Adenosine Deaminases Acting On RNA Research Articles

Abstract I006: Programmable RNA sensors for internal states and external cues in living cells

Abstract Cell and gene therapies against cancer could be empowered by sensors that produce proteins in response to specific intracellular markers (e.g., expressing a cytotoxic payload in response to oncogenes) or extracellular cues (e.g., expressing a chimeric antigen receptor only when T cells are in the tumor microenvironment). An emerging delivery modality is in vitro transcribed mRNA, which holds the promise of superior safety and affordability than more conventional DNA vectors. While synthetic biology tools have predominantly relied on DNA-level regulations, here I will share two sensor designs uniquely compatible with mRNA delivery. First, for intracellular markers, a promising strategy is sensing mRNA transcripts. It is now straightforward to obtain single-cell transcriptomics, which are highly informative of cell type/state. If we can build RNA sensors to output arbitrary proteins in direct response to specific RNAs, we will access virtually any cell type/state for research and therapy. Furthermore, RNA base pairing would facilitate programmable designs. Previous RNA sensors relied on base pairing-induced conformational changes of RNA, and have met with limited success in mammalian cells. As an alternative strategy, we took advantage of endogenous ADAR (adenosine deaminases acting on RNA) enzymes that specifically edit adenosines to inosines (treated as guanosines during translation) in dsRNA, and created RADAR (RNA sensing using ADAR, Kaseniit et al., 2023). RADAR relies on the formation of dsRNA between the sensor RNA we introduce and the endogenous input RNA we’d like to detect; ADAR then edits a strategically positioned stop codon (UAG) into a tryptophan codon (UIG), enabling the translation of the downstream output coding sequence. We validated and optimized RADAR, and reported its compatibility with human/mouse transcriptomes. We showed that RADAR is programmable and modular. It also uniquely enables compact implementation of AND logic, important for integrating the inputs from multiple markers and unambiguously identifying cells of interest. We have since demonstrated RADAR in a variety of contexts including cancer lines with an elevated oncogene. Second, while transcripts mark cell types/states, extracellular ligands reflect the other key aspect that payload production could be conditioned on – the microenvironment. Although our field has plenty of synthetic receptors to offer when it comes to sensing ligands, all previously engineered modular ones require DNA-level operation. Inspired by the power of ADAR editing, we created LIDAR (Ligand-Induced Dimerization Activating RNA editing, Zhang & Mille et al., 2024). LIDAR brings an attenuated ADAR to the proximity of a stop codon in a ligand-dependent fashion, and is compatible with diverse inputs, outputs, and cellular contexts. We are in the process of demonstrating RADAR and LIDAR to improve cell therapies and enable non-invasive diagnostics. Citation Format: Xiaojing Gao. Programmable RNA sensors for internal states and external cues in living cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I006.

Charting and probing the activity of ADARs in human development and cell-fate specification.

Adenosine deaminases acting on RNA (ADARs) impact diverse cellular processes and pathological conditions, but their functions in early cell-fate specification remain less understood. To gain insights here, we began by charting time-course RNA editing profiles in human organs from fetal to adult stages. Next, we utilized hPSC differentiation to experimentally probe ADARs, harnessing brain organoids as neural specific, and teratomas as pan-tissue developmental models. We show that time-series teratomas faithfully recapitulate fetal developmental trends, and motivated by this, conducted pan-tissue, single-cell CRISPR-KO screens of ADARs in teratomas. Knocking out ADAR leads to a global decrease in RNA editing across all germ-layers. Intriguingly, knocking out ADAR leads to an enrichment of adipogenic cells, revealing a role for ADAR in human adipogenesis. Collectively, we present a multi-pronged framework charting time-resolved RNA editing profiles and coupled ADAR perturbations in developmental models, thereby shedding light on the role of ADARs in cell-fate specification.

IMMU-17. ADENOSINE DEAMINASE ACTING ON RNA (ADAR) SILENCES TUMOR-INTRINSIC INTERFERON RESPONSE IN DIFFUSE MIDLINE GLIOMA

Abstract INTRODUCTION Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children and is currently incurable. Clinical trials using immune checkpoint blockade (ICB) therapy have failed to show efficacy due to multifactorial mechanisms including limited lymphocyte infiltration, low immune checkpoint (IC) expression, and a low mutational burden. Adenosine deaminase acting on RNA (ADAR) is an RNA editor, which limits anti-tumor immunity by editing/preventing the sensing of endogenous double-stranded RNA (dsRNA). We have found that ADAR protein levels are significantly elevated in DMG patients, suggesting that ADAR’s immune-silencing function is relevant to DMG immune suppression. The onco-histone driver H3K27M promotes epigenetic dysregulation in DMGs, de-suppressing retroelement transcription, a major source of endogenous dsRNA. By inhibiting ADAR, we expect that the increased levels of dsRNA will stimulate intracellular type-1 interferon (IFN) responses, induce chemokine secretion, and promote antitumor CD8+ cytotoxicity. METHODS Human DMG cells and patient-derived glioma spheres were treated with silencing RNA to transiently knockdown (KD) ADAR levels for assays involving in vitro viability and proliferation. Affected pathways were identified using immunoblotting and quantitative PCR. RESULTS Our data shows that ADAR protein levels are elevated in DMG patient proteomic datasets and cell lines. ADAR-depleted patient-derived DMG cell lines exhibit significantly less proliferation compared to control siRNA and ADAR-depleted hTERT-astrocytes (Mann-Whitney test, P-value <0.0001, ****). CONCLUSION Our results demonstrate that DMG tumor cells have an ADAR selective dependency in vitro. In ongoing studies, we aim to determine if ADAR-depletion improves in-vivo survival in both immunodeficient and immunocompetent models in combination with immunotherapies against DMG.

LoDEI: a robust and sensitive tool to detect transcriptome-wide differential A-to-I editing in RNA-seq data

RNA editing is a highly conserved process. Adenosine deaminase acting on RNA (ADAR) mediated deamination of adenosine (A-to-I editing) is associated with human disease and immune checkpoint control. Functional implications of A-to-I editing are currently of broad interest to academic and industrial research as underscored by the fast-growing number of clinical studies applying base editors as therapeutic tools. Analyzing the dynamics of A-to-I editing, in a biological or therapeutic context, requires the sensitive detection of differential A-to-I editing, a currently unmet need. We introduce the local differential editing index (LoDEI) to detect differential A-to-I editing in RNA-seq datasets using a sliding-window approach coupled with an empirical q value calculation that detects more A-to-I editing sites at the same false-discovery rate compared to existing methods. LoDEI is validated on known and novel datasets revealing that the oncogene MYCN increases and that a specific small non-coding RNA reduces A-to-I editing.

ADAR3 modulates neuronal differentiation and regulates mRNA stability and translation.

ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.

Rational design of base, sugarand backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

The Effects of Oxidative Stress on RNA Editing with Insight into the Relationship Between Air Pollution and Alzheimer's Disease

Human interactions with the environment today add pressure on the world’s resources and ecosystems, which in turn harm human health. Many anthropogenic environmental changes, including climate change, biodiversity loss, and pollution, have accelerated morbidity and mortality. Research across multiple countries have found correlations between anthropogenic air pollution exposure and neurological degeneration, particularly with Alzheimer’s disease (AD). RNA editing induced by oxidative stress is one possible mechanism in which air pollution increases the risk of AD. To study this further, RNA adenosine-to-inosine (A-to-I) alterations were analyzed to identify a mechanism linking air pollution to neurological degeneration. These alterations occur in RNA, most prevalently in the central nervous system (CNS). Adenosine deaminase acting on RNA (ADAR) enzymes mediate A-to-I alterations and are essential for mammalian development and survival. ADAR mediated alterations have been associated with AD and the A-to-I occurrence frequency may increase in polluted environments that trigger increased oxidative stress. To test this possibility, wildtype (WT) and ADAR knockout (adr-2(-) Caenorhabditis elegans in their first larval (L1) developmental stage were exposed to juglone solution for 0, 15, and 60 minute experimental settings. For the two worm strains, WT and adr-2(-), the 0 minute treatment was the control, the 15 minute treatment represented acute oxidative stress exposure, and the 60 minute treatment represented chronic exposure. RNA was extracted from both groups and quantitative Real Time PCR (qRT-PCR) was used to measure the expression of gst-4, an Alzheimer’s associated gene (sel-12), and adr-2 normalized to the expression of gpd-3, the housekeeping gene that corrected for systemic errors. The gst-4 gene expression confirmed that oxidative stress occurred. Lack of adr-2 gene significantly decreased sel-12 expression at acute exposure, but adr-2 gene expression in WT worms did not significantly change across treatments. It remains to be determined whether oxidative stress impacts RNA editing of target mRNAs. Future studies are needed to explore the role of adr-2 in promoting sel-12 expression.

Structural impacts of two disease-linked ADAR1 mutants: a molecular dynamics study.

Adenosine deaminases acting on RNA (ADARs) are pivotal RNA-editing enzymes responsible for converting adenosine to inosine within double-stranded RNA (dsRNA). Dysregulation of ADAR1 editing activity, often arising from genetic mutations, has been linked to elevated interferon levels and the onset of autoinflammatory diseases. However, understanding the molecular underpinnings of this dysregulation is impeded by the lack of an experimentally determined structure for the ADAR1 deaminase domain. In this computational study, we utilized homology modeling and the AlphaFold2 to construct structural models of the ADAR1 deaminase domain in wild-type and two pathogenic variants, R892H and Y1112F, to decipher the structural impact on the reduced deaminase activity. Our findings illuminate the critical role of structural complementarity between the ADAR1 deaminase domain and dsRNA in enzyme-substrate recognition. That is, the relative position of E1008 and K1120 must be maintained so that they can insert into the minor and major grooves of the substrate dsRNA, respectively, facilitating the flipping-out of adenosine to be accommodated within a cavity surrounding E912. Both amino acid replacements studied, R892H at the orthosteric site and Y1112F at the allosteric site, alter K1120 position and ultimately hinder substrate RNA binding.

Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Spatial Visualization of A-to-I Editing in Cells Using Endonuclease V Immunostaining Assay (EndoVIA).

Adenosine-to-inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions, and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed endonuclease V (EndoV), a magnesium-dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces an endonuclease V immunostaining assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

Insights into ADAR gene complement, expression patterns, and RNA editing landscape in Chlamys farreri

Adenosine Deaminases Acting on RNA (ADARs) are evolutionarily conserved enzymes known to convert adenosine to inosine in double-stranded RNAs and participate in host-virus interactions. Conducting a meta-analysis of available transcriptome data, we identified and characterised eight ADAR transcripts in Chlamys farreri, a farmed marine scallop susceptible to Acute viral necrosis virus (AVNV) infections and mortality outbreaks. Accordingly, we identified six ADAR genes in the Zhikong scallop genome, revised previous gene annotations, and traced alternative splicing variants. In detail, each ADAR gene encodes a unique combination of functional domains, always including the Adenosine deaminase domain, RNA binding domains and, in one case, two copies of a Z-DNA binding domain. After phylogenetic analysis, five C. farreri ADARs clustered in the ADAR1 clade along with sequences from diverse animal phyla. Gene expression analysis indicated CF051320 as the most expressed ADAR, especially in the eye and male gonad. The other four ADAR1 genes and one ADAR2 gene exhibited variable expression levels, with CF105370 and CF051320 significantly increasing during early scallop development. ADAR-mediated single-base editing, evaluated across adult C. farreri tissues and developmental stages, was mainly detectable in intergenic regions (83 % and 85 %, respectively). Overall, the expression patterns of the six ADAR genes together with the editing and hyper-editing values computed on scallops RNA-seq samples support the adaptive value of ADAR1-mediated editing, particularly in the pre-settling larval stages.

RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

Small nuclear RNAs enhance protein-free RNA-programmable base conversion on mammalian coding transcripts.

Endogenous U small nuclear RNAs (U snRNAs) form RNA-protein complexes responsible for eukaryotic processing of pre-mRNA into mature mRNA. Previous studies have demonstrated the utility of guide-programmable U snRNAs in targeted exon inclusion and exclusion. We investigated whether snRNAs can also enhance conversion of RNA bases over state-of-the-art RNA targeting technologies in human cells. When compared to adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs, we find that guided A>I snRNAs consistently increase adenosine-to-inosine editing efficiency for genes with higher exon counts, perturb substantially fewer genes in the transcriptome, and localize more persistently to the nucleus where ADAR is expressed. A>I snRNAs can also edit pre-mRNA 3' splice sites to promote splicing changes. Finally, snRNA fusions to H/ACA box snoRNAs (U>Ψ snRNAs) increase targeted RNA pseudouridylation efficiency. Altogether, our results advance the protein-free RNA base conversion toolbox and enhance minimally invasive RNA targeting technologies to treat genetic diseases.

Site-specific regulation of RNA editing with ribose-modified nucleoside analogs in ADAR guide strands.

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here,we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.

F11R RNA trinucleotide over-edited by ADAR in gastric and colorectal cancers: Cross-cohort validation, gene expression regulation, and diagnostic significance

The F11 receptor (F11R) gene encoding junctional adhesion molecule A has been associated with gastric cancer (GC) and colorectal cancer (CRC), in which its role and regulation remain to be further elucidated. Recently F11R was also identified as a potential target of adenosine-to-inosine (A-to-I) mediated by the adenosine deaminases acting on RNA (ADARs). Herein, using RNA-Seq and experimental validation, our current study revealed an F11R RNA trinucleotide over-edited by ADAR, with its regulation of gene expression and clinical significance in four GC and three CRC cohorts. Our results found an over-edited AAA trinucleotide in an AluSg located in the F11R 3′-untranslated region (3′-UTR), which showed editing levels correlated with elevated ADAR expression across all GC and CRC cohorts in our study. Overexpression and knockdown of ADAR in GC and CRC cells, followed by RNA-Seq and Sanger sequencing, confirmed the ADAR-mediated F11R 3′-UTR trinucleotide editing, which potentially disrupted an RBM45 binding site identified by crosslinking immunoprecipitation sequencing (CLIP-seq) and regulated F11R expression in luciferase reporter assays. Moreover, the F11R trinucleotide editing showed promising predictive performance for diagnosing GC and CRC across GC and CRC cohorts. Our findings thus highlight both the potential biological and clinical significance of an ADAR-edited F11R trinucleotide in GC and CRC, providing new insights into its application as a novel diagnostic biomarker for both cancers.

ADR-2 regulates fertility and oocyte fate in C. elegans.

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

Deficiency of ADAR2 ameliorates metabolic-associated fatty liver disease via AMPK signaling pathways in obese mice

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease caused by hepatic steatosis. Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine RNA editing. However, the functional role of ADAR2 in NAFLD is unclear. ADAR2+/+/GluR-BR/R mice (wild type, WT) and ADAR2−/−/GluR-BR/R mice (ADAR2 KO) mice are fed with standard chow or high-fat diet (HFD) for 12 weeks. ADAR2 KO mice exhibit protection against HFD–induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, ADAR2 KO mice display reduced liver lipid droplets in concert with decreased hepatic TG content, improved hepatic insulin signaling, better pyruvate tolerance, and increased glycogen synthesis. Mechanistically, ADAR2 KO effectively mitigates excessive lipid production via AMPK/Sirt1 pathway. ADAR2 KO inhibits hepatic gluconeogenesis via the AMPK/CREB pathway and promotes glycogen synthesis by activating the AMPK/GSK3β pathway. These results provide evidence that ADAR2 KO protects against NAFLD progression through the activation of AMPK signaling pathways.

Target specificity in the ADAR editing landscape explains the unique immunoregulatory function of the interferon-inducible isoform ADAR1-p150

Abstract The adenosine deaminase acting on RNA (ADAR) family includes three enzymes, ADAR1-p150, ADAR1-p110, and ADAR2 that bind endogenous and pathogen-derived double-stranded RNA (dsRNA) and convert adenosine (A) to inosine (I). This process called A-to-I editing disrupts dsRNA structures and counteracts activation of innate immune sensors MDA-5 and PKR, preventing autoimmune disorders like Aicardi-Goutieres-Syndrome. We and others have shown that the interferon-inducible ADAR1-p150, but not the other ADARs, protects cells and organisms from aberrant innate immune activation by self-derived dsRNA, even though A-to-I editing is performed by all three ADARs. We hypothesized that ADAR1-p150 edits specific transcripts that cannot be edited by the other enzymes. Here we present a novel approach to quantify RNA editing in transcriptomic data. Our tool TSniffer detected more than 40,000 regions of A-to-I editing distributed over the entire human transcriptome, affecting 30% of protein-coding transcripts and 10% of long non-coding RNAs. Differential editing frequencies in cell lines with knock-outs for the different ADARs revealed transcripts exclusively edited by ADAR1-p150. In contrast, editing by ADAR1-p110 and ADAR2 was cooperative and more redundant. Our analyses fill gaps of knowledge about the A-to-I editing landscape, highlight the dominant immunoregulatory function of ADAR1-p150, and provide new insights in the role of aberrant A-to-I editing in disease development.

Abstract LB007: A systematic evaluation of the therapeutic potential of Endogenous-ADAR base editors in cancer prevention and treatment

Abstract Base editing encompasses techniques that efficiently alter specific nucleotides at the DNA or RNA level. Initially explored for inherited diseases, these techniques hold promise for addressing various genetically driven disorders caused by single nucleotide variants (SNVs). The precise programmability of base editors (BEs) for specific sequences allows customization for rare genetic variants, tailoring them to individual patients within affordability and delivery constraints. Cancer stems from the accumulation of mutations. However, the relevance of BEs in cancer therapy is doubted due to the limited types of mutations they can address within tumors. Yet, their untapped potential in the realm of cancer treatment invites exploration. BEs utilize a modified form of a deaminase enzyme to catalyze the conversion of one nucleotide to another by removing an amino group. A 'classic' BE consists of a deaminase, a Cas nuclease, and a guide RNA (gRNA) ensuring target specificity through Watson-Crick base pairing. An alternative RNA BE, known as Endogenous-ADAR, involves designing a gRNA to recruit native Adenosine Deaminases Acting on RNA (ADAR), responsible for extensive A-to-I(G) editing in mammals. Due to its reliance on ADAR capabilities, Endogenous-ADAR exclusively targets G>A SNVs at the RNA level. Notably, RNA editing, occurring before splicing, provides the flexibility to target all gene regions. Also, editing the RNA sequence is considered 'safer' in the event of an off-target error. Our objective is to systematically explore the potential of Endogenous-ADAR for cancer prevention and treatment. Our first approach evaluates Endogenous-ADAR's potential to revert germline mutations in cancer predisposition genes (CPGs) for cancer prevention. Focusing on CPGs recommended for pediatric genetic testing by NCBI, associated with cancer predisposition disorders, our findings indicate that among the 2,820 SNVs examined, 566 (20%) are suitable for Endogenous-ADAR. Remarkably, 88% of correctable variants show no off-target sites, indicating safe therapeutic targets. Further examining pathogenic high-penetrance germline variants in adults, 48 (20%) of the 239 SNVs examined are correctable using Endogenous-ADAR. Second, we estimate the potential to correct cancer driver mutations. Examining 5,913 driver mutations from 2,010 patients representing 36 cancer types, which underwent whole genome sequencing (WGS) in the PCWAG project, we find that 955 (16%) are suitable for Endogenous-ADAR. In 729 (36%) of patients, at least one driver mutation is correctable, and in 91 (5%), all known driver mutations are correctable. Aligned with the swift incorporation of WGS in oncology, our systematic exploration portrays a favorable landscape of correctable mutations in cancer. This points to the future possibility of leveraging the unique capabilities of base editing, particularly Endogenous-ADAR, for clinical cancer risk/prevention and treatment outcomes. Citation Format: Rona Merdler-Rabinowicz, Ariel Dadush, Sumeet Patiyal, Padma Sheila Rajagopal, Gulzar Daya, Alejandro Schäffer, Eli Eisenberg, Eytan Ruppin, Erez Y. Levanon. A systematic evaluation of the therapeutic potential of Endogenous-ADAR base editors in cancer prevention and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB007.