Abstract
We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1α 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 72 h 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 E1α protein expression were determined 10 days post-transduction. Both vectors increased E1α 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 E1α. 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 E1α 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.
Original language | English (US) |
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Pages (from-to) | 381-387 |
Number of pages | 7 |
Journal | Molecular Genetics and Metabolism |
Volume | 93 |
Issue number | 4 |
DOIs | |
State | Published - Apr 2008 |
Externally published | Yes |
Keywords
- AAV vector
- Dichloroacetate
- Gene therapy
- Pyruvate dehydrogenase deficiency
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Biochemistry
- Molecular Biology
- Genetics
- Endocrinology