Search Results (257)

Atherosclerosis is a complex disease characterized by pathological thickening of the arterial intima. The mechanisms underlying the induction and progression of atherosclerosis are convoluted and remain under active investigation, with key components such as lipid accumulation and local inflammation being identified. Several risk factors (e.g., metabolic disorders, genetic background, diet, infections) have been shown to exacerbate disease progression, but their roles as clinically relevant markers remain to be established. Despite the growing body of evidence on the molecular pathogenesis of atherosclerosis, there is no effective preventive treatment against the development of this disease. In this review, we focus on gene targets for gene therapy as a novel potential approach to cure and prevent atherosclerosis. We critically review recent research demonstrating the therapeutic potential of viral vector-based (adeno-associated virus (AAV) and lentivirus) gene therapy for the treatment of atherosclerosis. We also summarize alternative gene targets and approaches (e.g., non-coding RNA (ncRNA), micro RNA (miRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), CRISPR/Cas9) that aim to limit disease progression. We highlight the importance of local inflammation in the pathogenesis of atherosclerosis and propose gene targets with anti-inflammatory activity to inhibit the pathological inflammatory response. In addition, we provide perspectives on the future development of gene therapeutics and their potential applications. We anticipate that recent advances in gene therapy will help to identify new and effective targets to prevent atherosclerosis. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1–4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293–993 nt) and rep (1561–2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease’s molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes. Full article

Influenza A virus (IAV) is a highly diverse pathogen with genetic variability primarily driven by mutation and reassortment. Using next-generation sequencing (NGS), we characterised defective viral genomes (DVGs) generated during the serial passaging of influenza A/Puerto Rico/8/1934 (H1N1) virus in embryonated chicken eggs. Deletions were the most abundant DVG type, predominantly accumulating in the polymerase-encoding segments. Notably, we identified and validated a novel class of multisegment DVGs arising from intersegment recombination events, providing evidence that the IAV RNA polymerase can detach from one genomic template and resume synthesis on another. Multisegment recombination primarily involved segments 1–3 but also occurred between other segment pairings. In specific lineages, certain multisegment DVGs reached high frequencies and persisted through multiple passages, suggesting they are not transient by-products of recombination but may possess features that support stable maintenance. Furthermore, multisegment DVGs were shown to be encapsidated within virions, similar to deletion DVGs. The observation of recombination between segments with limited sequence homology underscores the potential for complex recombination to expand IAV genetic diversity. These findings suggest recombination-driven DVGs represent a previously underappreciated mechanism in influenza virus evolution. Full article

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (family Paramyxoviridae, order Mononegavirales), for which all basic replication processes are located in the cytoplasm. The HeV matrix (M) protein plays essential roles in viral assembly and budding at the plasma membrane, but also undergoes dynamic nuclear and nucleolar trafficking, accumulating in nucleoli early in infection, before relocalising to the plasma membrane. We previously showed that M targets sub-nucleolar compartments—the fibrillar centre (FC) and dense fibrillar component (DFC)—to modulate rRNA biogenesis by mimicking a process occurring during a nucleolar DNA-damage response (DDR). Here, we show that M protein sub-nucleolar localisation is regulated by ubiquitination, which controls its redistribution between the FC-DFC and granular component (GC). The mutagenesis of a conserved lysine (K258) reported to undergo ubiquitination, combined with the pharmacological modulation of ubiquitination, indicated that a positive charge at K258 is required for M localisation to the FC-DFC, while ubiquitination regulates subsequent egress from the FC-DFC to the GC. M proteins from multiple Henipaviruses exhibited similar ubiquitin-dependent sub-nucleolar trafficking, indicating a conserved mechanism. These findings reveal a novel mechanism regulating viral protein transport between phase-separated sub-nucleolar compartments and highlight ubiquitination as a key modulator of intra-nucleolar trafficking. Full article

Heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) is a multifunctional RNA-binding protein (RBP) that plays a central role in post-transcriptional regulation. Through its quasi-RNA recognition motifs and low-complexity domains, hnRNPH1 specifically binds guanine-rich RNA sequences, including G-quadruplex structures, to precisely modulate multiple aspects of RNA metabolism, such as alternative splicing, mRNA stability, translation, and subcellular localization. Accumulating evidence has implicated hnRNPH1 dysfunction in the pathogenesis of several human diseases. In cancer, hnRNPH1 often acts as a pro-tumorigenic factor, albeit in a context-dependent manner, influencing the alternative splicing of crucial oncogenes, mRNA stability, and tumor cell sensitivity to therapeutic agents. In the nervous system, hnRNPH1 is involved in neurodevelopment, neurodegenerative diseases, and drug addiction and plays an essential role in maintaining neuronal function and homeostasis. Furthermore, it exerts regulatory functions in reproductive system development and fertility and in non-neoplastic pathologies, including cardiovascular diseases, autoimmune disorders, and viral hepatitis. Given its pathophysiological significance, hnRNPH1 has emerged as a promising biomarker and therapeutic target. This review provides an overview of the structural basis and core molecular function of hnRNPH1. Its mechanisms of action and pathological significance in various diseases have also been detailed. Additionally, this review summarizes the current therapeutic strategies targeting hnRNPH1, discusses the associated challenges, outlines optimization approaches, and considers future research directions. Overall, this review aims to deepen our understanding of hnRNPH1 biology and inspire the development of novel diagnostic and therapeutic interventions. Full article

Self-amplifying RNA-based (saRNA) technology represents the last frontier in using synthetic RNA in vaccinology. Typically, saRNA consists of positive-strand RNA molecules of viral origin (almost exclusively from alphaviruses) where the sequences of structural proteins are replaced with the open reading frame coding the antigen of interest. For in vivo delivery, they are complexed with lipid nanoparticles (LNPs), just like current COVID-19 vaccines based on synthetic messenger RNA (mRNA). Given their ability to amplify themselves inside the cell, optimal intracellular levels of the immunogenic antigen can be achieved by delivering lower amounts of saRNA molecules compared to mRNA-based vaccines. However, the excessive intracellular accumulation of saRNA may represent a relevant drawback since, as already described in alphavirus-infected cells, the recipient cell may react by incorporating excessive RNA molecules into extracellular vesicles (EVs). These EVs can shed and enter neighboring as well as distant cells, where the EV-associated saRNA can start a new replication cycle. This mechanism could lead to an unwanted and unnecessary spread of saRNA throughout the body, posing relevant safety issues. This perspective article discusses the molecular mechanisms through which saRNAs can be transmitted among different cells/tissues. In addition, a simple way to control the possible excessive saRNA intercellular propagation through the co-expression of an EV-anchored protein inhibiting the saRNA replication is proposed. Based on current knowledge, a safety improvement of saRNA-based vaccines appears to be mandatory for their usage in healthy humans. Full article

Vaccines remain one of the most effective tools in combating infectious diseases, though traditional platforms are constrained by limitations including suboptimal immunogenicity, safety concerns, and manufacturing complexity. Circular RNA (circRNA) vaccines have recently emerged as a novel vaccine modality, demonstrating unique advantages including high stability, low innate immunogenicity, and sustained antigen expression. Although early research has predominantly focused on viral targets, accumulating evidence now supports the application potential of circRNA vaccines against diverse pathogens, particularly antibiotic-resistant bacteria. Through encoding critical antigens or virulence factors, these circRNA vaccines demonstrate capability to induce protective immune responses, presenting a viable alternative to conventional antimicrobial strategies. This review highlights recent advances in circRNA vaccine development, spanning synthetic circularization techniques, delivery approaches, and immunological mechanisms. We emphasize their potential against viral, bacterial, fungal, and parasitic infections, while addressing current challenges and future research directions of this emerging platform. Collectively, these insights underscore circRNA’s multifaceted versatility and its expanding relevance in next-generation vaccine innovation. Full article

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, remains a global health challenge with limited therapeutic options and high mortality rates. Despite advances in understanding its molecular pathogenesis, the role of metabolic reprogramming in HCC progression and therapy resistance demands further exploration. Nicotinamide N-methyltransferase (NNMT), a metabolic enzyme central to NAD⁺ and methionine cycles, has emerged as a critical regulator of tumorigenesis across cancers. However, its tissue-specific mechanisms in HCC—particularly in the context of viral hepatitis and methionine cycle dependency—remain understudied. This review systematically synthesizes current evidence on NNMT’s dual role in HCC: (1) driving NAD⁺ depletion and homocysteine (Hcy) accumulation via metabolic dysregulation, (2) promoting malignant phenotypes (proliferation, invasion, metastasis, and drug resistance), and (3) serving as a prognostic biomarker and therapeutic target. We highlight how NNMT intersects with epigenetic modifications, immune evasion, and metabolic vulnerabilities unique to HCC. Additionally, we critically evaluate NNMT inhibitors, RNA-based therapies, and non-pharmacological strategies (e.g., exercise) as novel interventions. By bridging gaps between NNMT’s molecular mechanisms and clinical relevance, this review provides a roadmap for advancing NNMT-targeted therapies and underscores the urgency of addressing challenges in biomarker validation, inhibitor specificity, and translational efficacy. Our work positions NNMT not only as a metabolic linchpin in HCC but also as a promising candidate for precision oncology. Full article

The phosphodiester linkage is found in DNA, RNA and many signaling molecules, such as cyclic mononucleotide, cyclic dinucleotides (CDNs) and cyclic oligonucleotides (cONs). Enzymes that cleave the phosphodiester linkage (nucleases and phosphodiesterases) play important roles in cell persistence and fitness and have therefore become targets for various diseased states. While various inhibitors have been developed for nucleases and cyclic mononucleotide phosphodiesterases, and some have become clinical successes, there is a paucity of inhibitors of the recently discovered phosphodiesterases or ring nucleases that cleave CDNs and cONs. Inhibitors of bacterial c-di-GMP or c-di-AMP phosphodiesterases have the potential to be used as anti-virulence compounds, while compounds that inhibit the degradation of 3′,3′-cGAMP, cA₃, cA₄, cA₆ could serve as antibiotic adjuvants as the accumulation of these second messengers leads to bacterial abortive infection. In humans, 2′3′-cGAMP plays critical roles in antiviral and antitumor responses. ENPP1 (the 2′3′-cGAMP phosphodiesterase) or virally encoded cyclic dinucleotide phosphodiesterases, such as poxin, however, blunt this response. Inhibitors of ENPP1 or poxin-like enzymes have the potential to be used as anticancer and antiviral agents, respectively. This review summarizes efforts made towards the discovery and development of compounds that inhibit CDN phosphodiesterases and cON ring nucleases. Full article

The present Perspective analyzes the remarkable evolution of the Amyloid Cascade Hypothesis 2.0 (ACH2.0) theory of Alzheimer’s disease (AD) since its inception a few years ago, as reflected in the diminishing role of amyloid-beta (Aβ) in the disease. In the initial iteration of the ACH2.0, Aβ-protein-precursor (AβPP)-derived intraneuronal Aβ (iAβ), accumulated to neuronal integrated stress response (ISR)-eliciting levels, triggers AD. The neuronal ISR, in turn, activates the AβPP-independent production of its C99 fragment that is processed into iAβ, which drives the disease. The second iteration of the ACH2.0 stemmed from the realization that AD is, in fact, a disease of the sustained neuronal ISR. It introduced two categories of AD—conventional and unconventional—differing mainly in the manner of their causation. The former is caused by the neuronal ISR triggered by AβPP-derived iAβ, whereas in the latter, the neuronal ISR is elicited by stressors distinct from AβPP-derived iAβ and arising from brain trauma, viral and bacterial infections, and various types of inflammation. Moreover, conventional AD always contains an unconventional component, and in both forms, the disease is driven by iAβ generated independently of AβPP. In its third, the current, iteration, the ACH2.0 posits that proteolytic production of Aβ is suppressed in AD-affected neurons and that the disease is driven by C99 generated independently of AβPP. Suppression of Aβ production in AD seems an oxymoron: Aβ is equated with AD, and the later is inconceivable without the former in an ingrained Amyloid Cascade Hypothesis (ACH)-based notion. But suppression of Aβ production in AD-affected neurons is where the logic leads, and to follow it we only need to overcome the inertia of the preexisting assumptions. Moreover, not only is the generation of Aβ suppressed, so is the production of all components of the AβPP proteolytic pathway. This assertion is not a quantum leap (unless overcoming the inertia counts as such): the global cellular protein synthesis is severely suppressed under the neuronal ISR conditions, and there is no reason for constituents of the AβPP proteolytic pathway to be exempted, and they, apparently, are not, as indicated by the empirical data. In contrast, tau protein translation persists in AD-affected neurons under ISR conditions because the human tau mRNA contains an internal ribosomal entry site in its 5′UTR. In current mouse models, iAβ derived from AβPP expressed exogenously from human transgenes elicits the neuronal ISR and thus suppresses its own production. Its levels cannot principally reach AD pathology-causing levels regardless of the number of transgenes or the types of FAD mutations that they (or additional transgenes) carry. Since the AβPP-independent C99 production pathway is inoperative in mice, the current transgenic models have no potential for developing the full spectrum of AD pathology. What they display are only effects of the AβPP-derived iAβ-elicited neuronal ISR. The paper describes strategies to construct adequate transgenic AD models. It also details the utilization of human neuronal cells as the only adequate model system currently available for conventional and unconventional AD. The final alteration of the ACH2.0, introduced in the present Perspective, is that AβPP, which supports neuronal functionality and viability, is, after all, potentially produced in AD-affected neurons, albeit not conventionally but in an ISR-driven and -compatible process. Thus, the present narrative begins with the “omnipotent” Aβ capable of both triggering and driving the disease and ends up with this peptide largely dislodged from its pedestal and retaining its central role in triggering the disease in only one, although prevalent (conventional), category of AD (and driving it in none). Among interesting inferences of the present Perspective is the determination that “sporadic AD” is not sporadic at all (“non-familial” would be a much better designation). The term has fatalistic connotations, implying that the disease can strike at random. This is patently not the case: The conventional disease affects a distinct subpopulation, and the basis for unconventional AD is well understood. Another conclusion is that, unless prevented, the occurrence of conventional AD is inevitable given a sufficiently long lifespan. This Perspective also defines therapeutic directions not to be taken as well as auspicious ways forward. The former category includes ACH-based drugs (those interfering with the proteolytic production of Aβ and/or depleting extracellular Aβ). They are legitimate (albeit inefficient) preventive agents for conventional AD. There is, however, a proverbial snowball’s chance in hell of them being effective in symptomatic AD, lecanemab, donanemab, and any other “…mab” or “…stat” notwithstanding. They comprise Aβ-specific antibodies, inhibitors of beta- and gamma-secretase, and modulators of the latter. In the latter category, among ways to go are the following: (1) Depletion of iAβ, which, if sufficiently “deep”, opens up a tantalizing possibility of once-in-a-lifetime preventive transient treatment for conventional AD and aging-associated cognitive decline, AACD. (2) Composite therapy comprising the degradation of C99/iAβ and concurrent inhibition of the neuronal ISR. A single transient treatment could be sufficient to arrest the progression of conventional AD and prevent its recurrence for life. Multiple recurrent treatments would achieve the same outcome in unconventional AD. Alternatively, the sustained reduction/removal of unconventional neuronal ISR-eliciting stressors through the elimination of their source would convert unconventional AD into conventional one, preventable/treatable by a single transient administration of the composite C99/iAβ depletion/ISR suppression therapy. Efficient and suitable ISR inhibitors are available, and it is explicitly clear where to look for C99/iAβ-specific targeted degradation agents—activators of BACE1 and, especially, BACE2. Directly acting C99/iAβ-specific degradation agents such as proteolysis-targeting chimeras (PROTACs) and molecular-glue degraders (MGDs) are also viable options. (3) A circumscribed shift (either upstream or downstream) of the position of transcription start site (TSS) of the human AβPP gene, or, alternatively, a gene editing-mediated excision or replacement of a small, defined segment of its portion encoding 5′-untranslated region of AβPP mRNA; targeting AβPP RNA with anti-antisense oligonucleotides is another possibility. If properly executed, these RNA-based strategies would not interfere with the protein-coding potential of AβPP mRNA, and each would be capable of both preventing and stopping the AβPP-independent generation of C99 and thus of either preventing AD or arresting the progression of the disease in its conventional and unconventional forms. The paper is interspersed with “validation” sections: every conceptually significant notion is either validated by the existing data or an experimental procedure validating it is proposed. Full article

In this study, we address the role of c-ABL (cellular Abelson Tyr kinase) in the cytoskeletal rearrangements induced by DENV (Dengue virus) infection in mammalian cells. Using the specific inhibitor imatinib and targeted RNA interference, we show that c-ABL is necessary for viral entry and subsequent ENV (DENV envelope protein) accumulation in infected cells. In addition, c-ABL targeting attenuates F-actin reorganization induced by DENV infection. Together with the involvement of c-ABL in endothelial dysfunction induced by DENV and host secreted factors, our findings strongly suggest that c-ABL is a potential host-targeted antiviral that could control DENV infection and/or its evolution to more severe forms of the disease. Full article

Chilli veinal mottle virus (ChiVMV) severely compromises the quality and yield of solanaceous crops. The helper component protease (HCPro) of ChiVMV functions as a multifunctional RNA silencing suppressor that subverts host antiviral defenses through diverse strategies, However, the underlying mechanisms remain mechanistically unresolved. In this study, HCPro-overexpressing (HCPro-OX) and wild-type (WT) plants were inoculated with ChiVMV to monitor the physiological and molecular changes. Transcriptome analysis identified 11,815 differentially expressed genes (DEGs) under viral infection, among which 1115 genes were specifically regulated by HCPro. KEGG enrichment analysis revealed that the DEGs were significantly associated with plant hormone signal transduction pathways, indicating their crucial role in host–virus interactions. Furthermore, functional clustering of HCPro-regulated DEGs specifically identified key components in ethylene biosynthesis pathways. GO analysis of DEGs between virus-inoculated WT and HCPro-OX plants annotated ethylene biosynthesis-related genes NtACO and NtACS. qPCR validation confirmed that the expression of ethylene biosynthesis-related genes was suppressed by HCPro. Exogenous treatments with the ethylene precursor ACC demonstrated that ethylene suppressed viral accumulation, enhanced POD activity, and reduced the ROS accumulation induced by viral infection. In conclusion, our results demonstrate that HCPro promotes viral infection by suppressing ethylene biosynthesis, which in turn attenuates peroxidase activity, leading to ROS accumulation. Full article

A key contributor to the pathogenicity of viruses is their interaction with cellular defense mechanisms, including UPR (unfolded protein response) that counteracts the accumulation of misfolded proteins in the endoplasmic reticulum (known as ER stress). One of the UPR branches is mediated by the IRE1 (inositol-requiring enzyme 1) protein, which possesses protein kinase and RNase activities that facilitate the unconventional cytoplasmic splicing of XBP1 mRNA, leading to the upregulation of the XBP1 transcription factor. In this study, we demonstrate that Encephalomyocarditis Virus (Cardiovirus rueckerti) is able to suppress IRE1-dependent XBP1 activation. HeLa cells infection with EMCV resulted in the modulation of phosphorylated IRE1 levels throughout the infection cycle. Viral infection did not result in the accumulation of spliced XBP1 mRNA. Moreover, the addition of a chemical inducer of ER stress (dithiothreitol) to infected cells led to a markedly lower accumulation of spliced XBP1 mRNA as compared to the level of this mRNA in inducer-treated mock-infected cells. Thus, our results demonstrate the ability of picornaviruses to modulate another defensive activity of the host cell. Full article

Immunotherapy is an established and efficient treatment strategy for a variety of malignancies. It aims to boost the anticancer properties of one’s own immune system. Several immunotherapeutic options are available, but immune checkpoint blockers represent the most widely known and investigated. Anticancer vaccines represent an evolving area of immunotherapy that stimulate antigen-presenting cells, cytotoxic responses of CD8+ T cells, and the presence of memory T cells, among others. Over the years, different approaches for anticancer vaccines have been studied, such as mRNA and DNA vaccines, together with dendritic cell- and viral vector-based vaccines. Recently, an accumulating number of clinical studies have been performed to analyze the safety and potential efficacy of these agents. The aim of this review is to summarize recent advances regarding different types of therapeutic anticancer vaccines. Furthermore, it will discuss how recent advances in preclinical models can enhance clinical outcomes. Full article