Search Results (301)

Endothelial dysfunction is a key characteristic of many diseases, including atherosclerosis, hypertension, heart failure, stroke, cancer, acute respiratory distress syndrome (ARDS), peripheral vascular disease, coronavirus 2019 (COVID-19), and pulmonary arterial hypertension (PAH). To improve understanding of the roles of endothelial cells (ECs) in health and disease, EC-specific genome editing technologies have been developed in recent years. Therapeutic strategies that aim to restore a healthy endothelial monolayer include the inhibition of endothelial genes that cause EC injury and dysfunction and the induction or activation of endothelial genes that drive EC repair and regeneration. In this review, we describe established recombinase-mediated genetic modification technologies and emerging EC-specific genome editing technologies including viral and non-viral delivery of the CRISPR/Cas9 genome editing system, and we summarize the strengths and limitations of each technology. We then discuss possible avenues for future research, including the development of organ-specific EC genome editing technologies. In short, EC-specific genome editing technologies can be used to modulate gene expression selectively in ECs and even within a specific vascular bed and/or distinctive EC subtype, and, in doing so, greatly improve the understanding of vascular biology and help develop precision genetic medicine targeting the disease-causing vascular bed(s) to effectively treat diseases caused by vascular endothelial dysfunction. Full article

RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides—eteplirsen, golodirsen, viltolarsen, and casimersen—have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies—including base and prime editing—offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships. Full article

Next-generation vaccines are being developed to elicit durable and cross-protective immune responses against diverse pathogens, particularly those targeting the respiratory and enteric systems. By strategically engaging T cell-centric antigen design, mucosal immune engagement, and induction of trained innate immunity, these innovative platforms are expected to reshape the paradigm of immunoprophylaxis and to offer promising avenues for enhanced protection against complex infectious diseases. Conventional antibody-based vaccines, though effective against many infections, often lack the capacity to induce durable or cross-protective immunity at mucosal surfaces. Advances in antigen design, delivery platforms, and adjuvant technologies now facilitate precise activation of tissue-resident memory T cells and enhancement of mucosal secretory IgA responses, thereby achieving sterilizing immunity at barrier surfaces while reinforcing systemic immune protection. Advanced delivery platforms, including lipid nanoparticles, viral vectors, and nano or liposomal carriers, further refine antigen presentation, enhancing stability, targeting, and overall immunogenicity. Concurrently, progress in understanding trained innate immunity highlights opportunities to induce broad, non-antigen-specific protection through epigenetic and metabolic reprogramming of innate cells. The integration of these adaptive and innate mechanisms may enhance early pathogen control, limits transmission, and strengthens defense against variant and antimicrobial-resistant pathogens across diverse populations. However, translating these immunological insights into safe, scalable, and globally accessible vaccines remains a major challenge. This review explores the emerging conceptual framework of next-generation vaccines that demonstrate partial integration of these axes in preclinical models, though human translation and functional synergy require Phase II validation. It highlights progress toward next-generation vaccines leveraging integrated adaptive and innate immune reprogramming for superior protection against respiratory and enteric pathogens. Full article

Intervertebral disc degeneration (IVDD) is the leading cause of low back pain, a global public health burden for which current pharmacological and surgical treatments provide symptomatic relief but fail to reverse the underlying degenerative process. The uniquely avascular, hypoxic, acidic, and mechanically demanding disc microenvironment poses formidable barriers to the survival and function of therapeutic cells and genes, emphasizing the critical need for bioengineered delivery systems. In this review, we introduce the structure and microenvironment of the intervertebral disc, as well as the molecular mechanisms underlying IVDD. We then provide a critical comparative analysis of delivery platforms, including hydrogels, microspheres, nanoparticles, nanofibrous scaffolds, and viral and non-viral vectors, around five core delivery challenges: mechanical protection, retention and leakage prevention, targeted intracellular delivery, controlled release kinetics, and metabolic support. Furthermore, we examine the fabrication technologies and material considerations that determine platform performance, and we analyze the translational barriers that have impeded clinical adoption, such as the limitations of small-animal models and unresolved cell leakage. Finally, we highlight emerging strategies, including gene-cell combination therapy and endplate preconditioning, to accelerate the clinical translation of precision therapies for IVDD. Full article

Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 > HepG2 > Huh7 > non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells. Full article

Gene therapy relies on safe and efficient delivery systems, yet traditional viral vectors and synthetic polymers often fail to meet these requirements due to immunogenicity and biocompatibility concerns. This review highlights self-assembling short peptides as a highly programmable and biocompatible non-viral platform uniquely positioned to overcome these translational bottlenecks. To provide a comprehensive overview of next-generation gene delivery, we systematically trace the trajectory from fundamental chemistry to clinical applications. First, we elucidate the supramolecular interactions and mechanisms driving peptide–nucleic acid co-assembly. Second, we outline concrete design strategies, detailing how sequence engineering and environmental responsiveness dictate the formation of optimized nanomorphologies. Third, we critically analyze how these nanocarriers navigate critical physiological and intracellular barriers, with a specific focus on cellular uptake, endosomal escape, and cargo release. Finally, we demonstrate the platform’s versatility in emerging frontiers, particularly mRNA vaccines and CRISPR/Cas9 gene editing. We conclude by identifying current obstacles to clinical translation and proposing future directions centered on multifunctional integration and stimuli-responsive design. Full article

The liver’s anatomic position and immune specialization make it both a major target and a major filter for systemically delivered therapeutics. Because portal venous inflow exposes the liver early to gut-derived molecules and exogenous compounds, many intravenously administered agents, including gene-based medicines and their viral and non-viral delivery systems, preferentially enter and accumulate in hepatic tissue. This review synthesizes how core liver physiology and immunobiology influence the performance, safety, and clinical translation of genomic medicines in hepatology, and outlines near-term practice and research shifts likely to define a genomics-driven future in liver disease care. We review the hepatic microarchitecture relevant to therapeutic trafficking, including sinusoidal transit, the space of Disse, hepatocyte uptake, and hepatobiliary elimination, and highlight the gatekeeping roles of liver sinusoidal endothelial cells and Kupffer cells in clearing particulate material and shaping inflammatory signaling. We then discuss how these same features create both opportunities, such as efficient hepatic targeting, and constraints, including innate immune activation, vector clearance, and variable intrahepatic distribution, for DNA- and RNA-based platforms. Finally, we propose five actionable developments poised to move genomics from a niche tool to a routine component of hepatology practice: earlier genomic testing in unexplained liver disease, multidisciplinary hepatology genome rounds, a centralized liver-specific gene resource, genetics-aware clinical trial design, and expansion of genetic therapies. Integrating liver biology with genomic medicine is essential to improve diagnostic yield, personalize therapy, and accelerate translation of gene-based treatments while mitigating immunologic and delivery-related barriers. Full article

Background/Objectives: Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that is critical for intestinal immune homeostasis. Despite its therapeutic potential, systemic delivery of IL-10 has failed in clinical trials for inflammatory bowel disease (IBD), largely due to its poor localization and short half-life. Methods: We present a proof-of-concept study demonstrating that hydrodynamic delivery of a naked plasmid bearing the human IL-10 gene to ex vivo human colonic segments from Crohn’s disease patients results in localized IL-10 expression and modulation of inflammatory mediators. Results: Compared to venous administration, arterial delivery yielded significantly higher IL-10 mRNA and protein levels, as well as decreased IL-6 and TNF-α expression. Furthermore, nanoparticle tracing confirmed efficient tissue penetration via the arterial route. Conclusions: These findings establish arterial hydrodynamic delivery as a feasible, non-viral strategy for targeted gene therapy in IBD. Full article

Viral vectors such as adenovirus (Ad) and lentivirus (LV) are central to gene therapy owing to their transduction efficiency and broad applicability; however, their clinical translation is often limited by immunogenicity, rapid clearance, and reduced bioavailability. Non-enveloped Ad vectors are highly susceptible to neutralization by pre-existing antibodies, while enveloped LVs remain vulnerable to immune surveillance and off-target clearance. In this study, a biomimetic encapsulation strategy using erythrocyte-derived membranes (EDMs) is reported to enhance viral immune resilience and functional gene delivery. Ad-GFP and LV-mCherry were successfully encapsulated within EDM using an extrusion-based assembly approach, resulting in uniform membrane-coated particles with physicochemical properties characteristic of erythrocyte membranes. EDM encapsulation significantly enhanced in vitro transduction efficiency of both viral platforms across multiple cancer cell lines without compromising viral activity. Notably, EDM-Ad-GFP demonstrated robust protection against neutralizing antibodies, achieving significantly higher transduction of HEK293 cells in the presence of diluted human serum compared to unencapsulated Ad. These findings indicate that EDM encapsulation can effectively shield viral vectors from immune recognition while improving cellular uptake and transduction performance. Collectively, this work establishes EDM encapsulation as a versatile and scalable platform to enhance the efficacy, durability, and translational potential of viral gene delivery systems. Full article

Viral hemorrhagic fevers (VHFs) comprise a heterogeneous group of severe infectious diseases that continue to represent a major global health concern. Although many VHFs remain endemic to regions of Africa, Asia, and the Americas, their wide geographic distribution, together with increasing international travel and global trade, facilitates the importation of cases into non-endemic areas and raises the risk of secondary transmission under favorable ecological and epidemiological conditions. These infections are frequently associated with high case-fatality rates and impose a substantial social and economic burden, including pressure on healthcare systems, disruption of essential services, and long-term physical and psychological sequelae among survivors. Despite notable advances in recent years, therapeutic options for VHFs remain limited. Supportive care continues to represent the cornerstone of clinical management for most infections, while pathogen-targeted therapies are available only for a restricted number of diseases. Monoclonal antibody-based therapies have achieved the most significant regulatory success to date, particularly for Ebola virus disease. In parallel, several small-molecule antivirals have been investigated in preclinical and clinical settings, including during outbreak responses, although inconsistent efficacy and safety concerns have limited widespread approval. Vaccine development has progressed further, with licensed vaccines available for selected VHFs, including Ebola, yellow fever, and dengue, and multiple candidates based on diverse technological platforms advancing through clinical evaluation. In addition to summarizing current therapeutic and vaccine strategies, this review highlights pharmaceutical development considerations relevant to biologic therapeutics and selected vaccine platforms, including formulation stability, pharmacokinetic behavior, delivery routes, storage requirements, and logistical constraints affecting deployment during outbreak responses. Using a comparative cross-pathogen framework, the review synthesizes recent literature to identify translational gaps, regulatory challenges, and future priorities for the development of safer and more effective medical countermeasures against VHFs. Full article

Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies. Full article

Extracellular vesicles (EVs), particularly exosomes, have emerged as critical mediators of intercellular communication, yet the metabolite fraction of their cargo remains substantially underexplored relative to proteins and nucleic acids. This review synthesizes current knowledge on the exosomal metabolome as a functionally distinct intercellular signaling system with unique biophysical properties. We review the mechanisms proposed to govern metabolite encapsulation into exosomes, encompassing membrane transporter involvement, lipid raft partitioning, and binding to luminal proteins, and discuss the unresolved question of whether metabolite loading is selective or stochastic. Critically, we present a quantitative framework evaluating whether delivered metabolite quantities are sufficient to alter recipient cell metabolic pools, distinguishing receptor-mediated signaling from bulk substrate delivery. We also address methodological considerations including contamination artifacts and isolation-method biases that complicate interpretation of EV metabolomics data. Exosomal metabolites are reviewed across four functional categories: energy substrates (ATP, lactate, amino acids), signaling molecules (TCA cycle intermediates, eicosanoids, nucleotides), redox cofactors and antioxidants (NADH, glutathione), and oncometabolites. For each category, available evidence is critically appraised, distinguishing metabolites with direct mass spectrometric detection from those whose roles are inferred from parent-cell biology. The review examines the roles of exosomal metabolites in tumor-stroma metabolic symbiosis, immunometabolic regulation, inter-organ crosstalk in metabolic diseases including type 2 diabetes and non-alcoholic fatty liver disease, cancer metastasis, viral infections, and immune evasion. A quantitative framework is discussed to evaluate whether delivered metabolite quantities are sufficient to alter recipient cell metabolic pools, distinguishing receptor-mediated signaling from bulk substrate delivery. Technical challenges in exosomal metabolomics are reviewed, including the impact of isolation method on data quality, contamination artifacts, and current standardization gaps. Therapeutic implications of exosomal metabolite signaling are discussed, encompassing metabolite-loaded exosomes as therapeutic vehicles and exosomal metabolite loading as a pharmacological target. Integration of single-vesicle technologies with systems biology approaches is highlighted as a promising direction for advancing this field toward precision medicine applications in oncological and metabolic disorders. Full article

Background: Immunomodulatory compounds can modify or regulate the immune responses. Given that vaccine-induced immune responses can vary in magnitude and durability depending on antigen properties and adjuvant selection. Immunomodulators that enhance antigen-specific immune responses with low toxicity may complement existing adjuvant systems. Recent studies indicate that adenosine receptor–mediated signaling can modulate dendritic cell (DC) function through mechanisms distinct from classical pathogen-associated molecular pattern (PAMP)-driven Toll-like receptor pathways. Methods: In this context, the present study comparatively evaluates poly-(lactic-co-glycolic acid) (PLGA) microparticle–encapsulated β-L-adenosine (BLA MPs) alongside established FDA-approved adjuvants to assess their immunomodulatory potential under limited-antigen conditions. FDA-approved PLGA was used to encapsulate BLA in combination with multiple viral antigens, including H1N1 influenza, Zika virus, and canine coronavirus, to enable sustained delivery, antigen protection, and efficient uptake by antigen-presenting cells. Results: Physicochemical characterization demonstrated uniform particle size distribution, a low polydispersity index, and a stable negative surface charge. Release studies showed more than 50% payload release within 12 h, with release kinetics best described by the Korsmeyer–Peppas model. Cytotoxicity evaluation using DC2.4 cells confirmed that BLA MPs were non-cytotoxic at concentrations up to 250 μg/mL. Comparative in vitro immunological assessments revealed that BLA MPs induced dendritic cell activation, including upregulation of antigen-presenting and co-stimulatory molecules, at levels largely comparable to those observed with Alum- and MF59-based formulations across multiple antigen groups. Nitric oxide production remained within comparable ranges, indicating balanced immunostimulatory activity without excessive inflammatory signaling. In select conditions, co-formulation of BLA MPs with MF59 further enhanced DC activation, supporting its role as a complementary immunomodulatory component. Conclusion: These findings align with previously reported adenosine-dependent pathways involved in DC maturation and antigen presentation. Overall, this comparative study demonstrates that PLGA-encapsulated β-L-adenosine functions as an effective immunomodulatory agent, with performance comparable to that of established FDA-approved adjuvants across diverse vaccine antigens. Further in vivo studies are warranted to evaluate dose dependency, cytokine profiles, and antibody responses to define its role within combinatorial vaccine adjuvant strategies. Full article

Background/Objectives: Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by α-L-iduronidase (IDUA) deficiency, leading to progressive glycosaminoglycan (GAG) accumulation and severe joint involvement. Gene editing represents a promising alternative to restore localized enzyme production. Therefore, this study aimed to evaluate the feasibility, efficacy, and safety of in situ genome editing through intra-articular administration of a nonviral CRISPR/Cas9 system to increase localized IDUA expression in an MPS I mouse model. Methods: Cationic liposomes were formulated to deliver plasmids encoding the CRISPR/Cas9 system targeted to the ROSA26 locus along with an IDUA donor sequence. In vitro assays were performed in fibroblast-like synoviocytes (FLSs) isolated from MPS I mice to assess cytotoxicity, gene editing efficiency, and IDUA activity. In vivo, MPS I mice received intra-articular injections in the knee joints, either as a single dose (short-term study) or monthly for three months (long-term study). IDUA activity, GAG levels, and genome editing efficiency were evaluated in joint tissues, synovial fluid, serum, and major organs. Results: Gene-edited FLS showed sustained IDUA activity for up to 30 days with low cytotoxicity. In vivo, intra-articular administration resulted in a significant increase in IDUA activity in joint tissue and synovial fluid without detectable systemic IDUA. Long-term treatment led to persistent joint-localized IDUA activity, significant reductions (>50%) in GAG levels, and detectable genome editing in joint DNA. Conclusions: Intra-articular delivery of CRISPR/Cas9 via cationic liposomes enables safe and effective localized genome editing, representing a promising strategy for treating joint manifestations of MPS I. Full article

In this work, we designed non-viral gene delivery vector systems based on three poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] copolymers functionalized by primary, secondary, and tertiary amino groups. The impact of copolymer structure and composition was sought through the examination of basic physicochemical and biological parameters. The complexation ability of copolymers with plasmid DNA was studied by ethidium bromide quenching assay. The polyplex particles size and ζ-potential were determined by dynamic and electrophoretic light scattering. The release ability of copolymers was assessed by competitive displacement of DNA using dextran sulfate. The biological performance of amino-functionalized poly(2-ethyl-2-oxazoline)-ran-poly[2-(3-butenyl)-2-oxazoline] based gene delivery systems was evaluated, and their behavior under various environmental conditions, such as pH and ionic strength, was investigated. Cytotoxicity was assessed in two human lung-derived cell lines, and the ability of the copolymers to mediate plasmid DNA delivery and expression was examined. The resulting polyplex nanoparticles exhibited the ability to release DNA molecules and sensitivity to alterations in pH and ionic strength. All systems showed high biocompatibility and were able to mediate plasmid DNA delivery, resulting in detectable EGFP expression in vitro. The vector properties were found to be driven by a multifactorial interplay among hydrophobic character, thermoresponsive behavior, polymer mobility, charge accessibility, intracellular environmental responsiveness, secondary structure effects, etc. The copolymer bearing primary amino groups displayed a distinct balance between DNA binding and release, characterized by moderate complex stability and enhanced sensitivity to environmental changes. These findings provide mechanistic insight into how amino functionality and polymer structure influence the structure–property–behavior relationships of polyoxazoline-based non-viral gene delivery systems. Full article