UC San Diego

The discovery of RNA interference (RNAi) and the subsequent demonstration that synthetic short interfering RNA (siRNA) molecules could induce sequence-specific silencing of mRNA expression in human cells has opened the door to a new class of therapeutics. However, unlike small molecule drugs (<500 Da) that can diffuse passively across cell membranes, siRNAs are both too large (~14,000 Da) and too charged to enter cells unassisted. Consequently, delivery of siRNA is the major problem for the development of RNAi therapeutics.

To address the problem of siRNA delivery, our laboratory developed short interfering ribonucleic neutrals (siRNNs) whose anionic phosphate backbone is synthetically neutralized by bioreversible phosphotriester groups. The first generation of siRNNs utilized t-butyl-s-acyl-2-thioethyl (tBu-SATE) phosphotriester groups that proved too hydrophobic for biologic use. To improve siRNNs, we synthesized more hydrophilic hydroxyl-SATE (O-SATE) phosphotriester groups and used them to generate maximally neutral siRNNs for delivery by conjugated cationic peptide transduction domains (PTDs). Unfortunately, despite an overall cationic charge, PTD-siRNN conjugates were incapable of self-delivery in vitro. To solve this problem, we utilized conjugatable Aldehyde-SATE (A-SATE) phosphotriesters to make multivalent PTD-siRNN conjugates. Multivalent PTD-siRNN conjugates were capable of non-cytotoxic self-delivery and the induction of dose-dependent RNAi responses in vitro, a first for siRNNs. To study the function of siRNNs in vivo, we employed N-acetylgalactosamine (GalNAc), a hepatocyte-specific targeting domain. Single dose systemically administered GalNAc-siRNN conjugates induced extended dose-dependent RNAi responses in mice. This work constituted the first instance of in vivo target gene knockdown by siRNNs containing bioreversible neutralizing phosphotriester groups. Finally, we investigated small, double-stranded, left-handed hairpin (LHP) RNAs as alternative RNAi triggers for the application of RNN technology. Taken together, this work describes the development of readily adaptable, monomeric siRNA prodrugs (siRNNs) and opens a new avenue for RNAi therapeutics to treat human disease.