Nucleic acid modulators and theranostics for ADAR

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Project Summary The objectives of this MIRA application are to 1) in vivo profiling of heterogeneous Adenosine deaminase acting on RNA (ADAR) using RNA nano-reporters as a tool to understand ADAR biology and guide the design of ADAR- based therapy; 2) promoting endogenous ADAR modulation using chimeric ADAR aptamer-gRNA (guide RNA) and a type I interferon (IFN-I)-activating DNA oligonucleotide that induces ADAR1; 3) targeted delivery of albumin-hitchhiking DNA/RNA for endogenous ADAR-based gene therapy and immunotherapy; and 4) pilot testing of these theranostics in mouse models of Factor V Leiden (FVL) thrombophilia and metastatic melanoma. ADAR mediates RNA Adenosine-to-Inosine editing in metazoans. ADAR is an intriguing endogenous RNA editor for the gene therapy of diseases caused by pathogenic G->A mutations, such as FVL thrombophilia; moreover, ADAR1 inhibition in tumor cells sensitizes their immunotherapy. However, ADAR levels are highly heterogeneous and dynamic across individuals, tissues, and cell environments, making it pivotal for spatiotemporal ADAR profiling to study ADAR biology and design personalized ADAR-based therapy. To this end, we will develop and test an ADAR reporter for non-invasive real-time ADAR profiling in vivo, using an ADAR- activatable split luciferase mRNA reporter followed by bioluminescence imaging. Further, the often low endogenous ADAR levels limit ADAR editing efficacy, which impedes ADAR biological discovery and theranostic applications. To address this challenge and promote the RNA editing efficacy of endogenous ADAR, we will study two strategies: 1) using ADAR aptamer-gRNA chimera to promote ADAR binding and editing of RNA; and 2) using our novel IFN-I-activating DNA agonist for cyclic GMP-AMP synthase to elevate ADAR levels and promote ADAR modulatory efficacy. All these nucleic acid theranostics, such as aptamers, siRNA, and mRNA, hold great potential to reshape human medicine. Yet, naked DNA/RNA has limited clinical success thus far, largely due to poor pharmacokinetics (PK) and inability to enter cells, calling for mechanistic understanding and manipulation of the interactions between biological systems and DNA/RNA. Previously, we developed a molecular albumin hitchhiker and nanoparticles that promoted the PK and delivery of nucleic acids by up to 200 folds. Here, we will use lipid nanoparticles to deliver mRNA reporters for in vivo ADAR profiling; we will study targeted delivery of albumin-hitchhiking ADAR nucleic acid therapeutics, including aptamer-gRNA chimera for FVL thrombophilia gene therapy and siRNAADAR1 for advanced melanoma immunotherapy. We will engineer nucleic acid scaffolds to co-deliver synergistic nucleic acids at defined stoichiometry. We will delineate the engineering principles to improve the PK, (co-)delivery, safety, and therapeutic efficacy, and promote intracellular trafficking and target interaction; we will study the impact of targeting ligands (aptamers, peptides, N- acetylgalactosamine) on these outcomes. Overall, these studies would establish the guidelines to design nucleic acid tools to understand and modulate ADAR biology, and design personalized ADAR therapy. 1