Abstract 839: Transfer of siRNA Molecules via Gap Junctions in Heart Cells
Specific gene knock-down in post-natal cells by siRNA molecules has immense potential as a research and therapeutic tool. While direct effects have been clearly demonstrated in siRNA-transduced cells, we investigated a possible “bystander effect” whereby siRNAs transfer between transduced and non-transduced (NT) primary neonatal rat ventricular myocytes (NRVMs) via gap junctions. To enable siRNA expression in NRVMs, lentiviral vectors (LV) encoding short-hairpin (sh)RNAs, which are processed to siRNAs, were generated. Two populations of LV-transduced NRVMs were produced; one expressing GFP and the other co-expressing a second reporter plus a siRNA designed to knock-down GFP. Following 7 days co-culture of these populations, flow cytometry revealed a 35% reduction in the mean GFP fluorescence intensity and real-time PCR confirmed GFP mRNA knock-down. To explore dependence of this effect on gap junctions, we co-transduced cells with a LV encoding a dominant-negative connexin43 mutant (Cx43Δ) as a molecular gap junction blocker. Repeat co-cultures revealed abrogation of GFP fluorescence and mRNA knockdown effect, confirming gap junction dependence. To directly identify and quantify the presence of siRNA molecules in transduced and NT cells, we developed a real-time PCR-based assay with a cDNA intermediate generated from siRNA templates. Following co-culture, cell sorting and extraction of small RNA species, we found that 12% of the siRNAs produced in transduced cells are transferred to NT cells. Furthermore, this proportion is substantially reduced in the presence of Cx43Δ. To target an endogenous gene and demonstrate physiological effects of siRNA transfer, two NRVM populations were co-cultured. One was NT and the other transduced with a bi-cistronic LV encoding GFP and a shRNA targeting the SERCA gene. Calcium transient analysis in NT NRVMs revealed a 30% reduction in transient amplitude relative to control cultures. Again, co-transduction with LVCx43Δ abrogated this effect. In conclusion, LV-derived siRNA molecules are capable of transferring between cells of cardiac origin. This intercellular transfer is dependent on the presence of functional gap junctions and is capable of mediating knockdown of both exogenous and endogenous genes.