SiRNA Delivery Methods

Hamil, Alexander S, Dowdy, Steven F, Alexander S. Hamil, Steven F. Dowdy, Hamil, Alexander S., Dowdy, Steven F.

Due to their high potency (EC50 ~1 pM) and exquisite target selectivity for all expressed mRNAs, small interfering RNA (siRNA)-induced RNAi responses hold significant promise as a therapeutic modality. However, the size and high negative charge of siRNAs render them unable to enter cells without assistance from a delivery agent. Most current methods of siRNA delivery rely on encasing siRNA molecules in large nanoparticles or cationic liposomes. However, these approaches suffer from a number of problems, including a poor diffusion coefficient, cytotoxicity, and poor pharmacokinetics. To address the problem of siRNA in vivo delivery, we developed monomeric neutral RNAi prodrugs, termed siRibonucleic neutrals (siRNNs), that directly neutralize the phosphate backbone negative charge by synthesis with bioreversible phosphotriester groups that are enzymatically cleaved off in the cytoplasm of cells. Here we describe the synthesis and purification of siRNN conjugates that induce in vivo target gene knockdown following systemic delivery into mice.