A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate

Abstract

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1alpha subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1alpha protein expression were determined 10 days post-transduction. Both vectors increased E1alpha expression 40-60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1alpha. Exposure for 24h to 5mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1alpha protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.

Figures

Fig. 1

Fluorescence microscopical (A) and quantitative (B) analyses of transduction efficiency of ssAAV and scAAV vectors. The transduction efficiency was evaluated in human fibroblast cells at vector particles per cell (PPC) of 10,000. Transgene expression was evaluated 1 day, 3 days, 5 days and 10 days post-transduction and analyzed using NIH ImageJ. *P < 0.01 (group-paired t-test). Original magnification 200×

Fig. 2

Quantitative analyses of transduction efficiency of scAAV-EGFP serotype vectors in human fibroblast cells. Relative transduction efficiencies were evaluated at a vector particle per cell of 200. Transgene expression was evaluated 72 h post-transduction and analyzed using NIH ImageJ.

Fig. 3

Effect of scAAV serotypes 2 and 6, with or without DCA, on expression of PDH complex proteins. (A) Immunoblots showing relative changes in steady-state levels of PDH E1α, E1β, E2 and E2/E3bp after AAV vectors delivery and/or combination of DCA administration. Cell lysates (15 µg) from three healthy subjects (C1, C2 and C3) and three PDH deficiency patients (P1, P2 and P3) were separated on 10% SDS–PAGE gels and immunoblotted with monoclonal Abs against E1α, E1β, E2 and E2/E3bp. (B) Quantitative analysis of immunoblot data. Using ImageJ the pixel densities in each band (E2, E2/E3bp, E1α and E1β) from the gel in (A) were quantitated and the amount of protein was standardized to the amount of β-actin (E2, E2/E3bp, E1α and E1β/β-actin) in each lane, respectively. Data are means of two independent experiments.