Creation of the Next Generation RNAi Therapeutics and an in vivo model for their study

Abstract RNA interference (RNAi) – a natural biological mechanism by which eukaryotic cells control gene expression – can be used to target and silence with high specificity virtually any expressed gene, making it a potentially powerful therapeutic approach. Importantly, RNAi mechanisms utilize DNA/RNA sequences to specify its target, enabling fine-tuned silencing of a single gene belonging to a family of structurally similar proteins that typically share small molecule binding pockets. For these reasons, RNAi-based therapeutics have the potential to provide effective on-target benefits while avoiding off-target toxicities that are commonly seen with promiscuous protease and kinase small molecule inhibitors. Despite their therapeutic potential, the most challenging aspect of RNAi- based drug development involves achieving effective delivery to the cells or tissues of interest, while avoiding systemic off-target effects. Indeed, only a handful of RNAi-based drugs have received FDA approval to treat liver diseases since this is the most readily accessible tissue for this class of drugs. More recently, alternative delivery mechanisms, such as ligand-coupled oligonucleotides, lipid nanoparticles, viral vectors, and extracellular vesicles (EVs) show great promise with the potential to penetrate tissues and have cellular uptake where they can engage the endogenous RNAi machinery to induce gene silencing. However, a systematic method for evaluating these delivery modalities in vivo has yet to be implemented. To overcome this hurdle, we generated a murine model – the Optimus model, that contains a dual fluorescence reporter cassette where one reporter harbors unique RNAi responsive elements. Using this Optimus model, we aim to generate an effective pipeline to readily test an array of new RNAi-based therapeutic biologics generated by coupling our optimized siRNAs or artificial miRNA scaffolds to established delivery vehicles, beginning with three unique modalities: 1) peptide- based platform developed by SRI International for specific delivery to lung adenocarcinoma cells; 2) aptamer- based modality developed by Aptamer group for the silencing in activated hepatic stellate cells under liver fibrotic conditions; and 3) recombinant adeno-associated viruses (rAAV) we previously generated carrying ubiquitous and tissue-specific RNAi expression cassettes. Importantly, these delivery platforms have already demonstrated successful delivery of oligonucleotide payloads, both in vitro and preliminary in vivo. Our collaboration enables us to further test these new therapeutic biologics in live animals and assess their biodistribution, the features that promote cellular uptake and enable trafficking of RNAi payload for functional engagement, and the systemic off-target effects at very early stages of pre-clinical development. This research will define a new paradigm to accelerate the creation of novel RNAi-based (and potentially oligonucleotide-based) drugs and readily evaluate their therapeutic potential in live animals.