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ADAR

ADAR (Adenosine Deaminase Acting on ) is a family of enzymes that catalyze the hydrolytic of (A) to (I) in double-stranded (dsRNA) substrates, a process known as A-to-I . This editing alters the sequence and structure of molecules, effectively changing the as is recognized as (G) during , thereby diversifying protein isoforms and regulating processing pathways. Discovered in the late through studies on dsRNA unwinding and modification activities, ADAR enzymes are highly conserved across metazoans, originating from ancient tRNA-modifying deaminases like ADATs. In mammals, including humans, there are three primary ADAR genes encoding the protein family: ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are catalytically active, while ADAR3 is predominantly catalytically inactive and functions mainly in the , potentially acting as a competitor for substrates. ADAR1 exists in two main isoforms: the constitutive p110 form, which is nuclear and expressed in most tissues, and the interferon-inducible p150 isoform, which is cytoplasmic and plays a key role in innate immune responses by editing viral RNAs and preventing aberrant activation of antiviral pathways. ADAR2, expressed widely but most abundantly in the , often functions as a dimer and undergoes self-editing of its own pre-mRNA to regulate its activity. Structurally, all ADARs feature 2–3 double-stranded RNA-binding domains (dsRBDs) at the for substrate recognition and a conserved deaminase domain at the for , with ADAR1 uniquely containing Z-DNA-binding domains. The functions of enzymes extend beyond simple sequence alteration, influencing a wide array of biological processes. In protein-coding transcripts, selective editing recodes in channels (e.g., GluR-B subunit of receptors, reducing calcium permeability to prevent ) and receptors (e.g., serotonin 2C receptor), thereby fine-tuning neuronal signaling and . For non-coding RNAs, ADARs edit introns, UTRs, and repetitive elements like Alu sequences, affecting , (miRNA) biogenesis, and endogenous siRNA (esiRNA) pathways to modulate and immune responses. ADAR1p150, in particular, is essential for suppressing signaling by editing self-dsRNAs, averting autoimmune conditions, while ADAR2 is critical for normal development. studies reveal their indispensability: ADAR1 deficiency is embryonic lethal due to defects in hematopoiesis, and ADAR2 loss leads to progressive seizures and neurodegeneration in mice. Dysregulation or mutations in ADAR genes are implicated in various diseases, highlighting their clinical significance. Loss-of-function mutations in ADAR1 cause Aicardi-Goutières syndrome, a severe autoinflammatory disorder mimicking viral infection, and dyschromatosis symmetrica hereditaria, a pigmentary . Altered ADAR2 activity is associated with (ALS), epilepsy, and , often through disrupted editing of targets like GluR-B. In various cancers, including gliomas and , ADAR1 overexpression promotes tumor progression by editing transcripts and modulating immune responses. Emerging therapeutic strategies leverage ADARs for programmable RNA editing to correct disease-causing mutations, such as in and inherited retinal diseases, with some candidates, like WVE-006, entering Phase 1/2 clinical trials as of 2025, offering precise, transient modifications without altering the genome.

History

Discovery

The adenosine deaminase acting on (ADAR) enzymes were first identified in 1987 by Brenda L. Bass and Harold Weintraub, who detected an activity in extracts from Xenopus laevis oocytes that unwinds double-stranded structures through the site-specific of to in mRNA substrates. This revealed A-to-I as a novel mechanism, distinct from previously known processing events. The activity was developmentally regulated, appearing prominently in oocytes and early embryos, and was shown to require double-stranded (dsRNA) conformations for efficient catalysis, as single-stranded RNAs were poor substrates. In the early , research shifted to mammalian systems, where site-specific A-to-I editing was found in pre-mRNAs encoding subunits, such as the Q/R site in GluR-B transcripts from brain, altering calcium permeability and neuronal excitability. These findings prompted the of family members. The ADAR1 , encoding a dsRNA-specific , was cloned in from a cell cDNA library, revealing a protein with deaminase and dsRNA-binding domains capable of editing both viral and cellular RNAs. In 1995, the ADAR2 was cloned from brain tissue, identifying an enzyme with high specificity for the GluR-B Q/R site and confirming the existence of a multigene family of editing enzymes. Early biochemical assays further characterized the dsRNA substrate requirement, demonstrating that ADAR activity is stimulated by structured longer than ~30 base pairs and inhibited by single-stranded or mismatched regions, laying the foundation for understanding editing site selectivity. A major milestone came in 2000 with the identification of ADAR3, cloned from cDNA, which shares structural with ADAR1 and ADAR2 but lacks catalytic activity due to amino acid substitutions in the deaminase , suggesting a regulatory role.

Evolutionary Origins

The gene family represents a metazoan , with homologs present across diverse phyla but absent in , fungi, and non-metazoan eukaryotes such as and . This distribution indicates that ADAR enzymes evolved specifically within the last common ancestor (LCA) of extant metazoans, approximately 700 million years ago, coinciding with the emergence of multicellular life and enabling A-to-I as a novel post-transcriptional . Phylogenetic analyses confirm ADAR presence in early-branching lineages like sponges (Porifera) and ctenophores, with secondary losses in certain groups such as (where only an ADAR2 homolog persists) and placozoans. Recent studies as of 2023 further support ADAR homologs in ctenophores, reinforcing their origin in the metazoan LCA. ADAR1 and ADAR2 arose via from an ancestral ADAR in early metazoan , approximately 700 million years ago. This event allowed for functional specialization: ADAR2 primarily handles site-specific editing in neural transcripts, while ADAR1 focuses on global editing of non-coding RNAs. A further duplication of ADAR2 gave rise to the catalytically inactive ADAR3 in vertebrates, enhancing the family's repertoire without altering core editing capabilities. These events align with the , suggesting adaptive pressures from increasing organismal complexity drove ADAR paralog formation. Throughout metazoan evolution, the deaminase domain and double-stranded RNA-binding motifs (dsRBMs) have remained highly conserved as core structural features of proteins. The C-terminal deaminase domain, responsible for , exhibits strong across species, tracing back to ancestral tRNA deaminases (ADATs) that acquired dsRBMs for . dsRBMs, typically numbering two to three per protein, show phylogenetic in their ability to bind dsRNA structures, with variations in count (e.g., one in nematodes, three in some isoforms) reflecting lineage-specific adaptations rather than loss of function. This conservation underscores the domains' indispensable role in maintaining editing fidelity from to vertebrates. Comparative genomics has revealed ADAR family expansion in mammals, where the presence of three paralogs (ADAR1, ADAR2, ADAR3) correlates with heightened demands for in the complex mammalian . Unlike simpler metazoans with one or two ADAR genes, mammalian genomes retain the full set, enabling diverse editing patterns in neuronal ion channels and receptors that enhance and behavioral adaptability. This expansion, evident in genome assemblies of and , likely provided selective advantages for processing extensive neural transcriptomes, as inferred from cross-species alignments showing increased editing site density in brain-specific genes.

Molecular Structure

Gene Organization and Isoforms

The in humans consists of three members: ADAR1, ADAR2, and ADAR3, each encoded by distinct that arose from evolutionary duplication events, contributing to isoform diversity through alternative promoters and splicing. The ADAR1 (also known as ADAR) is located on 1q21.1 and spans approximately 46 kb, comprising 15 . It produces two major protein isoforms via alternative promoter usage and first selection: the p110 isoform (also called ADAR1-S or constitutive form), which is transcribed from a housekeeping promoter and localized primarily to the , and the p150 isoform (ADAR1-L or interferon-inducible form), driven by an interferon-stimulated response element (ISRE)-containing promoter that responds to type I interferons, allowing both and cytoplasmic localization. The p150 isoform includes an N-terminal Z-DNA binding domain absent in p110, enabling its unique subcellular shuttling and response to immune signaling. The ADAR2 gene (ADARB1) resides on chromosome 21q22.3, covering about 153 kb with 15 exons, and generates multiple isoforms primarily through alternative splicing at several sites. Four major splice variants have been identified, differing in the inclusion or exclusion of sequences that affect double-stranded RNA-binding motifs (dsRBMs) and nuclear export signals: for instance, skipping of exon 2 eliminates one dsRBM, yielding a truncated, inactive form, while variants incorporating an Alu-derived exon 5a insert additional residues in the deaminase domain, and C-terminal splicing events between exons 9 and 10 alter sequences potentially influencing nuclear export and localization signals. These variants exhibit varying editing efficiencies and tissue-specific expression, with full-length forms predominant in the brain. The ADAR3 (ADARB2) is positioned on 10p15.3, spanning roughly 560 kb, and encodes a catalytically inactive, pseudogene-like protein that functions primarily as an RNA-binding inhibitor rather than an editor. ADAR3 expression is highly restricted to the , particularly postnatally in neurons, where it competes with active ADARs for substrate binding without deaminase activity due to in its catalytic . No major isoforms beyond the primary transcript have been widely reported, emphasizing its role as a dominant-negative regulator in neural tissues.

Protein Domains and Active Site

ADAR proteins feature a modular characterized by N-terminal double-stranded RNA-binding domains (dsRBDs) and a C-terminal deaminase essential for . Human ADAR1 contains three dsRBDs for substrate , along with a Z-DNA-binding (Zα) unique to its longer p150 isoform, which facilitates binding to left-handed Z-DNA or Z-RNA structures. In contrast, ADAR2 and ADAR3 each possess two dsRBDs that enable specific interactions with double-stranded RNA substrates, positioning the enzyme for efficient editing. The resides within the conserved deaminase and centers on a ion tetrahedrally coordinated by 394, 451, 516, and a , which activates nucleophilic attack on the substrate . A critical glutamate residue at position 396 functions as a , facilitating the reaction by stabilizing the . hexakisphosphate (IP6) binds nearby, stabilizing the catalytic core and enhancing activity. ADAR1 additionally features a secondary -binding site involving 1081 and 1082, along with 988 and 1103, which supports structural integrity but is absent in ADAR2. Structural investigations using and NMR spectroscopy have provided atomic-level details of the deaminase domain and dsRBD-RNA interactions; for instance, the of the ADAR2 deaminase domain (PDB: 1ZY7) reveals the 's compact fold, while NMR studies of ADAR2 dsRBDs bound to (PDB: 2L3J) demonstrate minor groove recognition via α-helices acting as molecular rulers. Recent cryo-EM analyses of ADAR1- complexes, achieved at resolutions around 3 Å in the early 2020s and higher resolutions in 2025 (e.g., elucidating pre- states and mismatch tolerance), illustrate partial unwinding of dsRNA substrates and adenosine base flipping into the , highlighting conformational changes that enable selective . Unlike ADAR1 and ADAR2, ADAR3 is catalytically inactive due to key mutations in the deaminase domain, including substitutions at glutamate 527 (to ) and glutamine 549 (to ), which disrupt proton shuttling and coordination essential for activity. These alterations render the non-functional, positioning ADAR3 primarily as a regulatory protein that competes for substrates via its intact dsRBDs.

Dimerization and Regulation

ADAR proteins primarily function as homodimers, with dimerization playing a crucial role in their enzymatic activity. For ADAR2, homodimerization occurs through interfaces in the C-terminal deaminase , as revealed by structures from the , including a of an asymmetric homodimer comprising the deaminase domain and double-stranded RNA-binding motif 2 (dsRBM2) (PDB: 6VFF). This dimerization is RNA-binding independent and essential for efficient A-to-I editing, as mutations in the interface reduce activity on certain substrates. Similarly, ADAR1 forms stable homodimers critical for its editing function. There is evidence for potential heterodimer formation between ADAR1 and ADAR2, particularly under cellular conditions, where RNA-dependent interactions between the two enzymes have been observed in various cell lines using techniques like . These heterodimers may coordinate editing of specific transcripts, though their precise role in stress responses remains under . Post-translational modifications provide key regulatory control over ADAR activity. of ADAR2 by ζ (PKCζ) at serine residues 211 and 216, located in the linker region adjacent to the dsRBMs, enhances its editing efficiency, as demonstrated in cells where this modification promotes miR-200 editing and influences . For ADAR1, SUMOylation by SUMO-1 primarily affects nucleolar localization and reduces editing activity on certain substrates, with desumoylation restoring function. Additionally, both ADAR1 and ADAR2 exhibit auto-inhibitory mechanisms in their inactive states; for ADAR2, the N-terminal region, including sequences near the dsRBMs, sterically hinders the deaminase domain, and relief of this inhibition occurs upon dsRNA binding. Allosteric regulation further modulates ADAR activity based on substrate features. The length of dsRNA substrates influences binding affinity and editing efficiency, with optimal activity requiring duplexes of at least 20-30 base pairs to accommodate the dsRBMs and deaminase domain. Mismatch positioning within the dsRNA also allosterically affects site selectivity, as ADAR enzymes preferentially edit adenosines adjacent to mismatches or bulges, which induce conformational changes that position the target base in the active site.

Catalytic Mechanism

Biochemical Reaction

The ADAR enzymes catalyze the hydrolytic deamination of (A) to (I) in double-stranded substrates, a that effectively alters the RNA sequence as is recognized as (G) during . This reaction proceeds via the incorporation of a molecule, yielding and as products, and can be represented by the equation: \text{Adenosine} + \text{H}_2\text{O} \rightarrow \text{Inosine} + \text{NH}_3 The catalytic mechanism involves a two-step hydrolytic process centered on the ADAR deaminase domain. Initially, a conserved glutamate residue abstracts a proton from the N1 position of the adenine ring, polarizing the substrate and facilitating the subsequent nucleophilic attack. A water molecule, activated by coordination to a catalytic zinc ion bound by one histidine and two cysteine residues (with the fourth ligand being a water molecule), then attacks the C6 position of the adenine, forming a transient tetrahedral intermediate at this carbon. Elimination of the amino group as ammonia follows, resulting in the hypoxanthine ring of inosine. In ADAR2, inositol hexakisphosphate (IP6) binds in the enzyme core and is essential for catalytic activity. Mammalian enzymes exhibit optimal activity at neutral (6.5–7.0) and physiological (37°C), conditions that support efficient while requiring double-stranded structures for binding.

Substrate Specificity

enzymes exhibit a strong preference for double-stranded (dsRNA) substrates consisting of stems longer than 20 base pairs, as shorter duplexes fail to provide sufficient binding affinity for the enzyme's double-stranded RNA-binding motifs (dsRBMs). This length requirement ensures stable interaction, with editing occurring preferentially in dsRNA regions containing mismatches, bulges, or internal loops that promote site-selective rather than widespread modification. In contrast, single-stranded (ssRNA) or non-dsRNA structures are not recognized or edited by , as the enzymes lack affinity for unstructured or single-stranded regions. Editing efficiency within dsRNA duplexes is generally higher in central stem regions and decreases in close proximity to duplex termini due to binding constraints of the dsRBDs. ADAR1 and ADAR2 display distinct site selectivities: ADAR1 favors hyper-editing of long dsRNAs, such as those formed by Alu repetitive elements, where up to 50% of adenosines may be modified across extensive regions. Conversely, ADAR2 specializes in precise, single-site editing of specific neuronal transcripts, like the subunit GluR2 Q/R site, enabling targeted recoding events critical for synaptic function. Perfectly matched dsRNA, while a substrate for ADAR1-mediated hyper-editing, results in reduced site specificity and can inhibit selective editing by promoting non-discriminatory . For ADAR2, site selectivity is further guided by a of 5'-NAN-3', where the central A is the edited (N denoting any ), with preferences strongly influenced by a 5' flanking U (enhancing base flipping) and a 3' flanking G (stabilizing the ). This contributes to the enzyme's accuracy in neuronal contexts, where even minor deviations reduce efficiency. ADAR1 shows broader tolerance but overlaps with ADAR2 preferences, such as a 5' U > A > C > G order, though without strict 3' constraints. These sequence and structural determinants collectively ensure that ADAR editing is confined to appropriate dsRNA contexts, avoiding off-target modifications in cellular RNAs.

Editing Efficiency Factors

Several intracellular factors modulate the efficiency and fidelity of ADAR-mediated A-to-I by influencing substrate accessibility and enzyme competition. ATP-dependent RNA helicases, such as DDX6, interact with ADAR1 and ADAR2 to regulate levels, potentially by unwinding or dsRNA substrates to facilitate deaminase . Similarly, competition for dsRNA binding occurs between ADARs and other dsRNA-binding proteins like PKR, where PKR's higher affinity for certain dsRNA structures can reduce ADAR recruitment and thereby lower rates. Isoform-specific differences in editing efficiency arise from structural and regulatory variations among family members. The interferon-inducible p150 isoform exhibits high efficiency in hyper-editing Alu repeat elements, often targeting multiple adenosines within inverted Alu pairs in non-coding regions to extensively modify transcripts. In contrast, ADAR2 demonstrates site-selective editing with near-complete efficiency at specific recoding sites, such as the Q/R site in the GluA2 subunit of receptors, where it precisely converts a codon to to alter properties.00079-1) Environmental conditions like hypoxia and cellular stress further influence ADAR editing by inducing ADAR1 expression and enhancing overall activity. Under hypoxic conditions, ADAR1 levels increase, promoting more robust editing of hypoxia-responsive transcripts and supporting adaptive cellular responses. Stress signals similarly upregulate ADAR1, amplifying editing to modulate RNA stability and processing during physiological challenges. In vivo, ADAR editing frequencies span a wide range, typically from 10% to over 90% depending on the , , and , as quantified through high-throughput sequencing of edited transcripts. Dimerization of ADAR enzymes, particularly ADAR2, briefly stabilizes the dsRNA-substrate complex to support efficient catalysis at preferred sites.

Physiological Functions

RNA Editing in Development and Neurobiology

ADAR2 plays a critical role in embryonic development by mediating RNA editing at the glutamine/arginine (Q/R) site of the glutamate receptor subunit GluA2 (formerly GluR2), which converts a genomically encoded glutamine codon (CAG) to arginine (CGG), thereby reducing calcium permeability of AMPA receptors and preventing excitotoxic neuronal death. This editing event is nearly 100% efficient in mature neurons and is essential for controlling Ca²⁺ influx, as unedited GluA2-containing channels exhibit heightened Ca²⁺ permeability, leading to cell death under physiological conditions. Loss of ADAR2 in mouse models results in progressive neurodegeneration due to insufficient Q/R editing, underscoring its necessity for neuronal survival during development. In neurobiology, ADAR-mediated editing diversifies protein function beyond the Q/R site; for instance, editing of the serotonin 2C receptor (5-HT₂C R) pre-mRNA at multiple intronic and exonic sites alters in the second intracellular , reducing G-protein coupling efficiency and modulating signaling. Fully edited isoforms show diminished constitutive activity and altered affinity, influencing behaviors such as anxiety and feeding. ADAR2 exhibits high expression in the , particularly in regions involved in , where it ensures efficient editing of subunits to fine-tune synaptic transmission and . Recent studies using human (iPSC)-derived models have revealed time-resolved dynamics of ADAR activity during , showing progressive increases in editing efficiency at neural-specific sites from neural progenitor stages to mature neurons, highlighting ADAR's temporal regulation in human development. ADAR1 also contributes critically to embryonic development by editing endogenous double-stranded RNAs to suppress aberrant signaling, which is essential for maintaining function and enabling normal hematopoiesis. Conditional knockout studies in mice demonstrate that ADAR1 deficiency disrupts erythroid and myeloid , leading to embryonic due to severe defects in production.

Role in Innate Immunity

ADAR1, particularly its interferon-inducible p150 isoform, plays a central role in modulating innate immune responses by editing endogenous double-stranded RNAs (dsRNAs) to distinguish self from non-self nucleic acids. Upon stimulation by type I interferons, ADAR1 p150 is transcriptionally upregulated via pathways involving STAT2, leading to its cytoplasmic localization where it catalyzes A-to-I editing of self dsRNAs, such as those derived from Alu retroelements. This editing disrupts the structure and immunostimulatory potential of these dsRNAs, preventing their recognition by sensors like MDA5 (melanoma differentiation-associated protein 5) and PKR (protein kinase R), thereby averting aberrant activation of downstream signaling cascades that drive autoinflammation. The binding domain (Zα) of ADAR1 enhances this protective function by sensing and binding left-handed Z-RNA structures formed during immune stress, facilitating targeted of endogenous dsRNAs to suppress excessive responses. Recent insights highlight how this integrates with innate immune sensing pathways to promote of cellular RNAs and maintain immune . Loss-of-function mutations in ADAR1, particularly in the Zα or affecting the p150 isoform, disrupt this balance and trigger type I opathies, such as Aicardi-Goutières syndrome, characterized by constitutive signaling and autoinflammatory phenotypes. These genetic defects result in unedited dsRNA accumulation, hyperactivation of and ZBP1 (Z-DNA binding protein 1), and lethal -driven pathology, underscoring ADAR1's essential role in immune .

Pathological Implications

Neurodevelopmental and Neurodegenerative Disorders

Mutations in the ADAR1 gene are a primary cause of Aicardi-Goutières syndrome (AGS), a severe characterized by , calcifications in the , and chronic activation of type I signaling. These mutations disrupt ADAR1's function, leading to the accumulation of unedited double-stranded RNA that aberrantly activates the MDA5-STING pathway and triggers excessive production, resulting in autoinflammation and neurological damage in affected infants. For instance, the p.K999N mutation in ADAR1 has been shown to induce pathway activation specifically in the , exacerbating in mouse models of AGS. ADAR1 variants also underlie bilateral striatal necrosis (BSN), a rare neurodevelopmental condition presenting with acute , , and degeneration of the , often accompanied by skin changes such as freckle-like . This disorder arises from impaired in ADAR1, which fails to suppress responses and leads to selective neuronal loss in the , as evidenced by a type I signature in patient and tissue. Clinical reports of siblings with homozygous ADAR1 mutations highlight the role of editing defects in causing progressive and motor impairments that mimic but are distinguished by overactivation. In (ALS), a neurodegenerative disorder affecting motor neurons, diminished ADAR2 activity results in inefficient A-to-I editing at the Q/R site of the GluR2 subunit of receptors, promoting calcium influx and excitotoxic death of spinal motor neurons. This editing deficiency is observed in 56% of sporadic cases, where ADAR2 expression is reduced in vulnerable motor neurons, directly correlating with disease progression and neuronal loss. Recent studies have further elucidated interactions between ADAR2 and TDP-43, a key pathological protein in ; ADAR2 deficiency leads to TDP-43 mislocalization and aggregation in motor neurons, amplifying neurodegeneration in conditional models. Rescue experiments using AAV-mediated ADAR2 delivery have demonstrated normalization of GluR2 editing and prevention of TDP-43 pathology, underscoring ADAR2's protective role against motor neuron death.

Oncogenic Roles in Cancer

ADAR1 is frequently upregulated in various cancers, including , , liver, and esophageal carcinomas, contributing to tumorigenesis through both editing-dependent and independent mechanisms. This upregulation often occurs via activation of pathways such as /JAK2/ signaling, as observed in drug-resistant organoids where ADAR1 expression increases in response to chemotherapeutic agents like 5-fluorouracil and . Similarly, in tumor-associated macrophages of , ADAR1 elevation promotes drug resistance through the pathway, enhancing resistance in cancer cells. ADAR1 also edits transcripts of oncogenes, such as through its interaction with NEIL3 to generate circNEIL3 in pancreatic ductal ; this circular RNA sponges miR-432-5p, forming a feedback loop that upregulates ADAR1 and confers chemoresistance by promoting and epithelial-mesenchymal transition. In (HCC), ADAR2 acts as a tumor suppressor, with its suppression correlating with poor prognosis and tumor progression. Downregulation of ADAR2 occurs in approximately 50% of HCC cases, leading to an imbalance in that favors oncogenic outcomes. This suppression contributes to hyper-editing of microRNA precursors, such as miR-214 and miR-122, by ADAR enzymes, altering their maturation and target specificity to promote tumor growth; elevated ADAR2 levels in a subset of HCC samples verify this editing pattern as a marker of severity. The resulting editome imbalance, driven by ADAR1 overexpression and ADAR2 loss, hyper-edits sites like those in AZIN1 mRNA while hypo-editing others, exacerbating HCC . In melanoma, ADAR1 facilitates immune evasion by modulating transcripts in the , including those influencing expression to suppress T-cell responses. Recent 2024 analyses highlight how A-to-I editing in acidic s increases editing levels, promoting immunosuppressive conditions that aid melanoma progression; for instance, ADAR1 deficiency sensitizes tumors to by disrupting this evasion. Across cancers, ADAR-mediated A-to-I editing generates proteomic diversity by introducing non-synonymous mutations, such as recoding events that enhance ; for example, edited isoforms promote invasiveness in cells. Pan-cancer analyses of (TCGA) data reveal distinct A-to-I editing signatures in 17 tumor types, with elevated editing at coding sites correlating with aggressive phenotypes and poor survival, underscoring ADAR's role in tumor heterogeneity.

Dermatological and Infectious Diseases

ADAR1 mutations are the primary cause of dyschromatosis symmetrica hereditaria (DSH), an autosomal dominant pigmentary characterized by hyperpigmented and hypopigmented macules on , particularly on the face, arms, and legs, appearing in infancy or . These mutations impair the enzyme's activity, leading to defective A-to-I editing of double-stranded transcripts in melanocytes, which disrupts normal pigmentation patterns and results in the hallmark lesions. Specifically, loss of ADAR1 function affects the development and migration of neural crest-derived melanocytes, causing irregular production and distribution in . Analysis of patient cohorts has identified over 80 distinct ADAR1 missense variants in DSH, confirming their pathogenicity through functional assays showing diminished enzymatic activity and altered targets. In the context of infectious diseases, ADAR1 plays a dual role in HIV-1 infection by editing viral RNAs, including those encoding the Tat transactivator and regions near the Vif accessory protein, which modulates viral replication dynamics. This editing activity can enhance HIV-1 production in certain cell types, such as CD4+ T cells, by stabilizing viral transcripts and preventing recognition by host antiviral sensors, thereby supporting proviral persistence. Conversely, in macrophages, ADAR1-mediated editing inhibits post-transcriptional HIV-1 replication, contributing to the establishment and maintenance of viral latency by reducing productive infection and proviral integration. ADAR enzymes also influence other infections, including () pathogenesis, where ADAR editing of viral miRNAs affects neurovirulence by altering that promotes neuronal and severity. In bacterial infections, pathogen-derived double-stranded mimics or structured RNAs can trigger ADAR activation, leading to hyper-editing of host transcripts and modulation of innate immune responses, such as production, to control bacterial dissemination. Recent studies highlight 's involvement in chronic skin inflammations like , where reduced A-to-I editing of Alu elements in transcripts generates immunogenic double-stranded RNAs that exacerbate inflammation and disease progression. In psoriatic lesions, diminished activity correlates with increased expression of interferon-stimulated genes, linking editing defects to the hyperproliferative and inflammatory observed in affected skin.

Viral Interactions

Antiviral Mechanisms

enzymes, particularly ADAR1, contribute to host antiviral defense by editing viral double-stranded (dsRNA), introducing A-to-I hypermutations that manifest as A-to-G changes during reverse transcription. This hyper-editing disrupts viral genome integrity and reduces replication fitness. For instance, in measles virus (MeV) , ADAR1 p150 isoform targets the (P) gene, catalyzing extensive A-to-I edits that generate defective viral genomes, limiting viral spread in the and restricting cytopathic effects. These mutations impair essential functions, such as activity, thereby acting as a restriction factor during acute . The interferon-inducible ADAR1 p150 isoform further bolsters antiviral responses by editing endogenous self-RNAs to prevent aberrant activation of the cytosolic sensor during viral infection. By converting adenosines to inosines in self-dsRNAs, p150 disrupts filamentation and signaling, suppressing excessive type I production that could otherwise lead to immunopathology while allowing focused antiviral immunity. This editing-dependent mechanism ensures that host responses target viral dsRNA preferentially, integrating with innate immune pathways to control infection without self-attack. In specific viral contexts, ADAR-mediated editing impairs pathogen proteins and replication. For (HCV), ADAR1 induced by interferon-α edits the HCV replicon , introducing A-to-I mutations that destabilize the genome and trigger degradation by inosine-specific RNases, thereby reducing replicon persistence and viral RNA levels. Similarly, for (IAV), ADAR1 edits minor viral populations, generating hypermutated transcripts with premature stop codons or frameshifts in genes like NS1, which diminish viral fitness and limit transmission efficiency. Recent studies from 2023 have engineered ADAR1 variants, such as editing-deficient mutants, to dissect these mechanisms, revealing that enhanced editing potency in p150 specifically amplifies suppression of and PKR activation, thereby potentiating antiviral restriction in model infections. This host-virus dynamic exemplifies an , where viruses evolve countermeasures to evade activity. Adenoviruses, for example, express virus-associated I (VAI) RNA, a structured dsRNA mimic that competitively binds and inhibits the deaminase domain of , preventing viral RNA editing and allowing unimpeded replication. Such viral inhibitors underscore 's critical role in innate antiviral immunity.

Proviral Contributions

ADAR-mediated RNA editing can facilitate by enhancing viral and replication in certain contexts. In HIV-1, ADAR1 edits adenosines in the Tat coding sequence, contributing to increased viral infectious potential through an editing-dependent mechanism that boosts virion release and infectivity, although the specific edit at position A6036 does not alter the Tat protein's sequence. Similarly, in (KSHV), ADAR editing targets the ORF50 () transcript at sites such as the predicted recoding of to at 378 in the , potentially modulating RTA's transcriptional regulatory activity to support viral by influencing the balance between latent and lytic phases. ADAR1 also promotes replication of flaviviruses, such as , by viral substrates, including potential sites in structured regions that may enhance stability, alongside inhibiting PKR activation to reduce antiviral signaling and facilitate non-structural protein synthesis. This activity stabilizes viral RNAs, allowing for more efficient and propagation in host cells. For instance, ADAR1's interaction with viral components enables hyperediting or site-specific modifications that counteract host defenses while supporting viral fitness. A key proviral mechanism involves immune evasion, where ADAR editing alters viral antigens to diminish T-cell recognition. Edited viral proteins present modified epitopes that evade CD8+ T-cell surveillance, reducing cytotoxic responses and promoting persistence. Studies have detected A-to-I editing in SARS-CoV-2 viral transcripts, potentially contributing to viral evolution, though direct evidence for spike gene-specific editing driving T-cell immune escape remains limited and under investigation as of 2025. Recent 2025 analyses indicate SARS-CoV-2 infection induces alterations in ADAR editing patterns, which may influence viral persistence and host immune modulation. ADAR's roles exhibit duality, shifting from antiviral to proviral depending on infection context; under chronic conditions, elevated ADAR1 expression favors by suppressing innate immunity while enabling adaptive for long-term survival. This context-dependent behavior underscores ADAR's exploitation by viruses during persistent infections.

Therapeutic Applications

Engineered ADAR for Therapies

Engineered enzymes have emerged as a cornerstone for programmable therapies, leveraging the natural A-to-I activity of to correct pathogenic mutations at the transcript level. The REPAIR (RNA Editing for Programmable A to I Replacement) system, pioneered by the Zhang laboratory in 2017, fuses the deaminase domain of human ADAR2 to a catalytically inactive Cas13 (dCas13) protein, which is directed by a guide RNA (crRNA) to specific adenosine sites in target mRNAs. This enables precise, site-specific A-to-I edits without genomic intervention, demonstrating up to 35% editing efficiency in cellular models for disease-relevant transcripts. Applications include potential correction of point mutations in monogenic disorders such as (SCD), where editing the HBB mRNA could restore function, and Duchenne muscular dystrophy (DMD), targeting transcripts to ameliorate frameshift defects. Advancements in have focused on enhancing specificity and to address limitations like bystander editing. The SPRING (strand displacement-responsive ADAR system for RNA ) platform introduces a hairpin-structured with a blocking sequence that unfolds upon , promoting strand and restricting ADAR access to the intended , resulting in over 2.2-fold higher (up to 67%) and significantly reduced off-target activity compared to MS2-MCP-ADAR methods. Complementing this, engineered ADAR2 variants with mutated deaminase domains and improved nuclear localization signals have minimized transcriptome-wide off-target edits by up to 90% while maintaining on-target potency models. These innovations build on -directed to enable multiplexed for complex diseases. Clinical translation of ADAR-based therapies is progressing, with delivery optimized via adeno-associated viruses (AAV) for ocular and CNS targets or lipid nanoparticles (LNPs) for to improve tissue specificity and reduce . ProQR Therapeutics' Axiomer platform recruits endogenous ADAR using synthetic guide RNAs (editing ) for A-to-I and has advanced to clinical evaluation, with clinical trial authorization (CTA) received in 2025 for lead candidate AX-0810 targeting NTCP-mediated , marking the first entry into s for the platform. As of November 2025, the Phase 1 study is anticipated to commence, focusing on safety in healthy volunteers. Earlier ProQR retinal programs, such as QR-1123 (an antisense , not ADAR-based), received IND clearance in 2019 for adRP due to P23H RHO and remain in Phase 1/2 trials. Compared to CRISPR-Cas9 DNA editing, ADAR-based RNA approaches provide transient, reversible modifications that do not induce double-strand breaks, thereby avoiding risks of insertional mutagenesis, chromosomal rearrangements, or permanent off-target genomic changes. This reversibility allows for tunable expression via degradable guides, enhancing safety for non-integrative therapies in post-mitotic tissues like the retina or brain.

Inhibitors and Modulators

Small-molecule inhibitors targeting ADAR enzymes primarily focus on the to prevent . analogs such as 8-azaadenosine act as competitive inhibitors by mimicking the and interfering with binding and editing functions, with use in cellular assays at low micromolar concentrations (1-10 μM). These compounds have demonstrated suppression of ADAR1 activity in lines, leading to reduced and activation of innate immune pathways like PKR-mediated . However, early analogs like 8-azaadenosine and 8-chloroadenosine lack isoform specificity, inhibiting both ADAR1 and ADAR2, which limits their therapeutic window due to ADAR2's essential role in neuronal . Recent advances have introduced more selective ADAR1-targeted small molecules, particularly for and immune-related disorders. Rebecsinib, a first-in-class splicing modulator, selectively inhibits the production of the interferon-inducible ADAR1 p150 isoform by disrupting its , thereby reducing hyper-editing in stem cells and enhancing sensitivity to . Preclinical data from 2024 highlight a novel ADAR1 p150 inhibitor that promotes tumor cell death via and PKR activation, showing with immune checkpoint blockade in solid tumor models. These ADAR1-specific agents are being explored for interferonopathies like Aicardi-Goutières syndrome (AGS), where dysregulated editing contributes to , though their use requires careful dosing to avoid exacerbating IFN responses. Nucleic acid-based approaches, including antisense oligonucleotides (), provide another avenue for modulating ADAR1 in tumors. ADAR1 knockdown, including via ASOs targeting ADAR1 mRNA for RNase H-mediated degradation, has been shown to diminish oncogenic editing events that promote immune evasion in cancers like and . In preclinical tumor models, ADAR1 knockdown promotes antitumor immunity by increasing dsRNA accumulation and IFN signaling. Modulators of activity extend beyond inhibition to include enhancers for precise therapeutic . Allosteric small molecules that stabilize ADAR-substrate interactions are under development to boost efficiency in contexts, though most current enhancers rely on designs rather than purely chemical agents. Key challenges in ADAR and modulator development include achieving isoform selectivity, as ADAR1 p150 drives pathological in cancer while ADAR2 maintains functions, and off-target effects can trigger unintended IFN storms. Preclinical efficacy has been validated in models, where ADAR1 inhibition alleviated toxicity by normalizing aberrant patterns, and in AGS mouse models, where selective modulation mitigated without fully ablating activity.