Evaluating a novel delivery method for siRNA via gap junctions

Short interfering RNA (siRNA) has been suggested as an efficient suppressor of target genes in mammalian cells (Mittal 2004). The ability to regulate disease-associated genes makes it a potent therapeutic agent (Kim and Rossi 2007). There are still technical challenges, which must be solved before effective clinical application of RNA interference (RNAi). Among them, delivery of effective siRNA to the target cells or tissue is a vital and rate-limiting step in the development of RNAi based treatment. Being the most significant and widely distributed intercellular channels (Goldberg, Valiunas et al. 2004), the gap junction is a promising candidate for an siRNA delivery method. SiRNA delivery mediated by gap junction channels excludes the extracellular space; therefore gives more efficient target-directed delivery. Previous studies from our laboratory showed that siRNA could transfer from a delivery cell to a target cell via gap junction channels (Valiunas, Polosina et al. 2005). However, no functional evaluation of the target gene was performed. In the present study we chose the type 2 mouse hyperpolarization activated cyclic nucleotide-gated cation channel gene (mHCN2) as our functional target. We hypothesized that siRNA could be delivered via gap junctions and downregulate the function of the mHCN2 gene in single cells and in the recipient cells of cell pairs. Here, we report that: (1) siRNA for mHCN2 can significantly downregulate the function of its target gene in single cells. We made a HEK293-mHCN2 cell-line which stably expressed the mHCN2 gene. Our results showed that the mHCN2-induced current dramatically declined after transfection with mHCN2 siRNA. The maximum effect and the duration of the downregulation vary with exposure time to the transfection medium. The downregulation started at 6h after transfection started and lasted for at least 72 hours. (2) When HEK293-mHCN2 cells were co-cultured with siRNA transfected HEK293 cells, the mHCN2-induced current in the stable cell-line cells was reduced. The reduction started at 12h after co-culture and lasted for at least 72 hours. This result suggests that siRNA can be delivered between cells and efficiently downregulate the function of its target gene in the recipient cells. When we blocked gap junction channels during co-culture, the downregulation disappeared. This indicates that the gap junction channel is necessary for this cell-cell delivery system. To further examine the necessity of the gap junction channel, we co-cultured siRNA transfected N2A cells with HEK293-mHCN2 cells. N2A cells can't form gap junctions with HEK293 cells. And as we hypothesized, no change was observed in the mHCN2-induced current in the recipient cells. (3) When we upregulated gap junctional conductance between cells by phenylbutyrate (4PB), the effect of siRNA in the recipient cell of the cell pairs was accelerated. 12 hours after co-culture was initiated, while gap junctional conductance between cells was increased, 4PB induced a more significant downregulation of the mHCN2-induced current. 24 hours after co-culture was initiated, although conductance between cells showed a more dramatic increase due to 4PB, the downregulation of current magnitude was similar in the 4PB or the non-4PB treated group.;In conclusion, these results give us fundamental evidence which supports the hypothesis: siRNA can be delivered between cells via gap junction channels and downregulate the expression and function of its target gene in the recipient cell for an extended time period.