Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Nucleic Acid-Based Technologies Targeting Coronaviruses.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer.

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

Genetic analysis using targeted exome sequencing of 53 Vietnamese children with developmental and epileptic encephalopathies.

Developmental and epileptic encephalopathies (DEE) refers to a group of rare and severe neurodevelopmental disorders where genetic etiologies can play a major role. This study aimed to elucidate the genetic etiologies of a cohort of 53 Vietnamese patients with DEE. All patients were classified into known electroclinical syndromes where possible. Exome sequencing (ES) followed by a targeted analysis on 294 DEE-related genes was then performed. Patients with identified causative variants were followed for 6 months to determine the impact of genetic testing on their treatment. The diagnostic yield was 38.0% (20/53), which was significantly higher in the earlier onset group (<12 months) than in the later onset group (≥12 months). The 19 identified variants belonged to 11 genes with various cellular functions. Genes encoding ion channels especially sodium voltage-gated channel were the most frequently involved. Most variants were missense variants and located in key protein functional domains. Four variants were novel and four had been reported previously but in different phenotypes. Within 6 months of further follow-up, treatment changes were applied for six patients based on the identified disease-causing variants, with five patients showing a positive impact. This is the first study in Vietnam to analyze the genetics of DEE. This study confirms the strong involvement of genetic etiologies in DEE, especially early onset DEE. The study also contributes to clarify the genotype-phenotype correlations of DEE and highlights the efficacy of targeted ES in the diagnosis and treatment of DEE.

De novo homozygous variant of the SCN1A gene in a patient with severe Dravet syndrome complicated by acute encephalopathy.

Variants in the SCN1A gene have been identified in epilepsy patients with widely variable phenotypes and they are generally heterozygous. Here, we report a homozygous missense variant, NM_001165963.4: c.4319C>T (p.Ala1440Val), in the SCN1A gene which seemed to occur de novo together with a gene conversion event. It's highly possible that this variant, although located in a critical functional domain of protein Nav1.1, depending on the nature of the amino acid substitution, may not cause the complete loss of protein function. And the accumulated effect by having this variant on both alleles results in a Dravet syndrome phenotype which is more severe than average. This first report of a de novo homozygous variant in the SCN1A gene, therefore, provides a clear illustration of a complex genotype-phenotype relationship.

A mutation in GABRB3 associated with Dravet syndrome.

Dravet syndrome is a rare and severe type of epilepsy in infants. Approximately, 70-80% of patients with Dravet syndrome have mutations in SCN1A, the gene encoding the alpha-1 subunit of the sodium channel, while some simplex patients have variants in one of several other genes, including but not limited to GABRA1, SCN2A, STXBP1, GABRG2, and SCN1B. In this study, we performed exome sequencing in six patients with SCN1A-negative Dravet syndrome to identify other genes related to this disorder. In one affected individual, we detected a novel de novo heterozygous missense variant, c.695G>A, p.(Arg232Gln), in GABRB3, the gene encoding the ß3-subunit of the gamma-aminobutyric acid type A (GABAA) receptor, which mediates inhibitory signaling within the central nervous system. In summary, the data in this study identify GABRB3 as a candidate gene for Dravet syndrome.

Between hope and reality: treatment of genetic diseases through nucleic acid-based drugs.

Rare diseases (RD) affect a small number of people compared to the general population and are mostly genetic in origin. The first clinical signs often appear at birth or in childhood, and patients endure high levels of pain and progressive loss of autonomy frequently associated with short life expectancy. Until recently, the low prevalence of RD and the gatekeeping delay in their diagnosis have long hampered research. The era of nucleic acid (NA)-based therapies has revolutionized the landscape of RD treatment and new hopes arise with the perspectives of disease-modifying drugs development as some NA-based therapies are now entering the clinical stage. Herein, we review NA-based drugs that were approved and are currently under investigation for the treatment of RD. We also discuss the recent structural improvements of NA-based therapeutics and delivery system, which overcome the main limitations in their market expansion and the current approaches that are developed to address the endosomal escape issue. We finally open the discussion on the ethical and societal issues that raise this new technology in terms of regulatory approval and sustainability of production.

ZNF703 mRNA-Targeting Antisense Oligonucleotide Blocks Cell Proliferation and Induces Apoptosis in Breast Cancer Cell Lines.

The luminal B molecular subtype of breast cancers (BC) accounts for more than a third of BCs and is associated with aggressive clinical behavior and poor prognosis. The use of endocrine therapy in BC treatment has significantly contributed to the decrease in the number of deaths in recent years. However, most BC patients with prolonged exposure to estrogen receptor (ER) selective modulators such as tamoxifen develop resistance and become non-responsive over time. Recent studies have implicated overexpression of the ZNF703 gene in BC resistance to endocrine drugs, thereby highlighting ZNF703 inhibition as an attractive modality in BC treatment, especially luminal B BCs. However, there is no known inhibitor of ZNF703 due to its nuclear association and non-enzymatic activity. Here, we have developed an antisense oligonucleotide (ASO) against ZNF703 mRNA and shown that it downregulates ZNF703 protein expression. ZNF703 inhibition decreased cell proliferation and induced apoptosis. Combined with cisplatin, the anti-cancer effects of ZNF703-ASO9 were improved. Moreover, our work shows that ASO technology may be used to increase the number of targetable cancer genes.

Castration-Resistant Prostate Cancer: From Uncovered Resistance Mechanisms to Current Treatments.

Prostate cancer (PC) is the second most common cancer in men worldwide. Despite recent advances in diagnosis and treatment, castration-resistant prostate cancer (CRPC) remains a significant medical challenge. Prostate cancer cells can develop mechanisms to resist androgen deprivation therapy, such as AR overexpression, AR mutations, alterations in AR coregulators, increased steroidogenic signaling pathways, outlaw pathways, and bypass pathways. Various treatment options for CRPC exist, including androgen deprivation therapy, chemotherapy, immunotherapy, localized or systemic therapeutic radiation, and PARP inhibitors. However, more research is needed to combat CRPC effectively. Further investigation into the underlying mechanisms of the disease and the development of new therapeutic strategies will be crucial in improving patient outcomes. The present work summarizes the current knowledge regarding the underlying mechanisms that promote CRPC, including both AR-dependent and independent pathways. Additionally, we provide an overview of the currently approved therapeutic options for CRPC, with special emphasis on chemotherapy, radiation therapy, immunotherapy, PARP inhibitors, and potential combination strategies.

Prevalence and Characterization of Gastroenteritis Viruses among Hospitalized Children during a Pilot Rotavirus Vaccine Introduction in Vietnam.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) are the most common viral causes of gastroenteritis in children worldwide. From 2016 to 2021, we conducted a cross-sectional descriptive study to determine the prevalence of these viruses in hospitalized children under five years old in Nam Dinh and Thua Thien Hue provinces in Vietnam during the pilot introduction of the RV vaccine, Rotavin-M1 (POLYVAC, Hanoi, Vietnam). We randomly selected 2317/6718 (34%) acute diarrheal samples from children <5 years of age enrolled at seven sentinel hospitals from December 2016 to May 2021; this period included one year surveillance pre-vaccination from December 2016 to November 2017. An ELISA kit (Premier Rotaclone®, Meridian Bioscience, Inc., Cincinnati, OH, USA) was used to detect RV, and two multiplex real-time RT-PCR assays were used for the detection of NoV, SaV and HAstV. The prevalence of RV (single infection) was reduced from 41.6% to 22.7% (p < 0.0001) between pre- and post-vaccination periods, while the single NoV infection prevalence more than doubled from 8.8% to 21.8% (p < 0.0001). The SaV and HAstV prevalences slightly increased from 1.9% to 3.4% (p = 0.03) and 2.1% to 3.3% (p = 0.09), respectively, during the same period. Viral co-infections decreased from 7.2% to 6.0% (p = 0.24), mainly due to a reduction in RV infection. Among the genotypeable samples, NoV GII.4, SaV GI.1, and HAstV-1 were the dominant types, representing 57.3%, 32.1%, and 55.0% among the individual viral groups, respectively. As the prevalence of RV decreases following the national RV vaccine introduction in Vietnam, other viral pathogens account for a larger proportion of the remaining diarrhea burden and require continuing close monitoring.

HSP27/Menin Expression as New Prognostic Serum Biomarkers of Prostate Cancer Aggressiveness Independent of PSA.

The screening of PCa is based on two tests, the total PSA test and the rectal examination. However, PSA is not specific for PCa stage confirmation, leading in false positive result and involving PCa over-diagnosis and over-treatment. HSP27 and Menin have been found to be overexpressed in a wide range of human cancers. Recent studies showed how HSP27 interacts with and stabilizes Menin to lead PCa progression and treatment resistance. The purpose of our study was to evaluate the correlation of HSP27 and Menin molecular expression, and their prognosis value in PCa with respect to clinicopathological features. Elisa was employed to measure serum HSP27 and Menin concentrations in 73 PCa patients and 80 healthy individuals. Immunohistochemistry (IHC) was used to determine HSP27 and Menin tissue expression in 57 tumors and 4 Benign Prostatic Hyperplasia (BPH) tissues. Serum HSP27 expression correlated with its tissue expression in all PCa patients, whereas serum Menin expression correlated only with tissue expression in aggressive PCa patients. Moreover, the results showed a positive correlation between HSP27 and Menin either in serum (r = 0.269; p = 0.021) or in tissue (r = 0.561; p < 0.0001). In aggressive PCa, serum expression of HSP27 and Menin was positively correlated (r = 0.664; R = 0.441; p = 0.001). The correlation between HSP27 and Menin expression in tissue was found only in patients with aggressive PCa (r = 0.606; R = 0.367; p = 0.004). Statistical analysis showed that the expression of both biomarkers was positively correlated with the hormone resistance or sensitivity, tumor aggressiveness, metastasis, Gleason Score, death and did not significantly correlate with age and PSA. Survival was illustrated by Kaplan−Meier curves; increased HSP27 and Menin expression correlated with shorter survival of PCa patients (p = 0.001 and p < 0.0001, respectively). Accuracy in predicting aggressiveness was quantified by the Area Under the Curve (AUC) of Receiver Operating Characteristic (ROC). We demonstrated that the combination of HSP27/Menin was statistically greater than PSA; it achieved an AUC of 0.824 (95% CI, 0.730−0.918; p < 0.0001). However, HSP27/Menin/PSA combination decreased the diagnostic value with an AUC of 0.569 (95% CI, 0.428−0.710; p = 0.645). Our work suggests the potential role of HSP27/Menin as diagnostic and prognostic biomarkers.

Distributionally Robust and Multi-Objective Nonnegative Matrix Factorization.

Nonnegative matrix factorization (NMF) is a linear dimensionality reduction technique for analyzing nonnegative data. A key aspect of NMF is the choice of the objective function that depends on the noise model (or statistics of the noise) assumed on the data. In many applications, the noise model is unknown and difficult to estimate. In this paper, we define a multi-objective NMF (MO-NMF) problem, where several objectives are combined within the same NMF model. We propose to use Lagrange duality to judiciously optimize for a set of weights to be used within the framework of the weighted-sum approach, that is, we minimize a single objective function which is a weighted sum of the all objective functions. We design a simple algorithm based on multiplicative updates to minimize this weighted sum. We show how this can be used to find distributionally robust NMF (DR-NMF) solutions, that is, solutions that minimize the largest error among all objectives, using a dual approach solved via a heuristic inspired from the Frank-Wolfe algorithm. We illustrate the effectiveness of this approach on synthetic, document and audio data sets. The results show that DR-NMF is robust to our incognizance of the noise model of the NMF problem.

Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity.

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

Antisense Oligonucleotide-Based Therapeutic against Menin for Triple-Negative Breast Cancer Treatment.

The tumor suppressor menin has dual functions, acting either as a tumor suppressor or as an oncogene/oncoprotein, depending on the oncological context. Triple-negative breast cancer (TNBC) is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2) and is often a basal-like breast cancer. TNBC is associated with a dismal prognosis and an insufficient response to chemotherapies. Previously, menin was shown to play a proliferative role in ER-positive breast cancer; however, the functions of menin in TNBC remain unknown. Here, we have demonstrated that menin is expressed in various TNBC subtypes with the strongest expression in the TNBC Hs 578T cells. The depletion of menin by an antisense oligonucleotide (ASO) inhibits cell proliferation, enhances apoptosis in Hs 578T cells, highlighting the oncogenic functions of menin in this TNBC model. ASO-based menin silencing also delays the tumor progression of TNBC xenografts. Analysis of the menin interactome suggests that menin could drive TNBC tumorigenesis through the regulation of MLL/KMT2A-driven transcriptional activity, mRNA 3'-end processing and apoptosis. The study provides a rationale behind the use of ASO-based therapy, targeting menin in monotherapy or in combination with chemo or PARP inhibitors for menin-positive TNBC treatments.

Nucleoside-Lipid-Based Nanoparticles for Phenazine Delivery: A New Therapeutic Strategy to Disrupt Hsp27-eIF4E Interaction in Castration Resistant Prostate Cancer.

Heat shock protein 27 (Hsp27) has an established role in tumor progression and chemo-resistance of castration-resistant prostate cancer (CRPC). Hsp27 protects eukaryotic translation initiation factor 4E (eIF4E) from degradation, thereby maintaining survival during treatment. Phenazine derivative compound #14 was demonstrated to specifically disrupt Hsp27/eIF4E interaction and significantly delay castration-resistant tumor progression in prostate cancer xenografts. In the present work, various strategies of encapsulation of phenazine #14 with either DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate) and DOU-PEG2000 (5'-PEG2000-2',3'-dioleoyluridine) nucleolipids (NLs) were developed in order to improve its solubilization, biological activity, and bioavailability. We observed that NLs-encapsulated phenazine #14-driven Hsp27-eIF4E interaction disruption increased cytotoxic effects on castration-resistant prostate cancer cell line and inhibited tumor growth in castration-resistant prostate cancer cell xenografted mice compared to phenazine #14 and NLs alone. Phenazine #14 NL encapsulation might represent an interesting nanostrategy for CRPC therapy.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

Anal human papillomavirus prevalence and risk factors among men who have sex with men in Vietnam.

OBJECTIVES: Men who have sex with men (MSM) are at risk of human papillomavirus (HPV)-related cancers, while published data are scarce. This study determined HPV prevalence and risk factors in MSM in Vietnam to inform HPV prevention strategies in this key population. METHODS: A cross-sectional study of 799 MSM aged 16-50 years was conducted in Vietnam in 2017-2018. Information was collected on risk behaviours, and knowledge of HPV and anal cancer; rectal swabs were taken to detect anal HPV infection. An in-house polymerase chain reaction and Genoflow HPV array test kit were used for HPV detection and genotyping. RESULTS: The median age of the study participants was 25 years (range 18-52). Overall prevalence of any HPV and HPV16/18 infection was 32.3% and 11.0%, respectively. A higher prevalence of high-risk HPV infection to all 14 types tested was found in Ho Chi Minh City (30.9%) than in Hanoi (18.4%). High-risk HPV infection was associated with inconsistent condom use and history of engaging in sex under the influence of drugs (adjusted odds ratio (aOR), 2.27; 95% CI, 1.48-10.67), as well as having multiple sexual partners (aOR, 1.01; 95% CI, 1.00-1.02). CONCLUSIONS: High-risk anal HPV infections in Vietnamese MSM were significantly associated with risky sexual behaviours. A targeted HPV vaccination strategy would have substantial benefit for MSM in Vietnam.

SCN1A Gene Mutation and Adaptive Functioning in 18 Vietnamese Children with Dravet Syndrome.

BACKGROUND AND PURPOSE: Dravet syndrome is a rare and severe type of epilepsy in infants. The heterogeneity in the overall intellectual disability that these patients suffer from has been attributed to differences in genetic background and epilepsy severity. METHODS: Eighteen Vietnamese children diagnosed with Dravet syndrome were included in this study. SCN1A variants were screened by direct sequencing and multiplex ligation-dependent probe amplification. Adaptive functioning was assessed in all patients using the Vietnamese version of the Vineland Adaptive Behavior Scales, and the results were analyzed relative to the SCN1A variants and epilepsy severity. RESULTS: We identified 13 pathogenic or likely pathogenic variants, including 6 that have not been reported previously. We found no correlations between the presence or type of SCN1A variants and the level of adaptive functioning impairment or severity of epilepsy. Only two of nine patients aged at least 5 years had an adaptive functioning score higher than 50. Both of these patients had a low frequency of convulsive seizures and no history of status epilepticus or prolonged seizures. The remaining seven had very low adaptive functioning scores (39 or less) despite the variability in the severity of their epilepsy confirming the involvement of factors other than the severity of epilepsy in determining the developmental outcome. CONCLUSIONS: Our study expands the spectrum of known SCN1A variants and confirms the current understanding of the role of the genetic background and epilepsy severity in determining the developmental outcome of Dravet syndrome patients.