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

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• 作者：Zongchao Han ; Kristen Berendzen ; Li Zhong ; Ira Surolia ; Nitin Chouthai ; Weihong Zhao ; Njeri Maina ; Arun Srivastava ; Peter W. Stacpoole
• 关键词：Pyruvate dehydrogenase deficiency ; AAV vector ; Gene therapy ; Dichloroacetate
• 刊名：Molecular Genetics and Metabolism
• 出版年：2008
• 出版时间：April 2008
• 年：2008
• 卷：93
• 期：4
• 页码：381-387
• 全文大小：1136 K

文摘

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> 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 E1www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> protein expression were determined 10 days post-transduction. Both vectors increased E1www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> 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 E1www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0"">. 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 E1www.sciencedirect.com/scidirimg/entities/204e.gif"" alt=""greek small letter alpha"" title=""greek small letter alpha"" border=""0""> 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.