Exposure to siRNA-GalNAc Conjugates in Systems of the Standard Test Battery for Genotoxicity.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3+ human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 µg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.

Shielding of Lipid Nanoparticles for siRNA Delivery: Impact on Physicochemical Properties, Cytokine Induction, and Efficacy.

Formulation of short interfering RNA (siRNA) into multicomponent lipid nanoparticles (LNP) is an effective strategy for hepatic delivery and therapeutic gene silencing. This study systematically evaluated the effect of polyethylene glycol (PEG) density on LNP physicochemical properties, innate immune response stimulation, and in vivo efficacy. Increased PEG density not only shielded LNP surface charge but also reduced hemolytic activity, suggesting the formation of a steric barrier. In addition, increasing the PEG density reduced LNP immunostimulatory potential as reflected in cytokine induction both in vivo and in vitro. Higher PEG density also hindered in vivo efficacy, presumably due to reduced association with apolipoprotein E (ApoE), a protein which serves as an endogenous targeting ligand to hepatocytes. This effect could be overcome by incorporating an exogenous targeting ligand into the highly shielded LNPs, thereby circumventing the requirement for ApoE association. Therefore, these studies provide useful information for the rational design of LNP-based siRNA delivery systems with an optimal safety and efficacy profile.

Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.