutation, could be ‘rescued’ by alleles with distinct mutations, such as p.R93H. Using a yeast expression approach, we first established that the co-expression of the murine homologues of these mutations, Mut p.R91H and p.G715V, produced a recombinant Mut enzyme with improved kinetic parameters compared to either allele, supporting previous cell culture observations. We next hypothesized that we could recapitulate interallelic complementation in vivo using an rAAV vector to deliver the p.R91H Mut transgene to Mut-/-;TgINS-CBA-G715V mice. A single retro-orbital injection of (5e10 S137