Nucleic acid modulators and theranostics for ADAR

ADAR 的核酸调节剂和治疗诊断

• 批准号: 10276650
• 负责人: Guizhi Zhu
• 金额: $ 38.81万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-18 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276650
• 关键词: ADAR1; Acetylgalactosamine; Address; Adenosine; Agonist; Albumins; Biological; Biological Process; Biology; Cells; Chimera organism; Clinical; Cyclic GMP; DNA; Disease; Drug Kinetics; Engineering; Environment; GTP-Binding Protein alpha Subunits, Gs; Guide RNA; Guidelines; Human; Immunotherapy; Individual; Inosine; Interferon Type I; Interferons; Ligands; Luciferases; Mediating; Medicine; Messenger RNA; Metastatic Melanoma; Molecular; Mutation; Nucleic Acids; Oligonucleotides; Outcome; Pathogenicity; Peptides; RNA; RNA Binding; RNA Editing; Reporter; Safety; Small Interfering RNA; Testing; Thrombophilia; Time; Tissues; Treatment Efficacy; adenosine deaminase; aptamer; base; biological systems; bioluminescence imaging; design; efficacy testing; factor V Leiden; gene therapy; improved; in vivo; lipid nanoparticle; melanoma; mouse model; nano; nanoparticle; neoplastic cell; novel; nucleic acid delivery; nucleic acid-based therapeutics; scaffold; spatiotemporal; stoichiometry; success; targeted delivery; theranostics; therapeutic genome editing; tool; trafficking

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

项目摘要 该MIRA应用的目的是：1）在体内分析异源腺苷脱氨酶的作用， 使用RNA纳米报告分子作为工具来了解阿达尔生物学并指导阿达尔的设计- 2）使用嵌合阿达尔适体-gRNA（指导RNA）促进内源性阿达尔调节 和诱导ADAR 1的I型干扰素（IFN-I）活化DNA寡核苷酸; 3）靶向递送 用于内源性基于ADAR的基因治疗和免疫治疗的白蛋白搭便车DNA/RNA;以及4）试点 在凝血因子V Leiden（FVL）血栓形成倾向和转移性黑素瘤的小鼠模型中测试这些治疗诊断学。 阿达尔介导后生动物中RNA腺苷至肌苷编辑。阿达尔是一个有趣的内源性RNA编辑器 用于致病性G->A突变引起的疾病，如FVL血栓形成倾向的基因治疗;此外， 肿瘤细胞中的ADAR 1抑制使其免疫疗法敏感。然而，阿达尔水平很高， 在个体、组织和细胞环境中的异质性和动态性，使其成为 时空阿达尔分析以研究阿达尔生物学并设计基于ADAR的个性化治疗。本 最后，我们将开发和测试一种阿达尔报告基因，用于体内非侵入性实时阿达尔分析，使用阿达尔- 可激活的分裂荧光素酶mRNA报告子，随后进行生物发光成像。此外，经常低 内源性阿达尔水平限制了阿达尔编辑功效，这阻碍了阿达尔的生物学发现和治疗诊断。 应用.为了应对这一挑战并促进内源性阿达尔的RNA编辑功效，我们将 研究两种策略：1）使用阿达尔适体-gRNA嵌合体促进阿达尔结合和RNA编辑; 2)使用我们的新型IFN-1激活环GMP-AMP合酶的DNA激动剂来提高阿达尔水平， 促进阿达尔调节功效。所有这些核酸治疗诊断学，如适体、siRNA和mRNA， 拥有重塑人类医学的巨大潜力。然而，迄今为止，裸DNA/RNA的临床成功有限， 主要是由于不良的药代动力学（PK）和不能进入细胞，需要了解机制， 操纵生物系统和DNA/RNA之间的相互作用。在此之前，我们开发了一个 分子白蛋白搭便车和纳米颗粒，促进PK和核酸的输送高达200 褶皱在这里，我们将使用脂质纳米颗粒来传递mRNA报告基因，用于体内阿达尔分析;我们将研究 靶向递送白蛋白搭便车阿达尔核酸治疗剂，包括用于 FVL血栓形成基因疗法和siRNAADAR 1用于晚期黑色素瘤免疫疗法。我们将工程师 核酸支架以限定的化学计量共同递送协同核酸。我们将描绘出 改善PK、（共）递送、安全性和治疗功效，并促进细胞内 运输和目标相互作用;我们将研究靶向配体（适体，肽，N- 乙酰半乳糖胺）对这些结果的影响。总的来说，这些研究将建立设计核酸的指导方针， 酸性工具，以了解和调节阿达尔生物学，并设计个性化的阿达尔治疗。 1