Since 2019, there has been a growing focus on mRNA vaccines for infectious disease prevention, particularly following the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). mRNA vaccines offer advantages such as rapid production and the ability to induce robust cellular and antibody responses, which are essential for combating infections that require cell-mediated immunity, including Tuberculosis (TB). This review explores recent progress in TB mRNA vaccines and addresses several key areas: (1) the urgent need for new TB vaccines; (2) current advancements in TB vaccine development, and the advantages and challenges of mRNA technology; (3) the design and characteristics of TB mRNA vaccines; (4) the immunological mechanisms of TB mRNA vaccines; (5) manufacturing processes for TB mRNA vaccines; and (6) safety and regulatory considerations. This interdisciplinary review aims to provide insights for researchers working to address critical questions in TB mRNA vaccine development.

Fifty-eight million people worldwide are chronically infected with hepatitis C virus (HCV) and are at risk of developing cirrhosis and hepatocellular carcinoma (HCC). Direct-acting antivirals are highly effective; however, they are burdened by high costs and the unchanged risk of HCC and reinfection, making prophylactic countermeasures an urgent medical need. HCV high genetic diversity is one of the main obstacles to vaccine development. The protective role of the humoral response directed against the HCV E2 glycoprotein is well established, and broadly neutralizing antibodies play a crucial role in effective viral clearance.

This review explores the HCV targets and the different vaccination approaches, encompassing different expression systems, antigen selection strategies, and delivery methods, focusing on those aimed at eliciting a broad and effective humoral response. Our search criteria included the keywords 'HCV,' 'Hepatitis C,' and 'vaccine' using publicly available databases. Following the screening, 54 papers were selected.

The investigation of novel vaccine platforms beyond traditional approaches is necessary. While progress has been made in this direction, continued investigations on the HCV virology, immunology, and vaccinology are essential to surmount associated obstacles, heling in the development of an HCV vaccine that can benefit the global public health.

Forty years after the dystrophin gene was cloned, significant progress has been made in developing gene therapy approaches for Duchenne muscular dystrophy (DMD). The disorder has presented numerous challenges, including the enormous size of the gene (2.2 MB), the need to target muscles body wide, and immunogenic issues against both vectors and dystrophin. Among human genetic disorders, DMD is relatively common, and the genetics are complicated since one-third of all cases arise from a spontaneous new mutation, resulting in thousands of independent lesions throughout the locus. Many approaches have been pursued in the goal of finding an effective therapy, including exon skipping, nonsense codon suppression, upregulation of surrogate genes, gene replacement, and gene editing. Here, we focus specifically on methods using AAV vectors, as these approaches have been tested in numerous clinical trials and are able to target muscles systemically. We discuss early advances to understand the structure of dystrophin, which are crucial for the design of effective DMD gene therapies. Included is a summary of efforts to deliver micro-, mini-, and full-length dystrophins to muscles. Finally, we review current approaches to adapt gene editing to the enormous DMD gene with prospects for improved therapies using all these methods.

Locally advanced or metastatic melanoma is a cancer with a poor prognosis, characterised by its ability to respond to the induction of an immune response. Anti-tumour vaccination has been studied for many years, although initial results have sometimes been disappointing. The emergence of immune checkpoint inhibiting immunotherapies has dramatically changed the prognosis. Melanoma has emerged as a prime model for renewed research into anti-tumour vaccination. The aim of this review article is to provide an overview of recent developments and prospects for antigen vaccines in the treatment of melanoma. We will explore their mechanism of action, the results of recent clinical trials, and the limitations and challenges of this new therapeutic approach.

Cell membranes are thought of as barriers to extracellular RNA (exRNA) uptake. While naked exRNAs can be spontaneously internalized by certain cells, functional cytosolic delivery has been rarely observed. Here, we show that extracellular ribonucleases (RNases)-primarily from cell culture supplements-have obscured the study of exRNA functionality. When ribonuclease inhibitor (RI) is added to cell cultures, naked exRNAs can trigger pro-inflammatory responses in dendritic cells and macrophages, largely via endosomal Toll-like receptors (TLRs). Moreover, naked exRNAs can escape endosomes, engaging cytosolic RNA sensors. In addition, naked extracellular mRNAs can be spontaneously internalized and translated by various cell types in an RI-dependent manner. In vivo, RI co-injection amplifies naked-RNA-induced activation of splenic lymphocytes and myeloid leukocytes. Furthermore, naked RNA is inherently pro-inflammatory in RNase-poor compartments like the peritoneal cavity. These findings demonstrate that naked RNA is bioactive without requiring vesicular encapsulation, making a case for nonvesicular-exRNA-mediated intercellular communication.

Aptamers, short oligonucleotide sequences that bind specifically to cellular proteins and receptors, are emerging as versatile tools in molecular nanomedicine. Unlike passive tumor targeting via the enhanced permeability and retention (EPR) effect, aptamers enable precise drug delivery, enhancing therapeutic efficacy while minimizing side effects. Developed through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, aptamers offer compact size, robust structure, chemical versatility, and cost-effective synthesis. They serve as effective delivery vehicles for therapeutic molecules, including miRNA, siRNA, and small-molecule drugs, and function as antibody-like ligands for applications in cancer, diabetes, and autoimmune disorders. Since the approval of Macugen, the first aptamer targeting VEGF, aptamers have also shown promise as diagnostic sensors and theranostic agents. This review explores SELEX-derived aptamers in nanomedicine, focusing on their therapeutic and diagnostic roles, particularly in precision cancer therapies. It also addresses challenges such as degradation and clinical translation alongside prospects in vaccines, tissue engineering, and regenerative medicine.

Lipid nanoparticles (LNPs) have shown promising performance in mRNA delivery. Nevertheless, a thorough understanding of the relationship between mRNA delivery efficacy and the structure of LNPs remains imperative. In this study, we systematically investigated the effects of additional hydrophobic amines on the physicochemical properties of mRNA LNPs and their delivery efficacy. The results indicated that this influence depended on the chemical structure of the additional amines and the structure of the lipid carriers. The appropriate addition of the hydrophobic amine 2C8 to lipid carriers with structural 2C8 or 2C6 tails significantly increased their mRNA delivery efficiency. In contrast, the addition of hydrophobic amine C18 to LNPs resulted in a decrease in mRNA delivery efficiency, while the addition of hydrophobic amines 2C6 and C8, as well as alkanes C12' and C16', had relatively little effect on mRNA delivery. Further investigations demonstrated that the appropriate addition of 2C8 could reduce LNP size, moderate internal hydrophobicity and LNP stability, facilitate mRNA release, enhance cellular uptake, and improve intracellular transportation of LNPs, thereby achieving superior mRNA delivery efficiency. These findings highlight the important role of additional hydrophobic amines in mRNA delivery with LNPs and provide valuable insights for the advancement of mRNA delivery carriers.

RNA therapeutics came to global attention when mRNA-based vaccines provided an answer to the SARS-CoV-2 pandemic. The immense significance of this development notwithstanding, it is important to note that almost a decade prior to the pandemic, RNA drugs had made important inroads toward the amelioration of disease. The first class of RNA therapies to be introduced into clinical use were the antisense oligomers and siRNA drugs which generally induce a therapeutic effect by acting to brake or to modulate mRNA expression. RNA therapeutics is quickly becoming the fourth pillar of pharmacotherapy, and will have broad applications, including for the treatment of cardiovascular disease.

The United States (US) Food and Drug Administration (FDA) has approved several antisense oligomers (ASOs) and siRNA-based drugs to treat disorders associated with cardiovascular disease. In addition, multiple RNA-based drugs are in clinical trials to assess their safety and efficacy in patients with cardiovascular disorders, such as Zodasiran, a siRNA therapy that targets angiopoietin-like protein 3 (ANGPTL3) to reduce LDL cholesterol.

Because of limitless sequence choice; speed of design; and relative ease of synthesis, RNA drugs will be rapidly developed, will have broad applications, and will be generated at lower cost than other drug types. This review aims to highlight RNA therapies for cardiovascular diseases that are approved, and those that are under clinical evaluation.

mRNA-based therapies have emerged as a transformative tool in modern medicine, gaining significant attention following their successful use in COVID-19 vaccines. Delivery to the lungs offers several compelling advantages for mRNA delivery. The lungs are one of the most vascularized organs in the body, which provides an extensive surface area that can facilitate efficient drug transport. Local delivery to the lungs bypasses gastrointestinal degradation, potentially enhancing therapeutic efficacy. In addition, the extensive capillary network of the lungs provides an ideal target for systemic delivery. However, developing effective mRNA therapies for the lungs presents significant challenges. The complex anatomy of the lungs and the body's immune response to foreign particles create barriers to delivery. This review discusses key approaches for overcoming these challenges and improving mRNA delivery to the lungs. It examines both local and systemic delivery strategies aimed at improving lung delivery while mitigating off-target effects. Although substantial progress has been made in lung-targeted mRNA therapies, challenges remain in optimizing cellular uptake and achieving therapeutic efficacy within pulmonary tissues. The continued refinement of delivery strategies that enhance lung-specific targeting while minimizing degradation is critical for the clinical success of mRNA-based pulmonary therapies.

Messenger RNA (mRNA) vaccines have emerged as one of the most promising and rapidly evolving immunotherapeutic approaches due to their ease of production, demonstrated clinical efficacy, and high safety. The coronavirus disease 2019(COVID-19) pandemic has showcased the remarkable therapeutic potential of mRNA vaccines, prompting researchers to explore their use for cancer treatment. Preclinical studies and human clinical trials have indicated their substantial clinical applicability. However, current research faces several challenges, including the complexity of tumor antigen selection, vaccine stability, and the development of resistance. This review summarizes the optimization strategies for cancer mRNA vaccines in preclinical settings, the progress of clinical trials, and the challenges encountered while analyzing various delivery vehicle types, infusion methods, and application cases across different cancer types, highlighting key factors in vaccine design. The findings demonstrate that mRNA vaccines elicit specific immune responses and exhibit favorable safety and tolerability in clinical trials. Moreover, developing personalized neoantigen vaccines offers a novel direction for cancer immunotherapy. The unique contribution of this review lies in its comprehensive overview of the latest advancements in therapeutic mRNA vaccines for cancer treatment while identifying critical areas for future research to propel the field forward.

The treatment of advanced prostate cancer (PCa) primarily based on androgen deprivation therapy (ADT); however, patients inevitably progress to the castration-resistant prostate cancer (CRPC) stage. Despite the recent advancements in CRPC treatment with novel endocrine drugs that further inhibit androgen receptor signaling, resistance ultimately develops, underscoring the urgent need for new effective therapeutic strategies. Therapeutic cancer vaccines, a form of immunotherapy, exert anti-cancer effects by activating the host's immune system. Over the past few decades, various conventional therapeutic PCa vaccines based on cells, microbes, proteins, peptides, or DNA have been developed and tested in patients with advanced PCa. These attempts have largely failed to improve survival, with the sole exception of sipuleucel-T, which extended the median overall survival of asymptomatic or minimally symptomatic metastatic CRPC (mCRPC) patients by four months. The rapid development and high efficacy of mRNA vaccines during the COVID-19 pandemic have garnered worldwide attention. Compared to conventional vaccines, mRNA vaccines offer several unique advantages, including high production efficiency, low cost, high safety, strong immune response induction, and high adaptability and precision. These attributes make mRNA vaccines a promising frontier in the treatment of advanced PCa.

Vaccinology has revolutionized modern medicine, delivering groundbreaking solutions to prevent and control infectious diseases while pioneering innovative strategies to tackle non-infectious challenges, including cancer. Traditional vaccines faced inherent limitations, driving the evolution of next-generation vaccines such as subunit vaccines, peptide-based vaccines, and nucleic acid-based platforms. Among these, nucleic acid-based vaccines, including DNA and mRNA technologies, represent a major innovation. Pioneering studies in the 1990s demonstrated their ability to elicit immune responses by encoding specific antigens. Recent advancements in delivery systems and molecular engineering have overcome initial challenges, enabling their rapid development and clinical success. This review explores nucleic acid-based vaccines, including chemically modified variants, by examining their mechanisms, structural features, and therapeutic potential, while underscoring their pivotal role in modern immunization strategies and expanding applications across contemporary medicine.

Glioma is the most common primary brain tumor, with glioblastoma being the most lethal type due to its heterogeneous and invasive nature of the cancer. Current therapies have low curative success and are limited to surgery, radiotherapy, and chemotherapy. More than 50% of patients become resistant to chemotherapy, and tumor recurrence occurs in most patients following an initial course of therapy. Therefore, developing novel, effective strategies for glioma treatment is essential. Cancer vaccines are novel therapies that demonstrate advantages over conventional methods and, therefore, may be promising options for treating glioma.

This article provided a critical review of pre-clinical and clinical studies that explored appropriate tumor antigen candidates for developing mRNA vaccines and discussed their clinical application in glioma patients. Medline database, PubMed, and ClinicalTrials.gov were searched for glioma vaccine studies published before 2025 using related keywords.

mRNA vaccines are promising strategies for treating glioma because they are efficient, cost-beneficial, and have lower side effects than other types such as peptide or DNA-based vaccines.

The selection of an effective delivery carrier is crucial to assessing mRNA-based vaccines and therapeutics in vivo. Although lipid nanoparticles (LNPs) are commonly used for mRNA delivery, the LNP-mRNA formulation process is laborious and time-consuming and requires a high-cost microfluidic device. Instead, mixing with commercial reagents may simplify mRNA transfection into cells. However, their potential as in vivo carriers in intramuscular vaccination in mouse models remains unclear. In this study, we used three types of commercial RNA transfection reagents, MessengerMAX (MAX; liposome), TransIT-mRNA (IT; cationic polymer), and Invivofectamine (IVF; LNP), to produce nanoparticles directly by pipetting. The particle characteristics and mRNA delivery efficacy of the mRNA-transfection reagent mixtures were analyzed. Additionally, immune responses to vaccine efficacy and protective immunity of the mRNA mixtures as vaccine antigens were evaluated in a mouse model. Although MAX and IT showed high in vitro transfection efficiencies, their in vivo performances were limited. In contrast, IVF exhibited notable particle stability and homogeneity, making it a promising delivery carrier. Intramuscular IVF injection significantly enhanced both innate and adaptive immune responses with a robust systemic protein expression. Notably, when using SARS-CoV-2 Spike mRNA, IVF showed robust humoral immune responses, including production of IgG and neutralizing antibodies, thereby resulting in complete protection against SARS-CoV-2 infection. Therefore, these findings position IVF as an accessible and efficient mRNA carrier for evaluating mRNA vaccines and therapeutic efficacy in basic research.

Immobilized metal ion affinity chromatography (IMAC) is an effective method developed in the 1980s for the separation and purification of proteins. The system consists of a solid-phase matrix, a linking ligand, and a metal ion. The method is based on the ability of metal ions to bind specifically to certain specific amino acid residues of proteins, thereby selectively enriching and purifying proteins.

This review aims to describe current knowledge of fundamental principle of IMAC and summarize the supports, chelating ligands, and metal ions of IMAC. In addition, how IMAC technology is used in proteomics and DNA research are highlighted.

Over the past decades, IMAC has been extensively utilized as a predominant technique for protein enrichment in a variety of biological and medical research, such as disease diagnosis, tumor biomarker identification, protein purification, and nucleic acid research. In the future, IMAC should be integrated with other proteomics technologies to promote the applications of metalloproteomes in disease diagnosis, metallodrug development and clinical translation.

Self-amplifying RNA is synthetic nucleic acid engineered to replicate within cells without generating viral particles. Derived from alphavirus genomes, saRNA retains the non-structural elements essential for replication while replacing the structural elements with an antigen of interest. By enabling efficient intracellular amplification, saRNA offers a promising alternative to conventional mRNA vaccines, enhancing antigen expression while requiring lower doses. However, this advantage comes with challenges. In this review, we highlight the key limitations of saRNA technology and explore potential strategies to overcome them. By identifying these challenges, we aim to provide insights that can guide the future design of saRNA-based therapeutics, extending their potential beyond vaccine applications.

Messenger RNA (mRNA)-based therapy has revolutionized cancer research by enabling versatile delivery systems for therapeutic applications. The future of mRNA-based cancer therapies shows promise amidst challenges such as delivery efficiency, immunogenicity, and tumor heterogeneity. Recent progress has adapted various strategies such as design flexibility, scalable production, and targeted delivery capabilities to enhance the potential in personalized cancer therapy. Further research to optimize delivery for enhanced outcomes and efficacy in solid tumors is warranted. Therefore, we aim to explore the current landscape and future prospects of mRNA technology across various therapeutic platforms.

Over the past 20 years, intensive research has been conducted on the development of therapeutic mRNA, leading to numerous discoveries that have enabled its use in therapy. The main achievements in this field include increasing mRNA stability, reducing its immunogenicity (i.e., its ability to trigger an immune response), and solving the challenge of delivering mRNA into cells-all to achieve a therapeutic effect. The aim of this study was to review the scientific literature on the use of mRNA technology in the production of vaccines. Various methods of applying mRNA technology that could potentially be introduced into clinical practice in the future are described. A detailed analysis was conducted on the approved COVID-19 vaccines developed by Pfizer/BioNTech (New York, NY, USA) and Moderna (Kirkland, QC, Canada), as their introduction marked a groundbreaking moment in the advancement of mRNA technology. This study was based on the latest scientific literature from reputable publishers and medical databases such as PubMed and ClinicalTrials. In conclusion, mRNA technology is currently experiencing rapid development, significantly driven by the ongoing COVID-19 pandemic. The application of this technology holds great potential not only for vaccines against infectious diseases but also for cancer treatment. However, further research is necessary to facilitate its broader clinical implementation.

Prostate cancer has the second highest cancer mortality rate in the UK in males. Early prostate cancer is typically asymptomatic, with diagnosis at a locally advanced or metastatic stage. In addition, the inherent heterogeneity of prostate cancer tumours differs significantly in terms of genetic, molecular, and histological features. The successful treatment of prostate cancer is therefore exceedingly challenging. Immunotherapies, particularly therapeutic vaccines, have been widely used in preclinical and clinical studies to treat various cancers. Sipuleucel-T was the first cancer vaccine approved by the FDA for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), ushering in a new era of immunotherapy. In this review, the latest immunotherapy strategies for prostate cancer are considered with key tumour-associated antigens (TAA) and tumour-specific antigens (TSA) highlighted. The key components of mRNA vaccines include in vitro transcription, stability, and immunogenicity. Finally, strategies to circumvent in vivo mRNA degradation and approaches to optimise in vitro transcription (IVT) process are also discussed.

DNA vaccines are a novel form of vaccination that aims to harness genetic material to produce targeted immune responses. Nevertheless, their therapeutic application is hampered by low transfection efficacy, immunogenicity, and instability. Nanoparticle (NP) - based delivery systems are beneficial in enhancing DNA stability, increasing DNA uptake by antigen-presenting cells (APCs), and controlling antigen release. Some key progress includes the polymeric, lipid-based, and hybrid NPs and biocompatible carriers with inherent adjuvant effects. These systems have helped to enhance the antigen cross-presentation and T-cell activation significantly. In addition, biocompatible hybrid nanocarriers, antigen cross-presentation strategies, and next-generation sequencing (NGS) technologies are speeding up the identification of new antigens, while AI and machine learning are facilitating the development of efficient delivery systems. This review aims to assess how NPs have contributed to improving the effectiveness of DNA vaccines for treating diseases, cancer, and emerging diseases, as well as advancing the next generation of DNA vaccines.

mRNA technology has been successfully deployed to rapidly develop and mass-manufacture vaccines. Beyond vaccines, RNA-based therapeutics have potential for treatments for infectious diseases, cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. mRNA based vaccines and therapeutics work by translating exogenous mRNA into the target protein. Analytical methods for mRNA characterisation, lot release and stability testing of mRNA drug substance and drug product must be developed and performed to monitor critical quality attributes (CQAs). mRNA is a highly polar molecule due to its extensive negatively charged phosphodiester backbone. Its single stranded nature forms dynamic alternative secondary structures that can generate potential sample heterogeneity, creating challenges for the analysis and characterisation of this large biomolecule. In this review, we describe current analytical methods, focussing on high performance liquid chromatography in conjunction with both UV detection and mass spectrometry for the analysis and characterisation of mRNA. In particular, we describe recent developments covering a wide range of methods centred on liquid chromatography for the analysis of important CQAs including mRNA identity, mRNA integrity, 5' capping efficiency and poly(A) tail length and heterogeneity.

The rapid success of messenger (m) RNA vaccines against COVID-19 has pushed the mRNA to the forefront of drug research. The promise of mRNA-based therapeutics and vaccines in other areas is not new but is now emerging stronger. We review basic concepts, key historical aspects, and recent research on mRNA as a therapeutic molecule to fight infectious diseases and cancer. We also show a current patent perspective of this field. Altogether, we describe that the technology of mRNA as a therapeutic molecule is a rapidly moving field aiming for a bright future.

Autoimmune diseases (ADs) are a group of complex, chronic conditions characterized by disturbance of immune tolerance, with examples including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and psoriasis. These diseases have unclear pathogenesis, and traditional therapeutic approaches remain limited. However, advances in high-throughput histology technology and scientific discoveries have led to the identification of various pathogenic factors contributing to ADs. Coupled with improvements in RNA nucleic acid-based drug synthesis, design, and delivery, RNA-based therapies have been extensively investigated for their potential in treating ADs. This paper reviews the progress in the use of miRNAs, lncRNAs, circRNAs, siRNAs, antisense oligonucleotides (ASOs), aptamers, mRNAs, and other RNA-based therapies in ADs, focusing on their therapeutic potential and application prospects, providing insights for future research and clinical treatment of autoimmune diseases.

Advances in molecular medicine and biotechnology have demonstrated messenger RNA (mRNA)-based therapies to be a promising therapeutic modality for infectious diseases, genetic disorders, and cancers. However, key challenges persist, including low translation efficiency and short half-life of exogenous mRNA. The untranslated regions (UTRs) influence important parameters like mRNA stability and translation efficiency. This study adopted a combinatorial screening strategy to enhance exogenous mRNA translation efficiency by de novo designing 5'UTRs and combining them with multiple potential 3'UTRs. We designed a novel 5'UTR, 5UTR05, which exhibited comparable protein expression levels to the reference mRNA-1273 5'UTR that has been found to exhibit high expression in the COVID-19 vaccine development. Furthermore, the screening experiments found that combining 5UTR05 with both the 3'UTR of immunoglobulin heavy constant gamma 2 (IGHG2) and the 3'UTR of mitochondrially encoded 12S ribosomal RNA (mtRNR1) significantly improved mRNA translation efficiency, compared to individual 3'UTRs. Collectively, these findings provide valuable insights for UTR optimization strategies aimed at augmenting exogenous mRNA therapeutic translation. Continuing exploration of synergistic UTR combinations offers promise to advance customized mRNA constructs with optimized expression profiles tailored for diverse applications.

The 2023 annual scientific meeting of the French Society of Toxicologic Pathology (Société Française de Pathologie Toxicologique, SFPT), entitled "mRNA-based technologies: preclinical development and therapeutic applications," was held in Lyon (France) on May 25 to 26, 2023. The aim of the meeting was to discuss the biology, immunology, and preclinical development of messenger RNA (mRNA)-based vaccines and therapeutics, including immuno-oncology and rare diseases, as well as the regulatory aspect of the COVID-19 vaccines and an overview of the principles and applications of in situ hybridization techniques. This article presents the summary of five lectures along with selected figures, tables, and key literature references on this topic.

Intramuscular injection of plasmid DNA (pDNA) is a promising approach for gene therapy, but its efficiency is hindered by both extracellular and intracellular barriers. The extracellular matrix (ECM), including collagens and nucleases, obstructs pDNA penetration, while intracellular challenges include crossing the plasma membrane, escaping endosomes, and reaching the nucleus. Though non-viral carriers like polymers and cationic lipids have been developed, they often fail to address both barriers simultaneously, leading to poor gene transfer in vivo. Physical methods exist but may damage tissues and cause patient discomfort. Here, we introduce a Pluronic L64-Cupping (L/C) gene delivery system that enhances pDNA delivery by sequentially overcoming ECM diffusion, membrane permeabilization, and intracellular transfection. After intramuscular injection of the pDNA-Pluronic L64 mixture, negative pressure is applied to the injection site, significantly boosting reporter gene expression and sustaining it for at least 42 days. Additionally, this system effectively induces HBsAb production in mice, offering a safe, efficient, and cost-effective platform for both laboratory and clinical gene therapy applications.

Radiotherapy remains a cornerstone of cancer treatment, using high-energy radiation to induce DNA damage in tumor cells, leading to cell death. However, its efficacy is often hindered by challenges such as radiation resistance and side effects. As a powerful class of functional molecules, nucleic acid drugs (NADs) present a promising solution to these limitations. Engineered to target key pathways like DNA repair and tumor hypoxia, NADs can enhance radiotherapy sensitivity. NADs can also serve as delivery vehicles for radiotherapy agents such as radionuclides, improving targeting accuracy and minimizing side effects. This review explores the role of NADs in optimizing radiotherapy, highlighting their mechanisms, clinical applications, and synergies with radiotherapy, ultimately offering a promising strategy for improving patient outcomes in cancer therapy.

Nucleic acid therapeutics have become increasingly recognized in recent years for their capability to target both coding and non-coding sequences. Several types of nucleic acid modalities, including siRNA, mRNA, aptamer, along with antisense oligo, have been approved by regulatory bodies for therapeutic use. The field of nucleic acid therapeutics has been brought to the forefront by the rapid development of vaccines against COVID-19, followed by a number of approvals for clinical use including much anticipated CRISPR-Cas9. However, obstacles such as the difficulty of achieving efficient and targeted delivery to diseased sites remain. This review provides an overview of nucleic acid therapeutics and highlights substantial advancements, including critical engineering, conjugation, and delivery strategies, that are paving the way for their growing role in modern medicine.

Immunotherapy can treat cancer by boosting the immune system to mark and destroy cancer cells. Cancer vaccine is a promising therapeutic strategy in immunotherapy. Cancer vaccines are divided into four groups according to different preparation techniques: cell-based vaccine, virus-based vaccine, peptide-based vaccine, and nucleic acid-based vaccine. Cancer vaccines can be given with traditional treatments or another immunotherapy to give better results and overcome tumor resistance. The cancer vaccine is a promising immunotherapy that could stimulate the immune response to kill cancer cells and create immune surveillance. However, much work is still needed to identify neoantigens, optimize the vaccination platform, and develop combination therapy to improve the efficacy of immunotherapy. This review highlights the mechanism of action of cancer vaccines, the main four groups of cancer vaccines regarding their development, research progress, and clinical applications, and how to assess immune response following cancer vaccination.

The development of efficient and targeted methods for delivering DNA in vivo has long been a major focus of research. In this study, we introduce a gene delivery approach admitted by small metabolites (gDAM) for the efficient and targeted delivery of naked DNA into astrocytes in the adult brains of mice. gDAM uses a straightforward combination of DNA and small metabolites, including glycine, L-proline, L-serine, L-histidine, D-alanine, Gly-Gly, and Gly-Gly-Gly, to achieve astrocyte-specific delivery of naked DNA, resulting in transient and robust gene expression in these cells. Using gDAM, we successfully co-deliver the PiggyBac transposon and the CRISPR-Cas9 system to induce long-term overexpression of the oncogene EGFRvIII and knockout of tumor suppressor genes Nf1, Pten, and Trp53 in astrocytes, leading to the development of astrocyte-derived gliomas in immunocompetent mice. Furthermore, gDAM facilitates the delivery of naked DNA to peripheral glioma astrocytes. The overexpression of interferon-β and granulocyte-macrophage colony-stimulating factor in these peripheral glioma astrocytes significantly prolongs the overall survival of mice bearing 73C glioma cells. This approach offers a new perspective on developing gene delivery systems that specifically target astrocytes to meet the varied needs of both research and gene therapy. The innovative strategy behind gDAM is expected to provide fresh inspiration in the quest for DNA delivery to other tissues, such as skeletal muscle and skin.

mRNA vaccines utilize single-stranded linear DNA as a template for in vitro transcription. The mRNA is introduced into the cytoplasm via the corresponding delivery system to express the target protein, which then performs its relevant biological function. mRNA vaccines are beneficial in various fields, including cancer vaccines, infectious disease vaccines, protein replacement therapy, and treatment of rare diseases. They offer advantages such as a simple manufacturing process, a quick development cycle, and ease of industrialization. Additionally, mRNA vaccines afford flexibility in adjusting antigen designs and combining sequences of multiple variants, thereby addressing the issue of frequent mutations in pathogenic microorganisms. This paper aims to provide an extensive review of the global development and current research status of mRNA vaccines, with a focus on immunogenicity, classification, design, delivery vector development, stability, and biomedical application. Moreover, the study highlights current challenges and offers insights into future directions for development.

Over the last decades, messenger RNA (mRNA) has emerged as a promising therapeutic modality, enabling the delivery of genetic instructions to cells for producing therapeutic proteins or antigens. As such, mRNA-based therapies can be developed for a wide range of conditions, including infections, cancer, metabolic disorders, and genetic diseases. Nevertheless, using mRNA therapeutically requires chemical modifications to reduce immunostimulatory effects and nanotechnology to prevent degradation and ensure intracellular delivery. Lipid nanoparticles (LNPs) have become the most effective delivery platform for mRNA therapeutics, which are primarily employed for vaccine purposes following local administration and hepatic applications following systemic administration. Here, we review the state-of-the-art LNP-mRNA technology and discuss its potential for immunotherapy. We first outline the requirements for mRNA to be used therapeutically, including the role of LNP-mediated delivery. Next, we highlight LNP-mRNA immunotherapy approaches for vaccination, immuno-oncology, and autoimmune disorders. In addition, we discuss challenges that are limiting LNP-mRNA's widespread use, including tunable biodistribution and immunostimulatory effects. Finally, we provide an outlook on how implementing approaches such as library screening and machine learning will guide the development of next-generation mRNA therapeutics.

Directed evolution in mammalian cells offers a powerful approach for advancing synthetic biology applications. However, existing mammalian-based directed evolution methods face substantial bottlenecks, including host genome interference, small library size and uncontrolled mutagenesis. Here we engineered an orthogonal alphaviral RNA replication system to evolve RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells. This system generates a large, continuously diversified library of replicative RNAs through replicase-limited mode of replication and inducible mutagenesis. Using REPLACE, we engineered fluorescent proteins and transcription factors. Notably, cells equipped with REPLACE can undergo Darwinian adaptation, allowing them to evolve in response to both cell-extrinsic and cell-intrinsic challenges. Collectively, this work establishes a powerful platform for advancing mammalian synthetic biology and cell engineering applications through directed evolution.

mRNA technology has significantly changed the timeline for developing and delivering a new vaccine from years to months, as demonstrated by the development and approval of two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. Analytical methods are required to characterise mRNA therapeutics and underpin manufacturing development. In this study, we have developed and utilised partial RNase digests of mRNA using RNase T1 and RNase U2 in conjunction with an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of mRNA therapeutics. In conjunction with this, we have developed novel software engineered to optimise and streamline the visualisation and analysis of sequence mapping of mRNA using LC-MS/MS. We show that increased mRNA sequence coverage is obtained by combining multiple partial RNase T1 digests-44% and 37% individually, 64% together-or RNase T1 and U2 partial digests-73% and 52% individually, 88% combined. The developed software automates the process of combining digests, ensuring faster and more accurate analysis. Furthermore, the software provides additional information on sequence coverage by taking into account multiple overlapping oligoribonucleotide fragments to increase the confidence of the sequence mapping. Finally, the software enables powerful and accessible visualisation capabilities by generating spiral plots to quickly analyse the sequence maps in a single output from combined multiple partial RNase digests.

Ageing is a degenerative process characterised by a decline in physiological functioning of the organism. One of the core regulators of cellular ageing are telomeres, repetitive DNA sequences of TTAGGG that cap the ends of chromosomes and are maintained by the ribonucleoprotein complex, telomerase. Age-dependent progressive loss of the telomere ends eventually induces cell cycle arrest for the induction of either replicative senescence or apoptosis. It was previously established that overexpression of the 37 kDa/ 67 kDa laminin receptor (LRP/LR) increased telomerase activity and telomere length while concomitantly reducing senescence markers in aged normal cells in vitro. Therefore, it was hypothesized that elevating LRP/LR in vivo may increase telomerase activity and hinder the ageing process on an organism scale. To this end, aged C57BL/6J mice were treated/transfected to induce an overexpression of LRP::FLAG. Various physiological tests and histological analyses were performed to assess overall organism fitness as well as to discern the treatments' ability at reducing tissue degeneration and atrophy. It was found that mice overexpressing LRP::FLAG displayed improved physiological characteristics and markedly less tissue degeneration and atrophy when compared to control and non-treated mice. Alongside these improvements, certain organs displayed increased telomerase activity with a corresponding elongation in average telomere length. In addition the overexpression of LRP::FLAG significantly improved various proliferative and anti-ageing associated proteins while causing a concomitant decrease in senescence associated proteins. These findings are therefore indicative of a novel function of LRP/LR delaying the onset of senescence, while also promoting healthier ageing through elevating TERT and telomerase activity.

Over the last decade, mRNA vaccines development has shown significant advancement, particularly during the COVID-19 pandemic. This comprehensive review examines the efficacy of pivotal vaccines against emerging COVID-19 variants and strategies for enhancing vaccine effectiveness. It also explores the versatility of mRNA technology in addressing other infectious diseases such as influenza, respiratory syncytial virus, HIV, cytomegalovirus, Ebola, Zika, Rabies, and Nipah viruses. The analysis includes safety and clinical progress of mRNA vaccines and evaluates their potential in combination vaccine strategies. Additionally, it addresses challenges related to delivery and scalability while highlighting opportunities for future advancements in the field. Recent advances in mRNA optimization, biomaterial-based delivery and thermostable designs offer promising solutions. It is essential to gain insights into the evolving landscape of mRNA vaccine technology to maximize its vital role in addressing diverse viral threats, advancing vaccinology and enhancing public health preparedness for future pandemic.

Biological Standardization has been pivotal to the success of traditional biological products, such as vaccines, antitoxins, and immune globulins, by ensuring their quality and consistency across manufacturers worldwide. The principles of biological standardization have similarly supported the development and manufacture of safe and effective modern biological products, including hormone, therapeutic protein, and monoclonal antibody products, and continue to play a vital role in advancing new cutting-edge biological products, such as tissue, cellular, and gene-therapy products. Biological standardization started with the physical standards ensuring the reliability and suitability of methods used to test biological products and science of bioassays or biological methods and related biostatistics providing a framework for evaluating biological, functional activity or potency of these products. It expanded to include written standards defining the quality requirements for manufacturing and regulation of biological standards. Due to the shift in the biologics industry from public health to commercial-driven enterprises during the past 50 years, the biological standardization program has evolved to include the product-specific reference standards and harmonization of physical standards. The global success of conventional vaccines in controlling numerous deadly infectious diseases can largely be attributed to the availability of physical and written international standards developed through a strong biological standardization program. This article explores the evolution of biological standardization for more than a century, its scientific and regulatory principles, challenges from disruption in international standardization efforts, and future perspectives for the field.

The development of an effective HIV-1 vaccine remains a formidable challenge in biomedical research. Despite significant advancements in our understanding of HIV biology and pathogenesis, progress has been impeded by factors such as the virus's genetic diversity, high mutation rates, and its ability to establish latent reservoirs. Recent innovative approaches, including mosaic vaccines and mRNA technology to induce broadly neutralizing antibodies, have shown promise. However, the efficacy of these vaccines has been modest, with the best results achieving approximately 30% effectiveness. Ongoing research emphasizes the necessity of a multifaceted strategy to overcome these obstacles and achieve a breakthrough in HIV-1 vaccine development. This review summarizes current approaches utilized to further understand HIV-1 biology and to create a global vaccine. We discuss the impact of these approaches on vaccine development for other diseases, including COVID-19, influenza, and Zika virus. Additionally, we highlight the specific limitations faced with each approach and present the methods researchers employ to overcome these challenges. These innovative techniques, which have demonstrated preclinical and clinical success, have advanced the field closer to the ultimate goal of developing a global HIV-1 vaccine. Leveraging these advancements will enable significant strides in combating HIV-1 and other infectious diseases, ultimately improving global health outcomes.

Supercoiled (Sc) circular DNA, such as plasmids, has shown therapeutic potential since the 1990s, but is limited by bacterial modifications, unnecessary DNA sequences, and contaminations that may trigger harmful responses. To overcome these challenges, we have developed two novel scalable biochemical methods to synthesize unmodified Sc circular DNA. Linear DNA with two loxP sites in the same orientation is generated via PCR or rolling circle amplification. Cre recombinase then converts this linear DNA into relaxed circular DNA. After T5 exonuclease removes unwanted linear DNA, topoisomerases are employed to generate Sc circular DNA. We have synthesized EGFP-FL, a 2,002 bp mini-circular DNA carrying essential EGFP expression elements. EGFP-FL transfected human HeLa and mouse C2C12 cells with much higher efficiency than E. coli-derived plasmids. These new biochemical methods can produce unmodified Sc circular DNA, in length from 196 base pairs to several kilobases and in quantities from micrograms to milligrams, providing a promising platform for diverse applications.

Nucleic acid vaccines have emerged as crucial advancements in vaccine technology, particularly highlighted by the global response to the COVID-19 pandemic. The widespread administration of mRNA vaccines against COVID-19 to billions globally marks a significant milestone. Furthermore, the approval of an mRNA vaccine for Respiratory Syncytial Virus (RSV) this year underscores the versatility of this technology. In oncology, the combination of mRNA vaccine encoding neoantigens and immune checkpoint inhibitors (ICIs) has shown remarkable efficacy in eliciting protective responses against diseases like melanoma and pancreatic cancer. Although the use of a COVID-19 DNA vaccine has been limited to India, the inherent stability at room temperature and cost-effectiveness of DNA vaccines present a viable option that could benefit developing countries. These advantages may help DNA vaccines address some of the challenges associated with mRNA vaccines. Currently, several trials are exploring the use of DNA-encoded neoantigens in combination with ICIs across various cancer types. These studies highlight the promising role of nucleic acid-based vaccines as the next generation of immunotherapeutic agents in cancer treatment. This review will delve into the recent advancements and current developmental status of both mRNA and DNA-based cancer vaccines.

Following the coronavirus disease 2019 (COVID-19) pandemic, mRNA technology has made significant breakthroughs, emerging as a potential universal platform for combating various diseases. To address the challenges associated with mRNA delivery, such as instability and limited delivery efficacy, continuous advancements in genetic engineering and nanotechnology have led to the exploration and refinement of various mRNA structural modifications and delivery platforms. These achievements have significantly broadened the clinical applications of mRNA therapies. Despite the progress, the understanding of the interactions in mRNA delivery systems remains limited. These interactions are complex and multi-dimensional, occurring between mRNA and vehicles as well as delivery materials and helper ingredients. Resultantly, stability of the mRNA delivery systems and their delivery efficiency can be both significantly affected. This review outlines the current state of mRNA delivery strategies and summarizes the interactions in mRNA delivery systems. The interactions include the electrostatic interactions, hydrophobic interactions, hydrogen bonding, π-π stacking, coordination interactions, and so on. This interaction understanding provides guideline for future design of next-generation mRNA delivery systems, thereby offering new perspectives and strategies for developing diverse mRNA therapeutics.

Due to their widespread geographic distribution and frequent outbreaks, mosquito-borne flaviviruses, such as DENV (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and West Nile virus (WNV), are considered significant global public health threats and contribute to dramatic socioeconomic imbalances worldwide. The global prevalence of these viruses is largely driven by extensive international travels and ecological disruptions that create favorable conditions for the breeding of Aedes and Culex species, the mosquito vectors responsible for the spread of these pathogens. Currently, vaccines are available for only DENV, YFV, and JEV, but these face several challenges, including safety concerns, lengthy production processes, and logistical difficulties in distribution, especially in resource-limited regions, highlighting the urgent need for innovative vaccine approaches. Nucleic acid-based platforms, including DNA and mRNA vaccines, have emerged as promising alternatives due to their ability to elicit strong immune responses, facilitate rapid development, and support scalable manufacturing. This review provides a comprehensive update on the progress of DNA and mRNA vaccine development against mosquito-borne flaviviruses, detailing early efforts and current strategies that have produced candidates with remarkable protective efficacy and strong immunogenicity in preclinical models. Furthermore, we explore future directions for advancing nucleic acid vaccine candidates, which hold transformative potential for enhancing global public health.

Immuno-oncology has witnessed remarkable advancements in the past decade, revolutionizing the landscape of cancer therapeutics in an encouraging manner. Among the diverse immunotherapy strategies, mRNA vaccines have ushered in a new era for the therapeutic management of malignant diseases, primarily due to their impressive impact on the COVID-19 pandemic. In this comprehensive review, we offer a systematic overview of mRNA vaccines, focusing on the optimization of structural design, the crucial role of delivery materials, and the administration route. Additionally, we summarize preclinical studies and clinical trials to provide valuable insights into the current status of mRNA vaccines in cancer treatment. Furthermore, we delve into a systematic discussion on the significant challenges facing the current development of mRNA tumor vaccines. These challenges encompass both intrinsic and external factors that are closely intertwined with the successful application of this innovative approach. To pave the way for a more promising future in cancer treatments, a deeper understanding of immunological mechanisms, an increasing number of high-quality clinical trials, and a well-established manufacturing platform are crucial. Collaborative efforts between scientists, clinicians, and industry engineers are essential to achieving these goals.

The COVID-19 pandemic has spotlighted the potential of in vitro transcribed (IVT) mRNA vaccines with their demonstrated efficacy, safety, cost-effectiveness, and rapid manufacturing. Numerous IVT mRNA vaccines are now under clinical trials for a range of targets, including infectious diseases, cancers, and genetic disorders. Despite their promise, IVT mRNA vaccines face hurdles such as limited expression levels, nonspecific targeting beyond the liver, rapid degradation, and unintended immune activation. Overcoming these challenges is crucial to harnessing the full therapeutic potential of IVT mRNA vaccines for global health advancement. This review provides a comprehensive overview of the latest research progress and optimization strategies for IVT mRNA molecules and delivery systems, including the application of artificial intelligence (AI) models and deep learning techniques for IVT mRNA structure optimization and mRNA delivery formulation design. We also discuss recent development of the delivery platforms, such as lipid nanoparticles (LNPs), polymers, and exosomes, which aim to address challenges related to IVT mRNA protection, cellular uptake, and targeted delivery. Lastly, we offer insights into future directions for improving IVT mRNA vaccines, with the hope to spur further progress in IVT mRNA vaccine research and development.

Nucleic acid-based vaccines are leading-edge tools in developing next-generation preventative care. Much research has been done to convert vaccine gene therapy from an invasive to a noninvasive administration approach. The lung's large surface area and permeability make the pulmonary route a promising noninvasive delivery option for vaccines, with systemic and local applications. This review summarizes the challenges and the approaches that have been carried out to optimize the delivery of nucleic acids through the pulmonary route for vaccination purposes in recent years, with a spotlight on gold nanoparticles (AuNPs). Nonviral delivery systems have been widely explored, and AuNPs with their unique properties are emerging as promising tools for nucleic acid vaccines due to surface functionalization with mucus-penetrating polymers and targeting moieties that can bypass the barriers in pulmonary delivery and successfully deliver nucleic acids to the cells of interest. However, while promising, several challenges remain including selectively overcoming the lungs' immunological surveillance and adhesive mucus.

White spot syndrome virus (WSSV) causes highly destructive infection in crustacean aquaculture, often resulting in 100% mortality within a week. However, there is lack of studies addressing the safety issues of WSSV vaccines in shrimps. In this study, WSSV VP28 mRNA vaccines were developed using codon deoptimization approach. These vaccines were administered to Litopenaeus vannamei shrimps at various dosages to access their safety and the shrimps' immune responses using quantification PCR (qPCR). The findings of this study indicate that the expression level of codon deoptimized VP28 mRNA vaccines are lower compared to the wild type VP28 vaccines, as observed through a comparison of bioinformatic predictions and experimental results. Additionally, the total haemocyte count (THC) in shrimps injected with codon deoptimized VP28 vaccine was higher than those injected with wild type VP28 vaccines. Furthermore, the expression of immune-related genes differed between codon deoptimized and wild type VP28 vaccines. In summary, the results suggest that 0.01 μg codon deoptimized VP28-D1 mRNA vaccine is the most promising WSSV mRNA vaccine, displaying low pathogenicity and expression in shrimps. To the best of our knowledge, this research represents the first attempt to attenuate WSSV using codon deoptimization method and development of a potential mRNA vaccine for shrimp purpose. The study addresses an important gap in shrimp vaccine research, offering potential solutions for WSSV control in shrimps.