Abstract
Zinc finger protein transcription factors (ZFP TFs) can be designed to regulate the expression of any endogenous gene, and thus provide a powerful platform for drug target validation and human therapeutics development. To deliver ZFP TFs into target cells that are difficult to transfect and for long-term expression of therapeutic ZFP TFs in mammalian tissues, we produced adeno-associated viral (AAV) vectors that express ZFP TFs using the triple plasmid transfection method. Crude AAV lysates were used to infect a range of cell types including a human osteosarcoma cell line (U2OS), human primary uterine smooth muscle cells (UtSMC) and rat primary cardiomyoctes. We show here that AAV vectors are capable of efficiently delivering ZFP TFs that cause the down-regulation of the checkpoint kinase 2 (CHK2) or phospholamban gene in these cells. Our results indicate that AAV vectors are effective vehicles for delivering therapeutic ZFP TFs and provide a convenient tool for functional screening of ZFP TFs in hard-to-transfect cells. The AAV vector that expresses a phospholamban-repressing ZFP TF validated here is currently being tested in pre-clinical animal models of congestive heart failure.
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