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

[pyruvate dehydrogenase deficiency]

We determined the ability of self-complementary adeno-associated virus (scAAV ) vectors to deliver and express the pyruvate dehydrogenase E 1 alpha subunit gene (PDHA 1 ) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA 1 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 72 h later . The transduction efficiency of scAAV 2 and scAAV 6 vectors was 3 - to 5 -fold higher than that of the other serotypes , which were subsequently used to transduce fibroblasts with wild-type PDHA 1 cDNA under the control of the chicken beta -action (CBA ) promoter at a vector :cell ratio of 1000 . Total PDH -specific activity and E 1 alpha protein expression were determined 10 days post-transduction . Both vectors increased E 1 alpha 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 E 1 alpha . Exposure for 24 h to 5 mM 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 E 1 alpha 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 .