New Approach to Selective Targeting of Dendritic Cells by Sirna, Resulting in Long Term Graft Survival in Mouse Heart Transplantation Model

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Introduction: siRNA (small interfering RNA) is a 20-27 nucleotides long double stranded RNA capable of specifically targeting for degradation a complementary mRNA providing a new paradigm for targeted therapeutics. However, progress in realizing the promise of siRNA drugs has been hampered by the limitations of siRNA drug delivery. We found that siRNA having poly-dA extension at 5′-end of siRNA-sense strand is stably complexes with 1,3-beta-glucan (Schizophylan, SPG), which specifically delivers the cargo siRNA into Dendritic cells (DCs) through its receptor, Dectin-1, being expressed predominantly on the surface of DCs. Using an siRNA targeting the costimulator molecule CD40 (siCD40), we explored a possibility of selective siRNA delivery into DCs and examined the level of immune-regulatory activity in vitro and in vivo. Here we present for the first time successful gene delivery into DCs sufficient to show pharmacological activity of immunosuppression by siRNA leading to long term graft survival in mouse heart transplantation. Material and methods: Poly-dA(20-60mer)-attached siRNA was chemically synthesized, and complexed with SPG by our proprietary technology. Briefly, SPG is a triple helix 1,3-beta-glucan, and dissociates into corresponding single chain of ca. 150 KDa in alkaline solution, and was reconstituted back in a neutral condition in the presence of poly-dA-siRNA to form a complex (siRNA/SPG). We measured physicochemical and biological properties of siRNA/SPG. The siRNA introduced into Dectin-1 expressing cells was examined by confocal microscopic analysis. Biological activity was examined by quantitative PCR and FACS both in vitro and in vivo to delineate a dosing regimen in MHC fully mismatched mouse heart transplantation. Results: siRNA was successfully introduced into Dectin-1 expressing cells in sufficient amount to show biological activity. Treatment of either stimulator or responder splenocytes by siRNA/SPG complex dramatically suppressed lymphocytes proliferation in one way MLR. The same is true when siRNA/SPG complex was i.v. injected prior to spleen harvest for MLR. Biological activity was maintained up till 48 hours after introduction of siRNA, suggesting the possibility of every other day i.v. administration. Confocal molecular analysis indicated the presence of poly-dA-attached siRNA in the functional complex (RNA-Induced-Silencing-Complex Loading Complex, RLC), suggesting successful endosomal escape in a stable form. These data were consolidated to confirm long term graft survival in MHC fully mismatched mouse heterotopic heart transplantation (to be presented in a separate paper in this Congress). Conclusion: We conclude: Improved stability of siRNA is confirmed both in vitro and in vivo by forming siRNA/SPG complex. siRNA/SPG is docked and internalized through its receptor, Dectin-1, into DCs in sufficient amount and escapes from endosome to show functional activity in the cytoplasm. siRNA for CD40 (siCD40) showed sufficient immune-regulatory activity both in vitro and in vivo system. Our basic research was exemplified in mouse heterotopic heart transplantation of B10-to-CBA and Balb-to-B6 strain combinations, just 7 times i.v. administration from day -3 to day+7 of heart transplantation to show long term graft survival (to be presented in another paper in this Congress). Our results provide a new innovative way of siRNA delivery into targeting cells.

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