Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction.

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.

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.

Monogenic disorders are a group of human diseases caused by mutations in single genes. While some disease-altering treatments offer relief and slow the progression of certain conditions, the majority of monogenic disorders still lack effective therapies. In recent years, gene therapy has appeared as a promising approach for addressing genetic disorders. However, despite advancements in gene manipulation tools and delivery systems, several challenges remain unresolved, including inefficient delivery, lack of sustained expression, immunogenicity, toxicity, capacity limitations, genomic integration risks, and limited tissue specificity. This review provides an overview of the plasmid-based gene therapy techniques and delivery methods currently employed for monogenic diseases, highlighting the challenges they face and exploring potential strategies to overcome these barriers.

CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.

Bladder cancer (BC) accounts for about 4% of all malignancies. Non-muscle-invasive BC, 75% of cases, is treated with transurethral resection and adjuvant intravesical instillation, while muscle-invasive BC warrants cisplatin-based perioperative chemotherapy. Although immune-checkpoint inhibitors, antibody drug conjugates and targeted agents have provided dramatic advances, metastatic BC remains a generally incurable disease and clinical trials continue to vigorously evaluate novel molecules. Cancer vaccines aim at activating the patient's immune system against tumor cells. Several means of delivering neoantigens have been developed, including peptides, antigen-presenting cells, virus, or nucleic acids. Various improvements are constantly being explored, such as adjuvants use and combination strategies. Nucleic acids-based vaccines are increasingly gaining attention in recent years, with promising results in other malignancies. However, despite the recent advantages, numerous obstacles persist. This review is aimed at describing the different types of cancer vaccines, their evaluations in UC patients and the more recent innovations in this field.

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

RNA therapeutics, such as mRNA, siRNA, and CRISPR-Cas9, present exciting avenues for treating diverse diseases. However, their potential is commonly hindered by vulnerability to degradation and poor cellular uptake, requiring effective delivery systems. Lipid nanoparticles (LNPs) have emerged as a leading choice for in vivo RNA delivery, offering protection against degradation, enhanced cellular uptake, and facilitation of endosomal escape. However, LNPs encounter numerous challenges for targeted RNA delivery in vivo, demanding advanced particle engineering, surface functionalization with targeting ligands, and a profound comprehension of the biological milieu in which they function. This review explores the structural and physicochemical characteristics of LNPs, in-vivo fate, and customization for RNA therapeutics. We highlight the quality-by-design (QbD) approach for targeted delivery beyond the liver, focusing on biodistribution, immunogenicity, and toxicity. In addition, we explored the current challenges and strategies associated with LNPs for in-vivo RNA delivery, such as ensuring repeated-dose efficacy, safety, and tissue-specific gene delivery. Furthermore, we provide insights into the current clinical applications in various classes of diseases and finally prospects of LNPs in RNA therapeutics.

Cell death is a central process for organismal health. Pyroptosis, namely pyroptotic cell death, is recognized as a critical type that disrupts membrane and triggers pro-inflammatory cytokine secretion via gasdermins, providing a robust form of cytolysis. Meanwhile, along with the thorough research, a great deal of evidence has demonstrated the dual effects of pyroptosis in host defense and inflammatory diseases. More importantly, the recent identification of abundant gasdermin-like proteins in bacteria and fungi suggests an ancient origin of pyroptosis-based regulated cell death in the life evolution. In this review, we bring a general overview of pyroptosis pathways focusing on gasdermin structural biology, regulatory mechanisms, and recent progress in induction and inhibition strategies for disease treatment. We look forward to providing an insightful perspective for readers to comprehend the frame and challenges of the pyroptosis field, and to accelerating its clinical application.

Originally devised for cancer control, mRNA vaccines have risen to the forefront of medicine as effective instruments for control of infectious disease, notably their pivotal role in combating the COVID-19 pandemic. This review focuses on fundamental aspects of the development of mRNA vaccines, e.g., tumor antigens, vector design, and precise delivery methodologies, - highlighting key technological advances. The recent, promising success of personalized mRNA vaccines against pancreatic cancer and melanoma illustrates the potential value for other intractable, immunologically resistant, solid tumors, such as glioblastoma, as well as the potential for synergies with a combinatorial, immunotherapeutic approach. The impact and progress in human cancer, including pancreatic cancer, head and neck cancer, bladder cancer are reviewed, as are lessons learned from first-in-human CAR-T cell, DNA and dendritic cell vaccines targeting glioblastoma. Going forward, a roadmap is provided for the transformative potential of mRNA vaccines to advance cancer immunotherapy, with a particular focus on the opportunities and challenges of glioblastoma. The current landscape of glioblastoma immunotherapy and gene therapy is reviewed with an eye to combinatorial approaches harnessing RNA science. Preliminary preclinical and clinical data supports the concept that mRNA vaccines could be a viable, novel approach to prolong survival in patients with glioblastoma.

Immunotherapy has revolutionized cancer management, with antibody-based treatments leading the charge due to their superior pharmacodynamics, including enhanced effectiveness and specificity. However, these therapies are hampered by limitations such as prolonged half-lives, poor tissue and tumor penetration, and minimal oral bioavailability. Additionally, their immunogenic nature can cause adverse effects. Consequently, the focus is shifting towards small-molecule-based immunotherapies, which potentially overcome these drawbacks. Emerging as a promising alternative, small molecules offer the benefits of therapeutic antibodies and immunomodulators, often yielding synergistic effects when combined. Recent advancements in small-molecule cancer immunotherapy are notable, featuring inhibitors, agonists, and degraders that act as immunomodulators. This article delves into the current landscape of small-molecule immunotherapy in cancer treatment, highlighting novel agents targeting key pathways such as Toll-like receptors (TLR), PD-1/PD-L1, chemokine receptors, and stimulators of interferon genes (STING). The review emphasizes newly discovered molecular entities and their modulatory roles in tumorigenesis, many of which have progressed to clinical trials, that aims to provide a comprehensive snapshot of the evolving frontier in cancer treatment, driven by small-molecule immunomodulators.

Vaccination has proven to be an effective means of controlling pathogens in animals. Since the introduction of veterinary vaccines in the 19th century, several generations of vaccines have been introduced. These vaccines have had a positive impact on global animal health and production. Despite, the success of veterinary vaccines, there are still some pathogens for which there are no effective vaccines available, such as African swine fever. Further, animal health is under the constant threat of emerging and re-emerging pathogens, some of which are zoonotic and can pose a threat to human health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the need for new vaccine platforms that are safe and efficacious, but also importantly, are adaptable and can be modified rapidly to match the circulating pathogens. mRNA vaccines have been shown to be an effective vaccine platform against various viral and bacterial pathogens. This review will cover some of the recent advances in the field of mRNA vaccines for veterinary species. Moreover, various mRNA vaccines and their delivery methods, as well as their reported efficacy, will be discussed. Current limitations and future prospects of this vaccine platform in veterinary medicine will also be discussed.

Traditional therapeutic approaches such as chemotherapy and radiation therapy have burdened cancer patients with onerous physical and psychological challenges. Encouragingly, the landscape of tumor treatment has undergone a comprehensive and remarkable transformation. Emerging as fervently pursued modalities are small molecule targeted agents, antibody-drug conjugates (ADCs), cell-based therapies, and gene therapy. These cutting-edge treatment modalities not only afford personalized and precise tumor targeting, but also provide patients with enhanced therapeutic comfort and the potential to impede disease progression. Nonetheless, it is acknowledged that these therapeutic strategies still harbour untapped potential for further advancement. Gaining a comprehensive understanding of the merits and limitations of these treatment modalities holds the promise of offering novel perspectives for clinical practice and foundational research endeavours. In this review, we discussed the different treatment modalities, including small molecule targeted drugs, peptide drugs, antibody drugs, cell therapy, and gene therapy. It will provide a detailed explanation of each method, addressing their status of development, clinical challenges, and potential solutions. The aim is to assist clinicians and researchers in gaining a deeper understanding of these diverse treatment options, enabling them to carry out effective treatment and advance their research more efficiently.

Cancer remains one of the global leading causes of death and various vaccines have been developed over the years against it, including cell-based, nucleic acid-based, and viral-based cancer vaccines. Although many vaccines have been effective in in vivo and clinical studies and some have been FDA-approved, there are major limitations to overcome: (1) developing one universal vaccine for a specific cancer is difficult, as tumors with different antigens are different for different individuals, (2) the tumor antigens may be similar to the body's own antigens, and (3) there is the possibility of cancer recurrence. Therefore, developing personalized cancer vaccines with the ability to distinguish between the tumor and the body's antigens is indispensable. This paper provides a comprehensive review of different types of cancer vaccines and highlights important factors necessary for developing efficient cancer vaccines. Moreover, the application of other technologies in cancer therapy is discussed. Finally, several insights and conclusions are presented, such as the possibility of using cold plasma and cancer stem cells in developing future cancer vaccines, to tackle the major limitations in the cancer vaccine developmental process.

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

In the realm of cancer immunotherapy, a profound evolution has ushered in sophisticated strategies that encompass both traditional cancer vaccines and emerging viral vaccines. This comprehensive Review offers an in-depth exploration of the methodologies, clinical applications, success stories, and future prospects of these approaches. Traditional cancer vaccines have undergone significant advancements utilizing diverse modalities such as proteins, peptides, and dendritic cells. More recent innovations have focused on the physiological mechanisms enabling the human body to recognize and combat precancerous and malignant cells, introducing specific markers like peptide-based anticancer vaccines targeting tumor-associated antigens. Moreover, cancer viral vaccines, leveraging engineered viruses to stimulate immune responses against specific antigens, exhibit substantial promise in inducing robust and enduring immunity. Integration with complementary therapeutic methods, including monoclonal antibodies, adjuvants, and radiation therapy, has not only improved survival rates but also deepened our understanding of viral virulence. Recent strides in vaccine design, encompassing oncolytic viruses, virus-like particles, and viral vectors, mark the frontier of innovation. While these advances hold immense potential, critical challenges must be addressed, such as strategies for immune evasion, potential off-target effects, and the optimization of viral genomes. In the landscape of immunotherapy, noteworthy innovations take the spotlight from the use of immunomodulatory agents for the enhancement of innate and adaptive immune collaboration. The emergence of proteolysis-targeting chimeras (PROTACs) as precision tools for cancer therapy is particularly exciting. With a focus on various cancers, from melanoma to formidable solid tumors, this Review critically assesses types of cancer vaccines, mechanisms, barriers in vaccine therapy, vaccine efficacy, safety profiles, and immune-related adverse events, providing a nuanced perspective on the underlying mechanisms involving cytotoxic T cells, natural killer cells, and dendritic cells. The Review also underscores the transformative potential of cutting-edge technologies such as clinical studies, molecular sequencing, and artificial intelligence in advancing the field of cancer vaccines. These tools not only expedite progress but also emphasize the multidimensional and rapidly evolving nature of this research, affirming its profound significance in the broader context of cancer therapy.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

Ribonucleic acids (RNAs), including the messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), play important roles in living organisms and viruses. In recent years, the RNA-based technologies including the RNAs inhibiting other RNA activities, the RNAs targeting proteins, the RNAs reprograming genetic information, and the RNAs encoding therapeutical proteins, are useful methods to apply in prophylactic and therapeutic vaccines. In this review, we summarize and highlight the current application of the RNA therapeutics, especially on mRNA vaccines which have potential for prevention and treatment against human and animal infectious diseases.

Despite the effectiveness of vaccination in reducing or eradicating diseases caused by pathogens, there remain certain diseases and emerging infections for which developing effective vaccines is inherently challenging. Additionally, developing vaccines for individuals with compromised immune systems or underlying medical conditions presents significant difficulties. As well as traditional vaccine different methods such as inactivated or live attenuated vaccines, viral vector vaccines, and subunit vaccines, emerging non-viral vaccine technologies, including viral-like particle and nanoparticle vaccines, DNA/RNA vaccines, and rational vaccine design, offer new strategies to address the existing challenges in vaccine development. These advancements have also greatly enhanced our understanding of vaccine immunology, which will guide future vaccine development for a broad range of diseases, including rapidly emerging infectious diseases like COVID-19 and diseases that have historically proven resistant to vaccination. This review provides a comprehensive assessment of emerging non-viral vaccine production methods and their application in addressing the fundamental and current challenges in vaccine development.

As a non-viral transfection method, ultrasound and microbubble-induced sonoporation can achieve spatially targeted gene delivery with synergistic immunostimulatory effects. Here, we report for the first time the application of sonoporation for improving DNA vaccination performance. This study developed a new microbubble design with nanoscale DNA/PEI complexes loaded onto cationic microbubbles to attain significant increases in DNA-loading capacity (0.25 pg per microbubble) and in vitro transfection efficiency. Using live-cell imaging, we revealed the membrane perforation and cellular delivery characteristics of sonoporation. Using luciferase reporter gene for in vivo transfection, we showed that sonoporation increased the transfection efficiency by 40.9-fold when compared with intramuscular injection. Moreover, we comprehensively optimized the sonoporation protocol and further increased the transfection efficiency by 43.6-fold. Immunofluorescent staining results showed that sonoporation effectively activated the MHC-II+ immune cells. Using a hepatitis B DNA vaccine, sonoporation induced significantly higher serum antibody levels when compared with intramuscular injection, and the antibodies sustained for 56 weeks. In addition, we recorded the longest reported expression period (400 days) of the sonoporation-delivered gene. Whole genome resequencing confirmed that the gene with stable expression existed in an extrachromosomal state without integration. Our results demonstrated the potential of sonoporation for efficient and safe DNA vaccination.

The history of DNA vaccine began as early as the 1960s with the discovery that naked DNA can transfect mammalian cells in vivo. In 1992, the evidence that such transfection could lead to the generation of antigen-specific antibody responses was obtained and supported the development of this technology as a novel vaccine platform. The technology then attracted immense interest and high hopes in vaccinology, as evidence of high immunogenicity and protection against virulent challenges accumulated from several animal models for several diseases. In particular, the capacity to induce T-cell responses was unprecedented in non-live vaccines. However, the technology suffered its major knock when the success in animals failed to translate to humans, where DNA vaccine candidates were shown to be safe but remained poorly immunogenic, or not associated with clinical benefit. Thanks to a thorough exploration of the molecular mechanisms of action of these vaccines, an impressive range of approaches have been and are currently being explored to overcome this major challenge. Despite limited success so far in humans as compared with later genetic vaccine technologies such as viral vectors and mRNA, DNA vaccines are not yet optimised for human use and may still realise their potential.

Ovarian Cancer (OC) is the most common gynecological malignancy and the eighth most diagnosed cancer in females worldwide. Presently, it ranks as the fifth leading cause of cancer-related mortality among patients globally. Major factors contributing to the lethality of OC worldwide include delayed diagnosis, chemotherapy resistance, high metastatic rates, and the heterogeneity of subtypes. Despite continuous efforts to develop novel targeted therapies and chemotherapeutic agents, challenges persist in the form of OC resistance and recurrence. In the last decade, CRISPR-Cas-based genome editing has emerged as a powerful tool for modifying genetic and epigenetic mechanisms, holding potential for treating numerous diseases. However, a significant challenge for therapeutic applications of CRISPR-Cas technology is the absence of an optimal vehicle for delivering CRISPR molecular machinery into targeted cells or tissues. Recently, extracellular vesicles (EVs) have gained traction as potential delivery vehicles for various therapeutic agents. These heterogeneous, membrane-derived vesicles are released by nearly all cells into extracellular spaces. They carry a molecular cargo of proteins and nucleic acids within their intraluminal space, encased by a cholesterol-rich phospholipid bilayer membrane. EVs actively engage in cell-to-cell communication by delivering cargo to both neighboring and distant cells. Their inherent ability to shield molecular cargo from degradation and cross biological barriers positions them ideally for delivering CRISPR-Cas ribonucleoproteins (RNP) to target cells. Furthermore, they exhibit higher biocompatibility, lower immunogenicity, and reduced toxicity compared to classical delivery platforms such as adeno-associated virus, lentiviruses, and synthetic nanoparticles. This review explores the potential of employing different CRISPR-Cas systems to target specific genes in OC, while also discussing various methods for engineering EVs to load CRISPR components and enhance their targeting capabilities.

The development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation. To improve the efficacy of these therapeutics and minimize side effects, researchers can focus on the features of mRNA itself or the properties of the delivery agent to achieve the desired response. The tools considered for mRNA manipulation can be improved in terms of targetability, tunability, and translatability to medicine. While ongoing studies are dedicated to improving conventional approaches, innovative approaches can also be considered to unleash the full potential of mRNA-based therapeutics. Here, we discuss the opportunities that emerged from introducing synthetic biology to mRNA therapeutics. It includes a discussion of modular self-assembled mRNA nanoparticles, logic gates on a single mRNA molecule, and other possibilities.

Protein subunit vaccines have been used as prophylactic vaccines for a long time. The well-established properties of these vaccines make them the first choice for the coronavirus disease 2019 (COVID-19) outbreak. However, it is not easy to develop a protein vaccine that induces cytotoxic T lymphocyte responses and requires a longer time for manufacturing, which limits the usage of this vaccine type. Here, we report the combination of a recombinant spike (S)-trimer protein with a DNA vaccine-encoded S protein as a novel COVID-19 vaccine. The recombinant S protein was formulated with different adjuvants and mixed with the DNA plasmid before injection. We found that the recombinant S protein formulated with the adjuvant aluminum hydroxide and mixed with the DNA plasmid could enhance antigen-specific antibody titers, neutralizing antibody titers. We further evaluated the IgG2a/IgG1 isotype and cytokine profiles of the specific boosted T-cell response, which indicated that the combined vaccine induced a T-helper 1 cell-biased immune response. Immunized hamsters were challenged with severe acute respiratory syndrome coronavirus 2, and the body weight of the hamsters that received the recombinant S protein with aluminum hydroxide and/or the DNA plasmid was not reduced. Alternatively, those that received control or only the DNA plasmid immunization were reduced. Interestingly, after the third day of the viral load in the lungs, the viral challenge could not be detected in hamsters immunized with the recombinant S protein in aluminum hydroxide mixed with DNA (tissue culture infectious dose < 10). The viral load in the lungs was 109 , 106 , and 107 for the phosphate-buffered saline, protein in aluminum hydroxide, and DNA-only immunizations, respectively. These results indicated that antiviral mechanisms neutralizing antibodies play important roles. Furthermore, we found that the combination of protein and DNA vaccination could induce relatively strong CD8+ T-cell responses. In summary, the protein subunit vaccine combined with a DNA vaccine could induce strong CD8+ T-cell responses to increase antiviral immunity for disease control.

Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These mAbs are developed from naturally occurring antibodies and target specific epitopes of single molecules, minimizing off-target effects. Antibodies can also be designed to target particular pathogens or modulate immune function by activating or suppressing certain pathways. Despite their benefit for patients, the production and administration of monoclonal antibody therapeutics are laborious, costly, and time-consuming. Administration often requires inpatient stays and repeated dosing to maintain therapeutic levels, limiting their use in underserved populations and developing countries. Researchers are developing alternate methods to deliver monoclonal antibodies, including synthetic nucleic acid-based delivery, to overcome these limitations. These methods allow for in vivo production of monoclonal antibodies, which would significantly reduce costs and simplify administration logistics. This review explores new methods for monoclonal antibody delivery, including synthetic nucleic acids, and their potential to increase the accessibility and utility of life-saving treatments for several diseases.

The CRISPR-Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.

Vaccines represent one of the most significant advancements in public health since they prevented morbidity and mortality in millions of people every year. Conventionally, vaccine technology focused on either live attenuated or inactivated vaccines. However, the application of nanotechnology to vaccine development revolutionized the field. Nanoparticles emerged in both academia and the pharmaceutical industry as promising vectors to develop future vaccines. Regardless of the striking development of nanoparticles vaccines research and the variety of conceptually and structurally different formulations proposed, only a few of them advanced to clinical investigation and usage in the clinic so far. This review covered some of the most important developments of nanotechnology applied to vaccine technologies in the last few years, focusing on the successful race for the preparation of lipid nanoparticles employed in the successful anti-SARS-CoV-2 vaccines.

The coronavirus disease 2019 (COVID-19) pandemic has infected 673,010,496 patients and caused the death of 6,854,959 cases globally until today. Enormous efforts have been made to develop fundamentally different COVID-19 vaccine platforms. Nucleic acid-based vaccines consisting of mRNA and DNA vaccines (third-generation vaccines) have been promising in terms of rapid and convenient production and efficient provocation of immune responses against the COVID-19. Several DNA-based (ZyCoV-D, INO-4800, AG0302-COVID19, and GX-19N) and mRNA-based (BNT162b2, mRNA-1273, and ARCoV) approved vaccine platforms have been utilized for the COVID-19 prevention. mRNA vaccines are at the forefront of all platforms for COVID-19 prevention. However, these vaccines have lower stability, while DNA vaccines are needed with higher doses to stimulate the immune responses. Intracellular delivery of nucleic acid-based vaccines and their adverse events needs further research. Considering re-emergence of the COVID-19 variants of concern, vaccine reassessment and the development of polyvalent vaccines, or pan-coronavirus strategies, is essential for effective infection prevention.

Toxoplasma gondii (T. gondii) is not only a threat to the public health but it also poses adverse impacts on the livestock industry. This study aimed to develop a recombinant vaccine composed of T. gondii microneme protein 6 (TgMIC6) and T. gondii rhoptry protein 18 (TgROP18).The vaccine was delivered with a novel vector, named analogous hyaluronic acid chitosan nanoparticle-hydrogel (AHACNP-HG) and its immune protection was evaluated.

The recombinant MIC6 and ROP18 proteins were obtained by affinity chromatography and loaded onto AHACNP-HG by magnetic stirring. The characterizations of AHACNP-HG were investigated, including its structure, rheological property, nanoparticle size and zeta potential, its ability to release protein in vitro and toxicology in vivo. The immunological and anti-infection effects of AHACNP-HG/rMIC6/rROP18 were examined in the mice model.

AHACNP-HG presented a characteristic of composite system and possessed biosecurity with excellent protein control-release property. AHACNP-HG/rMIC6/rROP18 vaccine enhanced a mixed Th1/Th2 cellular immune response accompanied by an increased level of the cytokines, IFN-γ and IL-10. It also provoked a stronger humoral immune response. Additionally, after challenge with T. gondii tachyzoite, AHACNP-HG/rMIC6/rROP18 inoculation prolonged the survival time of mice.

Our data indicated that mixed rMIC6 and rROP18 induced strong immune response and played a certain protective role in controlling T. gondii infection, and the novel adjuvant AHACNP-HG improved modestly some immunogenicity properties in mouse model, which indicated that it can be used as a novel delivery system in vaccine development.

Ticks can seriously affect human and animal health around the globe, causing significant economic losses each year. Chemical acaricides are widely used to control ticks, which negatively impact the environment and result in the emergence of acaricide-resistant tick populations. A vaccine is considered as one of the best alternative approaches to control ticks and tick-borne diseases, as it is less expensive and more effective than chemical controls. Many antigen-based vaccines have been developed as a result of current advances in transcriptomics, genomics, and proteomic techniques. A few of these (e.g., Gavac® and TickGARD®) are commercially available and are commonly used in different countries. Furthermore, a significant number of novel antigens are being investigated with the perspective of developing new anti-tick vaccines. However, more research is required to develop new and more efficient antigen-based vaccines, including on assessing the efficiency of various epitopes against different tick species to confirm their cross-reactivity and their high immunogenicity. In this review, we discuss the recent advancements in the development of antigen-based vaccines (traditional and RNA-based) and provide a brief overview of recent discoveries of novel antigens, along with their sources, characteristics, and the methods used to test their efficiency.

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.

Therapeutic applications of synthetic mRNA were proposed more than 30 years ago, and are currently the basis of one of the vaccine platforms used at a massive scale as part of the public health strategy to get COVID-19 under control. To date, there are no published studies on the biodistribution, cellular uptake, endosomal escape, translation rates, functional half-life and inactivation kinetics of synthetic mRNA, rates and duration of vaccine-induced antigen expression in different cell types. Furthermore, despite the assumption that there is no possibility of genomic integration of therapeutic synthetic mRNA, only one recent study has examined interactions between vaccine mRNA and the genome of transfected cells, and reported that an endogenous retrotransposon, LINE-1 is unsilenced following mRNA entry to the cell, leading to reverse transcription of full length vaccine mRNA sequences, and nuclear entry. This finding should be a major safety concern, given the possibility of synthetic mRNA-driven epigenetic and genomic modifications arising. We propose that in susceptible individuals, cytosolic clearance of nucleotide modified synthetic (nms-mRNAs) is impeded. Sustained presence of nms-mRNA in the cytoplasm deregulates and activates endogenous transposable elements (TEs), causing some of the mRNA copies to be reverse transcribed. The cytosolic accumulation of the nms-mRNA and the reverse transcribed cDNA molecules activates RNA and DNA sensory pathways. Their concurrent activation initiates a synchronized innate response against non-self nucleic acids, prompting type-I interferon and pro-inflammatory cytokine production which, if unregulated, leads to autoinflammatory and autoimmune conditions, while activated TEs increase the risk of insertional mutagenesis of the reverse transcribed molecules, which can disrupt coding regions, enhance the risk of mutations in tumour suppressor genes, and lead to sustained DNA damage. Susceptible individuals would then expectedly have an increased risk of DNA damage, chronic autoinflammation, autoimmunity and cancer. In light of the current mass administration of nms-mRNA vaccines, it is essential and urgent to fully understand the intracellular cascades initiated by cellular uptake of synthetic mRNA and the consequences of these molecular events.

The group B coxsackieviruses (CVBs) exist in six serotypes (CVB1 to CVB6). Disease associations have been reported for most serotypes, and multiple serotypes can cause similar diseases. For example, CVB1, CVB3, and CVB5 are generally implicated in the causation of myocarditis, whereas CVB1 and CVB4 could accelerate the development of type 1 diabetes (T1D). Yet, no vaccines against these viruses are currently available. In this review, we have analyzed the attributes of experimentally tested vaccines and discussed their merits and demerits or limitations, as well as their impact in preventing infections, most importantly myocarditis and T1D.

After receiving emergency approval during the COVID-19 pandemic, mRNA vaccines have taken center stage in the quest to enhance future vaccination strategies for both infectious diseases and cancer. Indeed, they have significantly overshadowed another facet of genetic vaccination, specifically DNA vaccines. Nevertheless, it is important to acknowledge that both types of genetic vaccines have distinct advantages and disadvantages that set them apart from each other.

In this work, we delve extensively into the history of genetic vaccines, their mechanisms of action, their strengths, and limitations, and ultimately highlight ongoing research in key areas for potential enhancement of both DNA and mRNA vaccines.

Here, we assess the significance of the primary benefits and drawbacks associated with DNA and mRNA vaccination. We challenge the current lines of thought by highlighting that the existing drawbacks of DNA vaccination could potentially be more straightforward to address compared to those linked with mRNA vaccination. In our view, this suggests that DNA vaccines should remain viable contenders in the pursuit of the future of vaccination.

The vaccines being used against COVID-19 are composed of either non-viral or viral nanoparticles (NPs). Nanotechnology-based vaccine technology was studied for its potentially transformative advancement of medicine.

NPs protect the encapsulated mRNA in vaccines, thereby enhancing the stability of the ribonucleic acids and facilitating their intact delivery to their specific targets. Compared to liposomes, lipid nanoparticles (LNPs) are unique and, through their rigid morphology and better cellular penetrability, render enhanced cargo stability. To explore nanotechnology-mediated vaccine delivery and its potential in future pandemics, we assessed articles from various databases, such as PubMed, Embase, and Scopus, including editorial/research notes, expert opinions, and collections of data from several clinical research trials. In the current review, we focus on the nanoparticulate approach of the different SARS-CoV-2 vaccines and explore their success against the pandemic.

The mRNA-based vaccines, with their tremendous efficacy of ~95% (under phase III-IV clinical trials) and distinct nanocarriers (LNPs), represent a new medical front alongside DNA and siRNA-based vaccines.

Prophylactic vaccines against human papillomavirus (HPV) have proven efficacy in those who have not been infected by the virus. However, they do not benefit patients with established tumors. Therefore, the development of therapeutic options for HPV-related malignancies is critical. Third-generation vaccines based on nucleic acids are fast and simple approaches to eliciting adaptive immune responses. However, techniques to boost immunogenicity, reduce degradation, and facilitate their capture by immune cells are frequently required. One option to overcome this constraint is to employ delivery systems that allow selective antigen absorption and help modulate the immune response. This review aimed to discuss the influence of these different systems on the response generated by nucleic acid vaccines. The results indicate that delivery systems based on lipids, polymers, and microorganisms such as yeasts can be used to ensure the stability and transport of nucleic acid vaccines to their respective protein synthesis compartments. Thus, in view of the limitations of nucleic acid-based vaccines, it is important to consider the type of delivery system to be used-due to its impact on the immune response and desired final effect.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced COVID-19 is a complicated disease. Clinicians are continuously facing difficulties to treat infected patients using the principle of repurposing of drugs as no specific drugs are available to treat COVID-19. To minimize the severity and mortality, global vaccination is the only hope as a potential preventive measure. After a year-long global research and clinical struggle, 165 vaccine candidates have been developed and some are currently still in the pipeline. A total of 28 candidate vaccines have been approved for use and the remainder are in different phases of clinical trials. In this comprehensive report, the authors aim to demonstrate, classify and provide up-to-date clinical trial status of all the vaccines discovered to date and specifically focus on the approved candidates. Finally, the authors specifically focused on the vaccination of different types of medically distinct populations.

One of the greatest challenges in the treatment of cancer is tumor heterogeneity which results in differential responses to chemotherapy and drugs that work through a single pathway. A therapeutic agent that targets cancer cells for death through multiple mechanisms could be advantageous as a broad inhibitor for many types of cancers and the heterogeneous alterations they possess. Several viral proteins have been exploited for antiproliferative and apoptotic effect in cancer cells by disrupting critical survival pathways. Here, we report the use of the non-structural protein on the S segment (NSs) gene from the Rift Valley fever virus (RVFV) to induce cancer cell death. NSs has immune evasion functions in the context of RVFV with many of these functions affecting proliferation pathways and DNA damage signaling, which could be leveraged against cancer cells. We find that expression of NSs in multiple cancer cell lines leads to a rapid decline in cell viability and induction of apoptosis. Interestingly, we observed reduced toxicity in normal cells suggesting cancer cells may be more susceptible to NSs-mediated cell death. To enhance specificity of NSs for use in hepatocellular carcinoma, we incorporated four miR-122 binding sites in the 3' untranslated region (UTR) of the NSs mRNA to achieve cell type specific expression. Observations presented here collectively suggest that delivery of the NSs gene may provide a unique therapeutic approach in a broad range of cancers.

The novel SARS-CoV-2, responsible for the COVID-19 pandemic, is the third zoonotic coronavirus since the beginning of the 21 first century, and it has taken more than 6 million human lives because of the lack of immunity causing global economic losses. Consequently, developing a vaccine against the virus represents the fastest way to finish the threat and regain some "normality."

Here, we provide information about the main features of the most important vaccine platforms, some of them already approved, to clear common doubts fostered by widespread misinformation and to reassure the public of the safety of the vaccination process and the different alternatives presented.

Articles published in open access databases until January 2022 were identified using the search terms "SARS-CoV-2," "COVID-19," "Coronavirus," "COVID-19 Vaccines," "Pandemic," COVID-19, and LMICs or their combinations.

Traditional first-generation vaccine platforms, such as whole virus vaccines (live attenuated and inactivated virus vaccines), as well as second-generation vaccines, like protein-based vaccines (subunit and viral vector vaccines), and third-generation vaccines, such as nanoparticle and genetic vaccines (mRNA vaccines), are described.

SARS-CoV-2 sequence information obtained in a record time provided the basis for the fast development of a COVID-19 vaccine. The adaptability characteristic of the new generation of vaccines is changing our capability to react to emerging threats to future pandemics. Nevertheless, the slow and unfair distribution of vaccines to low- and middle-income countries and the spread of misinformation are a menace to global health since the unvaccinated will increase the chances for resurgences and the surge of new variants that can escape the current vaccines.