Jin Billy Li

Adenosine-to-inosine RNA editing mediated by endogenous ADAR enzymes represents a promising therapeutic strategy because it can correct mutations at the transcript level without inducing permanent changes to the genome. However, existing systems face challenges, particularly the limited editing efficiency, which limits their practical applications. Here we introduce MARRS (multivalent ADAR1-recruiting RNA editing system)—an efficient and specific RNA editing platform that leverages engineered multivalent RNA aptamers to robustly recruit endogenous ADAR1 enzymes. By optimizing aptamers targeting the Zα domain of ADAR1 p150 and embedding them in a circular RNA scaffold, MARRS enables near-complete adenosine-to-inosine editing of RNA targets in cultured cells and mouse models, achieving efficiencies up to 100% in vitro and 84% in vivo. MARRS also exhibits remarkable versatility across a range of transcript targets while maintaining high specificity with minimal off-target effects. Operating without the need for exogenous enzymes, the MARRS system offers a safe, efficient and programmable platform.

Across biology, organisms have retained a mechanism to diversify the RNA transcriptome through RNA editing. Mediated by ADAR (adenosine deaminase acting on RNA) enzymes, Adenosines in double-stranded RNA (dsRNA) structures can be edited to Inosines (adenosine-to-inosine edit). Although this can change the amino acid sequence if it occurs in a coding sequence of mRNA, the majority of RNA editing in mammalian cells is found in noncoding repetitive elements. These repetitive elements have a predisposition to form long dsRNA structures that can mimic a dsRNA virus. Since the initial discoveries of RNA editing over 30 years ago, investigators have now identified ADAR1 to play a crucial role in suppressing innate immune activation and type I interferon signaling. Through adenosine-to-inosine editing, these dsRNA change their conformational structures and evade activation of the innate immune dsRNA sensor, MDA5 (melanoma differentiation-associated gene 5; gene symbol IFIH1 ). In human disease, although rare loss-of-function variants of ADAR1 have been associated with severe autoimmune disease, there has also been a rapid advance in our understanding of this molecular pathway in common complex diseases. We now understand that common genetic variants can impact RNA editing frequencies, and variants that decrease RNA editing are associated with an increase in risk of numerous autoinflammatory disorders as well as coronary artery disease. This rapid advance in our understanding of the genetic determinants of RNA editing and coronary artery disease has been mirrored by new discoveries in molecular biology, where deficient RNA editing within the vascular wall and smooth muscle cell now highlights endogenous RNA sensing by MDA5 as a causal mechanism of coronary artery disease and other vascular disorders. Here, in this review, we provide a focused look at major advances in RNA editing and cardiovascular disease and put these discoveries into historical context with a goal to map the next steps to advance these molecular pathways to new therapeutic discovery.

BACKGROUND: ADAR1 (adenosine deaminase acting on RNA 1) catalyzes the conversion of adenosine to inosine in double stranded RNAs (dsRNAs), which is critical to prevent autoinflammatory responses mediated by activation of the type I IFN (interferon) signaling. We define the role of ADAR1-dependent RNA editing in IFNβ activation in pulmonary arterial smooth muscle cells (PASMCs) from idiopathic pulmonary arterial hypertension (IPAH), a devastating disease leading to right heart failure and death. METHODS: RNA editing levels were analyzed in PASMCs from IPAH patients versus healthy controls. A conditional transgenic mouse model, Adar1 ^{SMC - KO} , was generated by knocking out Adar1 selectively in Sma (smooth muscle actin)–expressing cells, followed by 3 weeks of hypoxic exposure to induce pulmonary hypertension (PH). RESULTS: PASMCs from patients with IPAH displayed decreased levels of ADAR1 mRNA and isoform p150 protein, accompanied by accumulated dsRNA compared with healthy PASMCs. ADAR1 knockdown in PASMCs upregulated MDA5 (melanoma differentiation-associated protein 5), PKR (protein kinase R), IFNβ, and IFN-stimulated genes. Compared with controls in vivo, hypoxic Adar1 ^{SMC -KO} mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles and increased endothelium leakage, resulting in elevated right ventricular systolic pressure and right ventricular hypertrophy. Mechanistically, Ifnβ signaling in Adar1 ^SMC-KO induced the recruitment of macrophages, enhancing pulmonary artery muscularization. Pharmacological treatment with Pkr-relevant inhibitor 2BAct decreased Ifnβ and macrophages, thus attenuating pulmonary hypertension (PH) in hypoxic Adar1 ^SMC-KO mice. CONCLUSIONS: Our study describes a fundamental molecular mechanism underlying the progression of PH. We highlight the detrimental role of innate immune responses, where smooth muscle cell and context-specific RNA editing, along with the sensing of dsRNA, mediate disease progression and excessive vascular remodeling. This finding suggests that targeting PKR could be the new therapeutic strategy for treating pulmonary arterial hypertension.

Endogenous long double-stranded RNAs (dsRNAs), which are not edited by the RNA editing enzyme ADAR1, may activate the antiviral dsRNA receptor MDA5 to trigger interferon-mediated immune responses. Among the large number of endogenous long dsRNAs, the key substrates that activate MDA5—termed as immunogenic dsRNAs—remain largely unidentified. Here we reveal that human immunogenic dsRNAs constitute a surprisingly small fraction of all cellular dsRNAs. We found that these immunogenic dsRNAs were highly enriched in mRNAs and depleted of introns, consistent with their role as cytosolic MDA5 substrates. We validated the MDA5-dependent immunogenicity of these dsRNAs, which was dampened following ADAR1-mediated RNA editing. Notably, immunogenic dsRNAs were enriched at genetic susceptibility loci associated with common inflammatory diseases, implying their functional importance. We anticipate that a focused analysis of immunogenic dsRNAs will enhance our understanding and treatment of cancer and inflammatory diseases, where the roles of dsRNA editing and sensing are increasingly recognized.

ADAR1 edits adenosine to inosine (A-to-I) in double-stranded RNA to prevent MDA5 sensing of cellular transcripts, its key physiological role. However, editing-independent functions of ADAR1 remain poorly understood. Using a series of Adar1 mutant mice rescued by loss of MDA5 and PKR, we investigated isoform-specific, editing-independent roles of ADAR1 in vivo . We found that the cytoplasmic ADAR1p150 isoform is essential for maintaining peripheral T cell numbers and differentiation of hematopoietic stem and progenitor cells (HSPCs). In bone marrow transplants, ADAR1p150 protein, but not its editing activity, was crucial for T cell regeneration and HSPC repopulation, demonstrating a cell-intrinsic function in hematopoiesis. Experiments with IFNβ-treatment of purified HSPCs in vitro and in vivo IFNAR1 neutralization revealed hypersensitivity to tonic type I interferon (IFN) in the absence of ADAR1p150. Using cell lines, we demonstrate that type I IFN activates the OAS-RNaseL pathway, leading to cell death. This study shows that tonic type I IFN induces immunogenic cellular RNAs in sterile conditions. ADAR1p150 suppresses immune sensing of self-dsRNAs through both editing-dependent (MDA5) and editing-independent (PKR, OAS-RNaseL) mechanisms. Thus, ADAR1p150 protein levels and activity combine to set the threshold for tolerance to self-derived dsRNAs.

Site-directed RNA editing is a promising and potentially safer alternative to genome editing. Previous methods have been developed that recruit the endogenously and ubiquitously expressed ADAR enzymes to initiate site-specific A-to-I edits, but often suffer from low efficacy or dependency on viral delivery. Chemically modified oligonucleotides may be a promising alternative, but the approach still lacks systematic in-depth studies. Furthermore, the best characterized platform uses stereo-pure backbone chemistry, which is not widely used, commercially unavailable and challenging to manufacture. Here, we report on single-stranded oligonucleotides of 30-60 nt length, which are fully chemically stabilized by applying commercially available, classical RNA drug modifications, like 2´-O-methyl, 2´-fluoro, and DNA on a stereo-random phosphate/phosphorothioate backbone. We demonstrate our so-called RESTORE 2.0 oligonucleotides to induce the correction of pathogenic point mutations, efficacy after GalNAc-mediated uptake into human primary hepatocytes, and proof of in-vivo efficacy in mice upon lipid nanoparticle-mediated delivery. The discovered design principles may increase the accessibility of site-directed RNA base editing to expand and support further research in this field.

Although genetic risk in coronary artery disease (CAD) is linked to changes in gene expression, recent discoveries have revealed a major role for A-to-I RNA editing in CAD. ADAR1 edits immunogenic double-stranded RNA (dsRNA), preventing activation of the dsRNA sensor MDA5 (IFIH1) and downstream interferon-stimulated gene signaling. Using human plaque analysis and human coronary artery smooth muscle cells (SMCs), here, we show that SMCs uniquely require RNA editing and that MDA5 activation regulates SMC phenotype. In a conditional SMC-specific Adar deletion mouse model on an atherosclerosis-prone background, combined with Ifih1 deletion and single-cell RNA sequencing, we demonstrate that ADAR1 preserves vascular integrity and limits atherosclerosis and calcification by suppressing MDA5 activation. Analysis of the Athero-Express carotid endarterectomy cohort further shows that interferon-stimulated gene expression correlates with SMC modulation, plaque instability and calcification. These findings reveal a fundamental mechanism of CAD, where cell type and context-specific RNA editing modulates genetic risk and vascular disease progression.

Endogenous, long double-stranded RNA (dsRNA) can resemble viral dsRNA and be recognized by cytosolic dsRNA sensors, triggering autoimmunity. Genetic studies of rare, inherited human diseases and experiments using mouse models have established the importance of adenosine-to-inosine RNA editing by the enzyme adenosine deaminase acting on RNA 1 (ADAR1) as a critical safeguard against autoinflammatory responses to cellular dsRNA. More recently, human genetic studies have revealed that dsRNA editing and sensing mechanisms are involved in common inflammatory diseases, emphasizing the broader role of dsRNA in modulating immune responses and disease pathogenesis. These findings have highlighted the therapeutic potential of targeting dsRNA editing and sensing, as exemplified by the emergence of ADAR1 inhibition in cancer therapy.

Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) by ADAR1 is an essential modifier of the immunogenicity of cellular dsRNA. The role of MDA5 in sensing unedited cellular dsRNA and the downstream activation of type I interferon (IFN) signaling are well established. However, we have an incomplete understanding of pathways that modify the response to unedited dsRNA. We performed a genome-wide CRISPR screen and showed that GGNBP2, CNOT10, and CNOT11 interact and regulate sensing of unedited cellular dsRNA. We found that GGNBP2 acts between dsRNA transcription and its cytoplasmic sensing by MDA5. GGNBP2 loss prevented induction of type I IFN and autoinflammation after the loss of ADAR1 editing activity by modifying the subcellular distribution of endogenous A-to-I editing substrates and reducing cytoplasmic dsRNA load. These findings reveal previously undescribed pathways to modify diseases associated with ADAR mutations and may be determinants of response or resistance to small-molecule ADAR1 inhibitors.

A-to-I RNA editing is a cellular mechanism that generates transcriptomic and proteomic diversity, which is essential for neuronal and immune functions. It involves the conversion of specific adenosines in RNA molecules to inosines, which are recognized as guanosines by cellular machinery. Despite the vast number of editing sites observed across the animal kingdom, pinpointing critical sites and understanding their in vivo functions remains challenging. Here, we study the function of an evolutionary conserved editing site in Drosophila , located in glutamate-gated chloride channel ( GluCl α). Our findings reveal that flies lacking editing at this site exhibit reduced olfactory responses to odors and impaired pheromone-dependent social interactions. Moreover, we demonstrate that editing of this site is crucial for the proper processing of olfactory information in projection neurons. Our results highlight the value of using evolutionary conservation as a criterion for identifying editing events with potential functional significance and paves the way for elucidating the intricate link between RNA modification, neuronal physiology, and behavior.