"Delivering Oligonucleotides as Phosphotriesters"


The delivery and targeting of nucleotide therapeutics is limited by the presence of excessive negative charge found on the phosphate backbone. Masking this charge as a lipophilic phosphotriester provides a potential solution to crossing the cell membrane and reduces the complexity of delivery moiety conjugation. Neutralizing charge with cationic lipids, proteins or through nano-particle based formulations have often generated off target effects, cytotoxicity and exceedingly complex CMC formulations.

Pro-Oligonucleotide Chemical Tools

The siRNN platform leverages the facile generation of phosphate diamidites to provide ready access to a highly diverse library of modifiers. The scheme above demonstrates examples with varying levels of steric bulk at the site of thioesterase cleavage, this particular set was used to evaluate intracellular reversal kinetics. The cost effective and scalable reactionseries begins with the thiolation of available chlorohydrins. The resulting thiols are selectively esterified at lowtemperature with an appropriate acid chloride and isolation is routinely achieved by distillation. Phosphinylation is accomplished by the addition of the thioester to tetraisopropyl chlorophosphoramidite and base resulting in quantitative conversion. The final products are routinely isolated by celite filtration or Et3N pretreated silica chromatography.

The TriPhos Therapeutics biocompatible phosphate modifications are reversed by intracellular mechanisms to return functional phosphodiesters. The cross-functional technology is independent of sequence and integrates into RNAi, miRNA, LNA and antisense designs. The constructs have demonstrated activity in RNAi model systems and can reduce phosphate charge by >50% without effecting solubility or duplexation profiles. 

The pro-oligos are endowed with improved pharmacological attributes including:

  •        nuclease stability 
  •        lower molecular weight when compared to alternate delivery systems
  •        synthetic scalability 
  •         reduced cellular toxicity and genotoxicity when compared to alternate trimester appoaches
  •        improved lipophilicity

    Charge neutralization enhances attachment of CPPs, steroids, antibodies and peptide hormones, without risking undesired intra-molecular ionic interactions. The siRNN platform opens unexplored targeting strategies and a new class of potential drugs.