Abstract
microRNA (miRNA) can play significant roles in regulating gene expression, which implicates its potential in gene therapy. However, effective delivery of miRNA remains a hurdle, thus entailing a vector system for efficient delivery. Since baculovirus mediates efficient gene transfer to mammalian cells, here we aimed to develop the baculovirus vectors to carry engineered pre-miRNA sequences to knock down selected genes. We first constructed baculoviruses that harbored the miRNA sequences targeting different regions of the egfp gene and demonstrated that these baculoviruses suppressed the EGFP expression in a sequence- and dose-dependent manner. The attenuation of EGFP expression was up to 10% and 11 % in the protein and mRNA levels. Osteoarthritis (OA) is a chronic, progressive joint disease within the cartilage and is accompanied by deregulated activities of cytokine such as TNF-a. To exploit the miRNA therapy for OA treatment, we constructed another baculovirus harboring the pre-miRNA sequences targeting TNF-a (BacTNF-miR) and showed that BacTNF-miR was effectively suppressed the mRNA and protein levels of TNF-a from the fibroblast-like synoviocytes isolated from the OA patients, without induction of appreciable apoptosis. To provide long-term suppression, we exploited the Sleeping Beauty system and constructed Bac-SB that expressed the SB transposase and BacT2dE-miR that harbored the destabilized EGFP (d2EGFP)-specific miRNA expression cassette flanked by the IR/DR sequences recognizable by the SB. Co-transduction of a cell line stably expressing d2EGFP with BacSB and BacT2dE-miR led to prolonged repression of the d2EGFP expression for up to 24 days. These data collectively proved that baculovirus is an effective vector for miRNA delivery and downregulation of the target gene in mRNA/protein level. Combination of the baculovirus vector with SB system further enabled sustained expression of miRNA, rendering it a feasible approach to treating such diseases as OA.