Synthesis of bioreversible, phosphotriester-modified siRNA oligonucleotides

The observation that synthetic siRNA molecules could induce RNAi responses following exogenous addition to human cells opened up an entirely new avenue for human disease intervention. Unfortunately, due a size of 1̃4,000 Da and inherent anionic character from the phosphodiester backbone, synthetic siRNA molecules fail to traverse the cellular membrane and display unfavorable pharmacokinetic patterns in vivo. The challenge has been to create safe and efficient delivery systems in order for siRNA therapeutics to begin to be translated into clinically- relevant settings. To date, the majority of siRNA delivery strategies have relied on nanoparticle formulations. Due to inherent problems of nanoparticle-based delivery systems, our laboratory has sought to design soluble, monomeric siRNA delivery strategies utilizing a class of small cationic peptides termed Peptide Transduction Domains (PTDs). Unfortunately, PTD- siRNA conjugation strategies have failed in enhancing the delivery of siRNA molecules due to PTD-neutralization from the strong anionic charge of the siRNA phosphodiester. In an attempt to circumvent the anionic properties of the siRNA backbone, our laboratory began novel oligonucleotide synthesis routes to mask phosphodiester linkages with bioreversible, phosphotriester modifications. Building upon the S-Acyl- ThioEthyl (SATE) phosphotriester linkage, a cytoplasmic thioesterase sensitive linkage used for small molecule pro -drugs, we have successfully synthesized a collection of phosphodiester-neutral siRNN oligonucleotides. SATE- modified siRNN molecules were shown to be efficiently synthesized and displayed robust RNAi responses in cells in culture following cytoplasmic conversion to RNAi- compatible phosphodiesters. However, creation of PTD-siRNN conjugates with the standard SATE modifications were futile due to the extensive hydrophobicity donated from the SATE modification. In order to circumvent this hydrophobicity barrier, we synthesized a collection of Amino- SATE (N-SATE) phosphotriester siRNN containing terminal primary amines to increase the solubility of PTD- siRNN conjugates. Indeed, N-SATE siRNNs have been successfully synthesized, induce efficient RNAi responses and have succeeded in overcoming hydrophobic issues associated with SATE siRNN oligonucleotides. Due to this increase in solubility, PTD-siRNN conjugation attempts are currently underway to determine the ability of PTD-siRNN to function as a viable siRNA delivery platfor