521. Gene Transfer of an Anti-CCR5 siRNA by Sleeping Beauty (SB) Transposon System

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The Sleeping Beauty transposon vector system is a non-viral gene delivery method. It is mediated by a transposase that employs a cut-and-paste mechanism. It is derived by repairing defective copies of Tc1/mariner family transposon elements. The utility of SB transposon system has been demonstrated in various mammalian cell lines for example by the gene transfer of a red fluorescence protein (RFP) in primary human lymphocytes. SiRNAs that mediate RNA interference have also been successfully introduced into cells using this system thus paving the way for permanent gene knock down using this novel non-viral gene transfer method. In our current studies we sought to introduce an anti-CCR5 siRNA into HIV-1 susceptible cells to down regulate this essential coreceptor involved in viral entry. A previously characterized short hairpin siRNA against CCR5 together with an RFP reporter was cloned into the SB gene transfer transposon and introduced into MAGI-CCR5 and GHOST-R3X4R5 cell lines. Since the levels of gene transfer were low, the stable transposed clones were enriched by FACS sorting for RFP expression. The enriched transgenic cells were cultured for many generations and monitored for RFP expression which appeared to be stable. To evaluate the siRNA mediated down regulation of CCR5, the transgenic cells were evaluated by FACS analysis. Both MAGI-CCR5 and GHOST-R3X4R5 cells showed a 90% reduction in CCR5 expression indicating that the siRNA is functional in this system. Stable transposition was also confirmed by southern blot hybridization testing of transposed cells with an RFP derived probe. To evaluate the HIV-1 resistance of the transgenic cells, they were challenged with a CCR5 tropic HIV-1 strain. Viral resistance was monitored by assaying for viral p24 antigen released into the supernatants at different days post viral challenge. Our results have shown significant viral resistance by the anti-CCR5 siRNA transgenic cells. These data indicate that a non-viral SB system can be utilized for transposition of siRNA coding genes to confer resistance against HIV.