RNA editing: Expanding the potential of RNA therapeutics.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

Correlations of Myeloperoxidase (MPO), Adenosine deaminase (ADA), C-C motif chemokine 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in the nasopharyngeal specimens with the diagnosis and severity of SARS-CoV-2 infections.

Cytokine dynamics in patients with coronavirus disease 2019 (COVID-19) have been studied in blood but seldomly in respiratory specimens. We studied different cell markers and cytokines in fresh nasopharyngeal swab specimens for the diagnosis and for stratifying the severity of COVID-19. This was a retrospective case-control study comparing Myeloperoxidase (MPO), Adenosine deaminase (ADA), C-C motif chemokine ligand 22 (CCL22), Tumour necrosis factor alpha (TNFα) and Interleukin-6 (IL-6) mRNA expression in 490 (327 patients and 163 control) nasopharyngeal specimens from 317 (154 COVID-19 and 163 control) hospitalized patients. Of the 154 COVID-19 cases, 46 died. Both total and normalized MPO, ADA, CCL22, TNFα, and IL-6 mRNA expression levels were significantly higher in the nasopharyngeal specimens of infected patients when compared with controls, with ADA showing better performance (OR 5.703, 95% CI 3.424-9.500, p < 0.001). Receiver operating characteristics (ROC) curve showed that the cut-off value of normalized ADA mRNA level at 2.37 × 10-3 had a sensitivity of 81.8% and specificity of 83.4%. While patients with severe COVID-19 had more respiratory symptoms, and elevated lactate dehydrogenase, multivariate analysis showed that severe COVID-19 patients had lower CCL22 mRNA (OR 0.211, 95% CI 0.060-0.746, p = 0.016) in nasopharyngeal specimens, while lymphocyte count, C-reactive protein, and viral load in nasopharyngeal specimens did not correlate with disease severity. In summary, ADA appears to be a better biomarker to differentiate between infected and uninfected patients, while CCL22 has the potential in stratifying the severity of COVID-19.

Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2.

Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory T cells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

Improved Durability to SARS-CoV-2 Vaccine Immunity following Coimmunization with Molecular Adjuvant Adenosine Deaminase-1.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only-immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain-specific memory B cells, and SARS-CoV-2-specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies.

Commentary on "Poor evidence for host-dependent regular RNA editing in the transcriptome of SARS-CoV-2".

Analysis of the SARS-CoV-2 transcriptome has revealed a background of low-frequency intra-host genetic changes with a strong bias towards transitions. A similar pattern is also observed when inter-host variability is considered. We and others have shown that the cellular RNA editing machinery based on ADAR and APOBEC host-deaminases could be involved in the onset of SARS-CoV-2 genetic variability. Our hypothesis is based both on similarities with other known forms of viral genome editing and on the excess of transition changes, which is difficult to explain with errors during viral replication. Zong et al. criticize our analysis on both conceptual and technical grounds. While ultimate proof of an involvement of host deaminases in viral RNA editing will depend on experimental validation, here, we address the criticism to suggest that viral RNA editing is the most reasonable explanation for the observed intra- and inter-host variability.

Virus-specific editing identification approach reveals the landscape of A-to-I editing and its impacts on SARS-CoV-2 characteristics and evolution.

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.

Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Detection of A-to-I RNA Editing in SARS-COV-2.

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.

ADAR Editing in Viruses: An Evolutionary Force to Reckon with.

Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This editing can be highly selective, affecting a specific site within a transcript, or nonselective, resulting in hyperediting. ADAR editing is important for regulating neural functions and autoimmunity, and has a key role in the innate immune response to viral infections, where editing can have a range of pro- or antiviral effects and can contribute to viral evolution. Here we examine the role of ADAR editing across a broad range of viral groups. We propose that the effect of ADAR editing on viral replication, whether pro- or antiviral, is better viewed as an axis rather than a binary, and that the specific position of a given virus on this axis is highly dependent on virus- and host-specific factors, and can change over the course of infection. However, more research needs to be devoted to understanding these dynamic factors and how they affect virus-ADAR interactions and viral evolution. Another area that warrants significant attention is the effect of virus-ADAR interactions on host-ADAR interactions, particularly in light of the crucial role of ADAR in regulating neural functions. Answering these questions will be essential to developing our understanding of the relationship between ADAR editing and viral infection. In turn, this will further our understanding of the effects of viruses such as SARS-CoV-2, as well as many others, and thereby influence our approach to treating these deadly diseases.

Analysis of SARS-CoV-2 haplotypes and genomic sequences during 2020 in Victoria, Australia, in the context of putative deficits in innate immune deaminase anti-viral responses.

The SARS-CoV-2 epidemic infections in Australia during 2020 were small in number in epidemiological terms and are well described. The SARS-CoV-2 genomic sequence data of many infected patients have been largely curated in a number of publicly available databases, including the corresponding epidemiological data made available by the Victorian Department of Health and Human Services. We have critically analysed the available SARS-CoV-2 haplotypes and genomic sequences in the context of putative deficits in innate immune APOBEC and ADAR deaminase anti-viral responses. It is now known that immune impaired elderly co-morbid patients display clear deficits in interferon type 1 (α/ß) and III (λ) stimulated innate immune gene cascades, of which APOBEC and ADAR induced expression are part. These deficiencies may help explain some of the clear genetic patterns in SARS-CoV-2 genomes isolated in Victoria, Australia, during the 2nd Wave (June-September, 2020). We tested the hypothesis that predicted lowered innate immune APOBEC and ADAR anti-viral deaminase responses in a significant proportion of elderly patients would be consistent with/reflected in a low level of observed mutagenesis in many isolated SARS-CoV-2 genomes. Our findings are consistent with this expectation. The analysis also supports the conclusions of the Victorian government's Department of Health that essentially one variant or haplotype infected Victorian aged care facilities where the great majority (79%) of all 820 SARS-CoV-2 associated deaths occurred. The implications of our data analysis for other localized epidemics and efficient coronavirus vaccine design and delivery are discussed.

The role of A-to-I RNA editing in infections by RNA viruses: Possible implications for SARS-CoV-2 infection.

RNA editing is a fundamental biological process with 2 major forms, namely adenosine-to-inosine (A-to-I, recognized as A-to-G) and cytosine-to-uracil (C-to-U) deamination, mediated by ADAR and APOBEC enzyme families, respectively. A-to-I RNA editing has been shown to directly affect the genome/transcriptome of RNA viruses with significant repercussions for viral protein synthesis, proliferation and infectivity, while it also affects recognition of double-stranded RNAs by cytosolic receptors controlling the host innate immune response. Recent evidence suggests that RNA editing may be present in SARS-CoV-2 genome/transcriptome. The majority of mapped mutations in SARS-CoV-2 genome are A-to-G/U-to-C(opposite strand) and C-to-U/G-to-A(opposite strand) substitutions comprising potential ADAR-/APOBEC-mediated deamination events. A single nucleotide substitution can have dramatic effects on SARS-CoV-2 infectivity as shown by the D614G(A-to-G) substitution in the spike protein. Future studies utilizing serial sampling from patients with COVID-19 are warranted to delineate whether RNA editing affects viral replication and/or the host immune response to SARS-CoV-2.

Ocular Surface Expression of SARS-CoV-2 Receptors.

PURPOSE: The spike proteins of SARS-CoV-2 interact with ACE2 or basigin/CD147 receptors, regulating human-to-human transmissions of COVID-19 together with serine protease TMPRSS2. The expression of these receptors on the ocular surface is unknown. MATERIAL AND METHODS: Gene expression of SARS-CoV-2 receptors was investigated in conjunctival epithelial cell samples and in ex-vivo cornea samples using microarray or transcriptome sequencing. RESULTS: ACE2 is expressed in conjunctival samples at a low level, while BSG and TMPRSS2 are expressed at intermediate levels in both conjunctiva and cornea. Other receptors such as ANPEP, AGTR2 are expressed at low level in the conjunctiva. Two RNA editing enzymes involved in antiviral responses, APOBEC3A, and ADAR-1 were also highly expressed. CONCLUSIONS: The ocular surface may represent an entry point for the SARS-CoV-2 in the human body. The conjunctiva and the cornea can adopt antiviral countermeasures which may explain the low prevalence of eye involvement.

Evidence for host-dependent RNA editing in the transcriptome of SARS-CoV-2.

The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.