Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality

Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.

LONG NON-CODING RNA REGULATES IFN-I RESPONSE AND SARS-CoV-2 REPLICATION

Background: Infection with SARS-CoV-2 triggers reprogramming through global transcriptomic changes that drive the development of Coronavirus disease 2019 (COVID-19). Although the expression and functions of proteincoding transcripts have been widely studied in SARS-CoV-2 infection, most of the transcriptome consists of non-protein-coding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs), which constitute a large proportion of the transcriptome, regulate immune responses and play prominent roles in health and disease. However, the impact of lncRNAs on SARS-CoV-2 infection is poorly understood. Our study will provide fundamental insights into the role of lncRNAs in SARS-CoV-2 infection. Method(s): We hypothesized that SARS-CoV-2-induced lncRNAs are critical regulators of viral replication and immune response. To test our hypothesis, we identified lncRNAs with significant differential expression in SARS-CoV-2 infected vs. uninfected cells across two cell types (A549-hACE2 and Calu) from published transcriptome data. We silenced the expression of the top lncRNA Bre- AS1 (BA) a human lung epithelial cell model (A549 cells stably expressing hACE2 and hTMPRSS2, A549AT) using lncRNA-specific ASO (lncsi) or negative control (NC) and compared viral replication in lncsi vs. NC cells. BA-silencing (BA-si) increased SARS-CoV-2 replication. and inhibited the expression of antiviral interferon-stimulated genes (ISG). (Tyr 705) pSTAT3 forms suppressor molecular complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) that inhibit ISG transcription. Using molecular methods such as gene-silencing, immunoprecipitation, western blot, and measuring promoter activity, we further show that Bre-AS1 inhibits the phosphorylation of STAT3 and enhances ISG transcription. Result(s): Our data show that cellular lncRNA, Bre-AS1 enhances antiviral interferon-stimulated genes (ISG) expression and inhibits replication of SARSCoV- 2. Our data show that Bre-AS1 inhibits the (Tyr705) phosphorylation of STAT3 that forms ISG repressor complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) and thus enhances ISG transcription. Conclusion(s): Cellular lncRNA Bre-AS1 enhances expression of antiviral interferon-stimulated genes and inhibits the replication of SARS-CoV-2. Our data show that cellular lncRNAs could play significant roles in immune response and viral propagation. Thus, unraveling the mechanisms of lncRNA-mediated regulation of virus replication and immune response may lead to identifying new, highly selective therapeutic targets Bre-AS1 inhibits STAT3 phosphorylation and enhances ISG transcription.

Sirna-A Potential Therapy for Covid-19 Infection

Background: The deadly virus COVID-19 has affected more than 1 crore people and claimed more than 5 lakh lives worldwide according to the World health organization. Though there are numerous treatment modalities available including anti-bacterials, antivirals, vaccines etc., none of them can be considered as effective cure for SARS CoV-2 virus as they are mostly non-specific in action. Aim(s): siRNA therapy can be considered as a significant treatment modality due to its specificity in action. The aim of this review is to explore siRNA as a potential treatment strategy for the treatment of COVID-19. Material(s) and Method(s): In this review we shall explore the targets of siRNA therapy which includes viral RNA-dependent RNA polymerase, helicase, protease and nucleoprotein N. siRNA related patents provide solutions for novel RNAi techniques, high expense of chemically synthetic siRNA, techniques for restraining SARS-CoV by disturbing RNA etc., siRNA-based drug delivery systems and limitations of nanocarrier delivery system were reviewed. siRNA is a gene silencer that targets highly conserved sequences which codes for protease 3CL (nsp5) and viral helicase (from 16-18 kbp). Conclusion(s): Thus, siRNA-based therapy is considered highly efficacious as it can hit the highly conserved regions of SARS-CoV-2 RNA.Copyright © 2023, Advanced Scientific Research. All rights reserved.

In silico identification of potential miRNAs -mRNA inflammatory networks implicated in the pathogenesis of COVID-19

COVID-19 has been found to affect the expression profile of several mRNAs and miRNAs, leading to dysregulation of a number of signaling pathways, particularly those related to inflammatory responses. In the current study, a systematic biology procedure was used for the analysis of high-throughput expression data from blood specimens of COVID-19 and healthy individuals. Differentially expressed miRNAs in blood specimens of COVID-19 vs. healthy specimens were then identified to construct and analyze miRNA-mRNA networks and predict key miRNAs and genes in inflammatory pathways. Our results showed that 171 miRNAs were expressed as outliers in box plot and located in the critical areas according to our statistical analysis. Among them, 8 miRNAs, namely miR-1275, miR-4429, miR-4489, miR-6721-5p, miR-5010-5p, miR-7110-5p, miR-6804-5p and miR-6881-3p were found to affect expression of key genes in NF-KB, JAK/STAT and MAPK signaling pathways implicated in COVID-19 pathogenesis. In addition, our results predicted that 25 genes involved in above-mentioned inflammatory pathways were targeted not only by these 8 miRNAs but also by other obtained miRNAs (163 miRNAs). The results of the current in silico study represent candidate targets for further studies in COVID-19.Copyright © 2023 Elsevier B.V.

Landscape Analysis of Quercetin: A Potential Candidate Against SARS-CoV-2

Fruit, vegetables, and green tea contain quercetin (a flavonoid). Some of the diet's most signifi-cant sources of quercetin are apples, onions, tomatoes, broccoli, and green tea. Antioxidant, anticancer, anti-inflammatory, antimicrobial, antibacterial, and anti-viral effects have been studied of quercetin. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, ribonucleic acid (RNA) polymer-ase, and other essential viral life-cycle enzymes are all prevented from entering the body by quercetin. Despite extensive in vitro and in vivo investigations on the immune-modulating effects of quercetin and vitamin C treatment. 3-methyl-quercetin has been shown to bind to essential proteins necessary to convert minus-strand RNA into positive-strand RNAs, preventing the replication of viral RNA in the cytoplasm. Quercetin has been identified as a potential SARS-CoV-2 3C-like protease (3CLpro) suppressor in recent molecular docking studies and in silico assessment of herbal medicines. It has been demonstrated that quercetin increases the expression of heme oxygenase-1 through the nuclear factor erythroid-related factor 2 (Nrf2) signal network. Inhibition of heme oxygenase-1 may increase bilirubin synthesis, an endoge-nous antioxidant that defends cells. When human gingival fibroblast (HGF) cells were exposed to lipo-polysaccharide (LPS), inflammatory cytokine production was inhibited. The magnesium (Mg+2) cation complexation improves quercetin free radical scavenging capacity, preventing oxidant loss and cell death. The main objective of this paper is to provide an overview of the pharmacological effects of quercetin, its protective role against SARS-CoV-2 infection, and any potential molecular processes.Copyright © 2022 Bentham Science Publishers.

RNAi Degrades the SARS-CoV-2 Spike Protein RNA for Developing Drugs to Treat COVID-19

COVID-19 is caused by severe acute respiratory SARS-CoV-2. Regardless of the availability of treatment strategies for COVID-19, effective therapy will remain essential. A promising approach to tackle the SARS-CoV-2 could be small interfering (si) RNAs. Here we designed the small hairpin RNA (named as shRNA688) for targeting the prepared 813 bp Est of the S protein genes (Delta). The conserved and mutated regions of the S protein genes from the genomes of the SARS-CoV-2 variants in the public database were analyzed. A 813 bp fragment encoding the most part of the RBD and partial downstream RBD of the S protein was cloned into the upstream red florescent protein gene (RFP) as a fusing gene in the pCMV-S-Protein RBD-Est-RFP plasmid for expressing a potential target for RNAi. The double stranded of the DNA encoding for shRNA688 was constructed in the downstream human H1 promoter of the plasmid in which CMV promoter drives enhanced green fluorescent protein (EGFP) marker gene expression. These two kinds of the constructed plasmids were co-transfected into HEK293T via Lipofectamine 2000. The degradation of the transcripts of the SARS-CoV-2 S protein fusing gene expressed in the transfected HEK293T treated by RNAi was analyzed by RT-qPCR with a specific probe of the targeted SARS-CoV-2 S protein gene transcripts. Our results showed that shRNA688 targeting the conserved region of the S protein genes could effectively reduce the transcripts of the S protein genes. This study provides a cell model and technical support for the research and development of the broad-spectrum small nucleic acid RNAi drugs against SARS-CoV-2 or the RNAi drugs for the other hazard viruses which cause human diseases. Copyright © Weiwei Zhang, Linjia Huang, Jumei Huang, Xin Jiang, Xiaohong Ren, Xiaojie Shi, Ling Ye, Shuhui Bian, Jianhe Sun, Yufeng Gao, Zehua Hu, Lintin Guo, Suyan Chen, Jiahao Xu, Jie Wu, Jiwen Zhang, Daxiang Cui, and Fangping Dai.

Halting the COVID-19 Widespread: A Audit on the Progresses of Determination, Treatment, and Control Measures

Efforts to prevent the transmission of the SARS-CoV-2 infection are critical in light of the ongoing worldwide spread of COVID-19. Recently developed diagnostic tools include CRISPR, IgG tests, spike protein detection, and artificial intelligence. RT-PCR has been replaced with point-of-care assays, which may be performed at the patient's bedside (RT-PCR). All of these options are available to treat the disease: antivirals and other antiparasitic agents, anti-inflammatory medications like interferon or convalescent plasma, monoclonal antibodies like gamma-globulin, and RNAi treatments like mesenchymal stem cell therapy are among the options (ECMO). More than a dozen different types of vaccines are now being tested in clinical studies. Furthermore, breakthrough technologies that are easily deployable and transportable.In addition, vaccination delivery technologies are being developed. The threat of a second wave of infection needs strict and reasonable control mechanisms to keep mortality to a minimal when governments begin to loosen their lockdown tactics. Research into COVID-19's advances in diagnostics and treatment may serve as a platform for future research that can lead to improved containment strategies. Copyright © 2023 Ubiquity Press. All rights reserved.

siRNA silencing of cellular entry proteins as a gene therapy strategy for Covid-19

SARS-CoV-2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid-19 vaccines have been developed but novel SARS-CoV-2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA-mediated silencing of Angiotensin-converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS-CoV-2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS-CoV-2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%-88% knockdown of ACE2 and TMPRSS2, as determined by qRT-PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS-CoV-2 Spike-pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one-way ANOVA). We are now working to confirm these results with live SARS-CoV-2 and to test lipid nanoparticle delivery of the siRNAs to air-liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS-CoV-2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS-CoV-2 variants that circumvent pre-existing immune responses.

The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides.

Nucleic acids play a central role in human biology, making them suitable and attractive tools for therapeutic applications. While conventional drugs generally target proteins and induce transient therapeutic effects, nucleic acid medicines can achieve long-lasting or curative effects by targeting the genetic bases of diseases. However, native oligonucleotides are characterized by low in vivo stability due to nuclease sensitivity and unfavourable physicochemical properties due to their polyanionic nature, which are obstacles to their therapeutic use. A myriad of synthetic oligonucleotides have been prepared in the last few decades and it has been shown that proper chemical modifications to either the nucleobase, the ribofuranose unit or the phosphate backbone can protect the nucleic acids from degradation, enable efficient cellular uptake and target localization ensuring the efficiency of the oligonucleotide-based therapy. In this review, we present a summary of structure and properties of artificial nucleic acids containing nucleobase, sugar or backbone modifications, and provide an overview of the structure and mechanism of action of approved oligonucleotide drugs including gene silencing agents, aptamers and mRNA vaccines.

Cheminformatics Modeling of Gene Silencing for Both Natural and Chemically Modified siRNAs.

In designing effective siRNAs for a specific mRNA target, it is critically important to have predictive models for the potency of siRNAs. None of the published methods characterized the chemical structures of individual nucleotides constituting a siRNA molecule; therefore, they cannot predict the potency of gene silencing by chemically modified siRNAs (cm-siRNA). We propose a new approach that can predict the potency of gene silencing by cm-siRNAs, which characterizes each nucleotide (NT) using 12 BCUT cheminformatics descriptors describing its charge distribution, hydrophobic and polar properties. Thus, a 21-NT siRNA molecule is described by 252 descriptors resulting from concatenating all the BCUT values of its composing nucleotides. Partial Least Square is employed to develop statistical models. The Huesken data (2431 natural siRNA molecules) were used to perform model building and evaluation for natural siRNAs. Our results were comparable with or superior to those from Huesken's algorithm. The Bramsen dataset (48 cm-siRNAs) was used to build and test the models for cm-siRNAs. The predictive r2 of the resulting models reached 0.65 (or Pearson r values of 0.82). Thus, this new method can be used to successfully model gene silencing potency by both natural and chemically modified siRNA molecules.

Use of Telemedicine and Home Infusion

The COVID-19 pandemic has further highlighted the management difficulties of NMDs patients and the necessity to continue the program of implementation of standard of care yet started in Europe with the definition of the ERNs and with different applications in the various European countries. It will be increasingly necessary to favor and further develop smart management care through the implementation of IT platforms, telemedicine services and other eHealth technologies. In the near future we are moving towards a health system that supports scientific research, strengthens prevention, and brings medicine home. The outbreak of COVID-19 has forced the health care system to undergo profound rearrangements in its services and facilities. In this setting, inpatient and outpatient services had to rethink and reorganize their activities to meet the needs of patients during the lockdown. The rapid and abrupt COVID-19 shutdowns and stay-at-home orders imposed challenges to routine clinical management and clinical trials. The opportunity for real-world evaluation and reduced patient burden are clear benefits to remote assessment and may provide a more robust understanding and characterization of disease impact in NMD. The quality of patient-physician relationship, the modality of remote clinical assessment and monitoring, and the administration of therapies are the key elements to be provided in neuromuscular Telemedicine. Telemedicine permits the maintenance of a close patient-physician relationship. Other important aspect is digital therapies: I.e., access in hospital settings to perform medical therapies that can be performed only and exclusively in protected settings (such as antisense oligonucleotides for SMA or Patisiran for genetic amyloidosis), but some treatments can be home-based, such as subcutaneous immunoglobulins, or enzyme replacement therapy for Pompe disease.

INHIBITION OF PROTEIN KINASE CK2 AND BCL-2: A NOVEL THERAPEUTIC STRATEGY TO ANTAGONIZE MANTLE CELL LYMPHOMA CELL GROWTH

Background: Mantle cell lymphoma (MCL) is a B-cell tumor which often relapses. BCR inhibitors (Ibrutinib, Acalabrutinib) and antiapoptotic BCL2-family members blockers BH3-mimetics (Venetoclax, ABT-199) are effective drugs to fight MCL. However, the disease remains incurable, due to therapy resistance, even to the promising Venetoclax and Ibrutinib combination. Therefore, there is a profound need to explore novel useful therapeutic targets. CK2 is a S/T kinase overexpressed in several solid and blood tumors. We demonstrated that CK2, operating through a 'non-oncogene addiction' mechanism promotes tumor cell survival, and counteracts apoptosis, by activating pro-survival signaling cascades, such as NF-κ B, STAT3 and AKT. CK2 could regulate also BCL2 family members. The CK2 chemical inhibitor CX-4945 (Silmitasertib, Sil) is already under scrutiny in clinical trials in relapsed multiple myeloma, solid tumors and COVID-19. Aims: In this work, we tested the effect of CK2 chemical inhibition or knock down on Venetoclax (Ven)-induced cytotoxicity in MCL pre-clinical models to effectively reduce MCL cell growth and clonal expansion. Methods: CK2 expression and BCR/BCL2 related signaling components were analyzed in MCL cells and control cells by Western blotting. CK2 and BCL2 inhibition was obtained with Sil and Ven, respectively and with CK2 gene silencing through the generation of anti-CK2 shRNA IPTG-inducible MCL cell clones. Survival, apoptosis, mitochondrial membrane depolarization and proliferation were investigated by FACS analysis of AnnexinV/PI and JC-10 staining. The synergic action of Ven and Sil was analyzed by the Chou-Talalay combination index (CI) method. CK2 knock down in vivo was obtained in xenograft NOD-SCID mouse models Results: CK2 inactivation (with Sil or CK2 silencing) determined a reduction in the activating phosphorylation of S529 p65/RelA and S473 and S129 AKT, important survival cascades for MCL. Sil or CK2 silencing caused BCL2 and related MCL1 protein reduction, causing cell death. Importantly, we confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown in MCL. Sil +Ven combination increased MCL cell apoptosis, as judged by the augmented frequency of Annexin V positive cells and expression of cleaved PARP protein, and JC-10 mitochondrial membrane depolarization, with respect to the single treatments. Captivatingly, Sil or CK2 gene silencing led to a substantial reduction of the Ven-induced increase of MCL-1, potentially counteracting a deleterious Ven-induced drawback. Analysis of cell cycle distribution confirmed an increased frequency of apoptotic cells in the sub G1 phase in CK2-silenced cells and a modulation of the other phases of the cell cycle. Remarkably, the calculated CI less than 1 suggested a strong synergic cell-killing effect between Sil and Ven, on all the cell lines tested, including those less sensitive or resistant to Ven Summary/Conclusion: We demonstrated that the simultaneous inhibition/knock down of CK2 and BCL2 synergistically cooperates in inducing apoptosis and cell cycle arrest of MCL malignant B-lymphocytes and has the potential of reducing MCL clonal growth, also counterbalancing mechanism of resistance that may arise with Ven. Therefore, CK2 is a rational therapeutic target for the treatment of MCL to be tested in combination with Ibrutinib or Ven.

RNA solutions to treat inborn errors of metabolism.

RNA-based therapies are a new, rapidly growing class of drugs that until a few years ago were being used mainly in research in rare diseases. However, the clinical efficacy of recently approved oligonucleotide drugs and the massive success of COVID-19 RNA vaccines has boosted the interest in this type of molecules of both scientists and industry, as wells as of the lay public. RNA drugs are easy to design and cost effective, with greatly improved pharmacokinetic properties thanks to progress in oligonucleotide chemistry over the years. Depending on the type of strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are more than a dozen RNA-based drugs approved for clinical use, including some for specific inborn errors of metabolism (IEM), and many other in different stages of development. New initiatives in n-of-1 RNA drug development offer new hope for patients with rare diseases and/or ultra-rare mutations. RNA-based therapeutics include antisense oligonucleotides, aptamers, small interfering RNAs, small activating RNAs, microRNAs, lncRNAs and messenger RNAs. Further research and collaborations in the fields of chemistry, biology and medicine will help to overcome major challenges in their delivery to target tissues. Herein, we review the mechanism of action of the different therapeutic approaches using RNA drugs, focusing on those approved or in clinical trials to treat IEM.

Applications, challenges and opportunities of RNA drugs

RNA-based therapies were considered a curiosity until recently, used mainly in research in rare diseases and personalised therapies. However, this research field has gained new momentum, as it has effectively increased the 'druggable' space;RNA drugs are easy to design and are cost effective, with greatly improved pharmacokinetic properties thanks to progress in oligonucleotide chemistry over the last few years. Moreover, the massive clinical scientific success of COVID-19 RNA vaccines has boosted the interest of both scientists and lay public for this type of molecules. Currently, there are more than a dozen RNA-based drugs approved for clinical use and many others in different stages of development. RNA-based therapeutics include antisense oligonucleotides, aptamers, small interfering RNAs, microRNAs and messenger RNAs. To date, the future of RNA therapeutics relies on overcoming the major challenges in delivery, requiring further research and collaborations in the fields of chemistry, biology and medicine.

A bioinformatic approach of targeting SARS-CoV-2 replication by silencing a conserved alternative reserve of the orf8 gene using host miRNAs.

The causative agent of the COVID-19 pandemic, the SARS-CoV-2 virus has yielded multiple relevant mutations, many of which have branched into major variants. The Omicron variant has a huge similarity with the original viral strain (first COVID-19 strain from Wuhan). Among different genes, the highly variable orf8 gene is responsible for crucial host interactions and has undergone multiple mutations and indels. The sequence of the orf8 gene of the Omicron variant is, however, identical with the gene sequence of the wild type. orf8 modulates the host immunity making it easier for the virus to conceal itself and remain undetected. Variants seem to be deleting this gene without affecting the viral replication. While analyzing, we came across the conserved orf7a gene in the viral genome which exhibits a partial sequence homology as well as functional similarity with the SARS-CoV-2 orf8. Hence, we have proposed here in our hypothesis that, orf7a might be an alternative reserve of orf8 present in the virus which was compensating for the lost gene. A computational approach was adopted where we screened various miRNAs targeted against the orf8 gene. These miRNAs were then docked onto the orf8 mRNA sequences. The same set of miRNAs was then used to check for their binding affinity with the orf7a reference mRNA. Results showed that miRNAs targeting the orf8 had favorable shape complementarity and successfully docked with the orf7a gene as well. These findings provide a basis for developing new therapeutic approaches where both orf8 and orf7a can be targeted simultaneously.

Role of NOX4 in Modulating Alveolar Type II (AT2) Epithelial Cell Phenotype and Mitochondrial Dynamics in Hyperoxic Acute Lung Injury

Introduction: Management of acute respiratory distress including COVID-19 pneumonia involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). AT2 cells are the most affected cell type in hyperoxia (HO). NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) in HO. NOX4, the only functionally active NOX present in mitochondria, and primarily produces H2O2 as well as mtROS has been shown to be involved in several human pathologies. Not much is known about NOX4-induced mitochondrial injury in HALI. The current study aims to determine the role of AT2 epithelial cell NOX4 in HALI and the impact of HO on the modulation of mtROS and mitochondrial dynamics in HALI. Methods: Nox4-/-Spc-Cre animals were generated using tamoxifen induction and the knockdown was validated. The Nox4- /-Spc-Cre knockout (KO) and wild type (WT) mice were exposed to room air (NO) or 95% O2 (HO) for 66h to study the structural and functional changes in the lung. Transmission Electron Microscopy (TEM) was used to study the HO-induced changes in mitochondria. Isolated primary AT2 and/ mouse lung epithelial (MLE) cell line was investigated for mtROS, mt dynamics and apoptosis. Mitochondrial injury was assessed in Nox4 WT and Nox4 silenced cells. Results: C57BL/6J WT animals subjected to HO for 66h showed increased expression of NOX4, determining the role of NOX4 in HALI. The H&E staining demonstrated significant HALI in Nox4 WT animals exposed to HO compared to Nox4 KO as determined by increased infiltration of neutrophils, alveolar wall thickening and presence of proteinaceous debris in the alveolar space. Further, increased BAL cell count and protein levels, increased AT2 cell death and elevation of the proinflammatory cytokine IL- 6 and the chemokine KC was seen in WT animals compared to Nox4 KO. Analysis of lung tissues by TEM showed mitochondrial swelling, cristae damage and mitophagy in AT2 cells due to HO. Changes in mt injury markers were also observed. HO-induced NOX4 increase in primary AT2/ MLE-12 cells resulted in increased mtROS production and apoptosis, which was reduced with Nox4 siRNA silencing. Conclusion: This study suggests that the HO induced NOX4 expression in mouse lung, and deletion of Nox4 gene in AT2 cells reduced mtROS production and apoptosis and protected the lungs from severe hyperoxic lung injury. These results suggest NOX4 as a potential target for the treatment of HALI.

Abstracts of the 37th International Congress of the ISBT, Virtual meeting, 4-8 June 2022

The proceedings contain 456 papers. The topics discussed include: DSLK and Kg are antithetical antigens in the RHAG blood group system;identification of a new low prevalence antigen in the RHAG glycoprotein;characterization of a novel high-prevalence antigen in the KEL blood group system;structural locations of single-nucleotide missense variants in the Kidd blood group on human urea transporter B;two new JK silencing alleles identified by a single molecule sequencer with 20 kb-long reads;nanopore sequencing to resolve Kidd blood group discrepancies;removal of HLA class-I antigens from platelets: increasing platelet availability for refractory patients;human neutrophil antigens and their clinical significance;a novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1 antibodies;blood supply chain management what has been learnt from the pandemic and others?;building towards organizational resilience: a case study of the organizational impacts and changes in Sanquin during COVID-19;and contingency planning in blood transfusion services African COVID-19 experience.

A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity

Plants have evolved defense mechanisms to suppress viral transcription and replication by transcriptional and post-transcriptional gene silencing mediated by virus-derived small interfering RNAs (vsiRNAs). Based on this response, virus-induced gene silencing (VIGS)-based technology has been developed to silence target genes on either host plants or insect pests. This mechanism could also be used for the silencing of genes of interest in the medical field. We used the VIGS vector pEuMV-YP:Krt18, which was obtained by inserting the Mus musculus (M. musculus) Krt18 sequence into pEuMV-YP:ΔAV1. The objective was to evaluate the capacity of pEuMV-YP:Krt18 to induce Nicotiana benthamiana (N. benthamiana) production of vsiRNAs of a specific sequence that belongs to neither the plant genome nor the wild virus genome, which were used to induce cross-kingdom gene silencing between plants and mammals. The percentage of vsiRNA for each viral gene was calculated from an sRNA library of N. benthamiana plants infected by pEuMV-YP: Krt18. When the vsiRNAs were characterized, it was found that they corresponded to all the genes of the pEuMV-YP:Krt18 vector. These vsiRNAs induced the silencing of the Krt18 gene in M. musculus macrophages, supporting the ability to use VIGS vectors in plants as biofactories for the production of sRNAs that induce gene silencing in mammals.