Search Results (530)

The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox’s effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway. Full article

Background: Transfection is vital for gene therapy, mRNA treatments, CAR-T cell therapy, and regenerative medicine. While viral vectors are effective, non-viral systems like lipid nanoparticles (LNPs) offer safer, more flexible alternatives. This work explores emerging non-viral transfection technologies to improve delivery efficiency and therapeutic outcomes. Methods: This review synthesizes the current literature and recent advancements in non-viral transfection technologies. It focuses on the mechanisms, advantages, and limitations of various delivery systems, including lipid nanoparticles, biodegradable polymers, electroporation, peptide-based carriers, and microfluidic platforms. Comparative analysis was conducted to evaluate their performance in terms of transfection efficiency, cellular uptake, biocompatibility, and potential for clinical translation. Several academic search engines and online resources were utilized for data collection, including Science Direct, PubMed, Google Scholar Scopus, the National Cancer Institute’s online portal, and other reputable online databases. Results: Non-viral systems demonstrated superior performance in delivering mRNA, siRNA, and antisense oligonucleotides, particularly in clinical applications. Biodegradable polymers and peptide-based systems showed promise in enhancing biocompatibility and targeted delivery. Electroporation and microfluidic systems offered precise control over transfection parameters, improving reproducibility and scalability. Collectively, these innovations address key challenges in gene delivery, such as stability, immune response, and cell-type specificity. Conclusions: The continuous evolution of transfection technologies is pivotal for advancing gene and cell-based therapies. Non-viral delivery systems, particularly LNPs and emerging platforms like microfluidics and biodegradable polymers, offer safer and more adaptable alternatives to viral vectors. These innovations are critical for optimizing therapeutic efficacy and enabling personalized medicine, immunotherapy, and regenerative treatments. Future research should focus on integrating these technologies to develop next-generation transfection platforms with enhanced precision and clinical applicability. Full article

Down syndrome (DS), stemming from the triplication of human chromosome 21, results in intellectual disability, with early mid-life onset of Alzheimer’s disease (AD) pathology. Early interventions to reduce cognitive impairments and neuropathology are lacking. One modality, maternal choline supplementation (MCS), has shown beneficial effects on behavior and gene expression in neurodevelopmental and neurodegenerative disorders, including trisomic mice. Loss of basal forebrain cholinergic neurons (BFCNs) and other DS/AD relevant hallmarks were observed in a well-established trisomic model (Ts65Dn, Ts). MCS attenuates these endophenotypes with beneficial behavioral effects in trisomic offspring. We postulate MCS ameliorates dysregulated cellular mechanisms within vulnerable BFCNs, with attenuation driven by novel gene expression. Here, choline acetyltransferase immunohistochemical labeling identified BFCNs in the medial septal/ventral diagonal band nuclei of the basal forebrain in Ts and normal disomic (2N) offspring at ~11 months of age from dams exposed to MCS or normal choline during the perinatal period. BFCNs (~500 per mouse) were microisolated and processed for RNA-sequencing. Bioinformatic assessment elucidated differentially expressed genes (DEGs) and pathway alterations in the context of genotype (Ts, 2N) and maternal diet (MCS, normal choline). MCS attenuated select dysregulated DEGs and relevant pathways in aged BFCNs. Trisomic MCS-responsive improvements included pathways such as cognitive impairment and nicotinamide adenine dinucleotide signaling, among others, indicative of increased behavioral and bioenergetic fitness. Although MCS does not eliminate the DS/AD phenotype, early choline delivery provides long-lasting benefits to aged trisomic BFCNs, indicating that MCS prolongs neuronal health in the context of DS/AD. Full article

Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant brain tumors, necessitating novel therapeutic approaches. Oncolytic treatments, particularly oncolytic viruses (OVs), have emerged as promising candidates by selectively infecting and lysing tumor cells while stimulating anti-tumor immunity. Various virus-based therapies are under investigation, including genetically engineered herpes simplex virus (HSV), adenovirus, poliovirus, reovirus, vaccinia virus, measles virus, and Newcastle disease virus, each exploiting unique tumor-selective mechanisms. While some, such as HSV-based therapies including G207 and Delytact^TM, have demonstrated clinical progress, significant challenges persist, including immune evasion, heterogeneity in patient response, and delivery barriers due to the blood–brain barrier. Moreover, combination strategies integrating OVs with immune checkpoint inhibitors, chemotherapy, and radiation are promising but require further clinical validation. Non-viral oncolytic approaches, such as tumor-targeting bacteria and synthetic peptides, remain underexplored. This review highlights current advancements while addressing critical gaps in the literature, including the need for optimized delivery methods, better biomarker-based patient stratification, and a deeper understanding of GBM’s immunosuppressive microenvironment. Future research should focus on enhancing OV specificity, engineering viruses to deliver therapeutic genes, and integrating OVs with precision medicine strategies. By identifying these gaps, this review provides a framework for advancing oncolytic therapies in GBM treatment. Full article

DNA connects the domains of genetic regulation and environmental interactions and plays a crucial role in neural development and cognitive function. The complex roles of genetic and epigenetic processes in brain development, synaptic plasticity, and higher-order cognitive abilities were reviewed in this study. Neural progenitors are formed and differentiated according to genetic instructions, whereas epigenetic changes, such as DNA methylation, dynamically control gene expression in response to external stimuli. These processes shape behavior and cognitive resilience by influencing neural identity, synaptic efficiency, and adaptation. This review also examines how DNA damage and repair mechanisms affect the integrity of neurons, which are essential for memory and learning. It also emphasizes how genetic predispositions and environmental factors interact to determine a person’s susceptibility to neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. Developments in gene-editing technologies, such as CRISPR, and non-viral delivery techniques provide encouraging treatment avenues for neurodegenerative disorders. This review highlights the fundamental role of DNA in coordinating the intricate interactions between molecular and environmental factors that underlie brain function and diseases. Full article

Background: The candidate therapeutic peptide TnP demonstrates broad, system-level regulatory capacity, revealed through integrated network analysis from transcriptomic data in zebrafish. Our study primarily identifies TnP as a multifaceted modulator of drug metabolism, wound healing, proteolytic activity, and pigmentation pathways. Results: Transcriptomic profiling of TnP-treated larvae following tail fin amputation revealed 558 differentially expressed genes (DEGs), categorized into four functional networks: (1) drug-metabolizing enzymes (cyp3a65, cyp1a) and transporters (SLC/ABC families), where TnP alters xenobiotic processing through Phase I/II modulation; (2) cellular trafficking and immune regulation, with upregulated myosin genes (myhb/mylz3) enhancing wound repair and tlr5-cdc42 signaling fine-tuning inflammation; (3) proteolytic cascades (c6ast4, prss1) coupled to autophagy (ulk1a, atg2a) and metabolic rewiring (g6pca.1-tg axis); and (4) melanogenesis-circadian networks (pmela/dct-fbxl3l) linked to ubiquitin-mediated protein turnover. Key findings highlight TnP’s unique coordination of rapid (protease activation) and sustained (metabolic adaptation) responses, enabled by short network path lengths (1.6–2.1 edges). Hub genes, such as nr1i2 (pxr), ppara, and bcl6aa/b, mediate crosstalk between these systems, while potential risks—including muscle hypercontractility (myhb overexpression) or cardiovascular effects (ace2-ppp3ccb)—underscore the need for targeted delivery. The zebrafish model validated TnP-conserved mechanisms with human relevance, particularly in drug metabolism and tissue repair. TnP’s ability to synchronize extracellular matrix remodeling, immune resolution, and metabolic homeostasis supports its development for the treatment of fibrosis, metabolic disorders, and inflammatory conditions. Conclusions: Future work should focus on optimizing tissue-specific delivery and assessing genetic variability to advance clinical translation. This system-level analysis positions TnP as a model example for next-generation multi-pathway therapeutics. Full article

Gene therapy is a groundbreaking strategy in regenerative medicine, enabling precise cellular behavior modulation for tissue repair. In situ nucleic acid delivery systems aim to directly deliver nucleic acids to target cells or tissues to realize localized genetic reprogramming and avoid issues like donor cell dependency and immune rejection. The key to success relies on biomaterial-engineered delivery platforms that ensure tissue-specific targeting and efficient intracellular transport. Viral vectors and non-viral carriers are strategically modified to enhance nucleic acid stability and cellular uptake, and integrate them into injectable or 3D-printed scaffolds. These scaffolds not only control nucleic acid release but also mimic native extracellular microenvironments to support stem cell recruitment and tissue regeneration. This review explores three key aspects: the mechanisms of gene editing in tissue repair; advancements in viral and non-viral vector engineering; and innovations in biomaterial scaffolds, including stimuli-responsive hydrogels and 3D-printed matrices. We evaluate scaffold fabrication methodologies, nucleic acid loading–release kinetics, and their biological impacts. Despite progress in spatiotemporal gene delivery control, challenges remain in balancing vector biocompatibility, manufacturing scalability, and long-term safety. Future research should focus on multifunctional “smart” scaffolds with CRISPR-based editing tools, multi-stimuli responsiveness, and patient-specific designs. This work systematically integrates the latest methodological advances, outlines actionable strategies for future investigations and advances clinical translation perspectives beyond the existing literature. Full article

Hydrogels have emerged as multifunctional biomaterials in cardiac surgery, offering promising solutions for myocardial regeneration, adhesion prevention, valve engineering, and localized drug and gene delivery. Their high water content, biocompatibility, and mechanical tunability enable close emulation of the cardiac extracellular matrix, supporting cellular viability and integration under dynamic physiological conditions. In myocardial repair, injectable and patch-forming hydrogels have been shown to be effective in reducing infarct size, promoting angiogenesis, and preserving contractile function. Hydrogel coatings and films have been designed as adhesion barriers to minimize pericardial adhesions after cardiotomy and improve reoperative safety. In heart valve and patch engineering, hydrogels contribute to scaffold design by providing bio-instructive, mechanically resilient, and printable matrices that are compatible with 3D fabrication. Furthermore, hydrogels serve as localized delivery platforms for small molecules, proteins, and nucleic acids, enabling sustained or stimuli-responsive release while minimizing systemic toxicity. Despite these advances, challenges such as mechanical durability, immune compatibility, and translational scalability persist. Ongoing innovations in smart polymer chemistry, hybrid composite design, and patient-specific manufacturing are addressing these limitations. This review aims to provide an integrated perspective on the application of hydrogels in cardiac surgery. The relevant literature was identified through a narrative search of PubMed, Scopus, Web of Science, Embase, and Google Scholar. Taken together, hydrogels offer a uniquely versatile and clinically translatable platform for addressing the multifaceted challenges of cardiac surgery. Hydrogels are poised to redefine clinical strategies in cardiac surgery by enabling tailored, bioresponsive, and functionally integrated therapies. Full article

Neuropathic pain is a significant global clinical issue that poses substantial challenges to both public health and the economy due to its complex underlying mechanisms. It has emerged as a serious health concern worldwide. Recent studies involving dorsal root ganglion (DRG) stimulation have provided strong evidence supporting its effectiveness in alleviating chronic pain and its potential for sustaining long-term pain relief. In addition to that, there has been ongoing research with clinical evidence relating to the role of small non-coding ribonucleic acids known as microRNAs in regulating gene expressions affecting pain signals. The signal pathway involves alterations in neuronal excitation, synaptic transmission, dysregulated signaling, and subsequent pro-inflammatory response activation and pain development. When microRNAs are dysregulated in the dorsal root ganglia neurons, they polarize macrophages from anti-inflammatory M2 to inflammatory M1 macrophages causing pain signal generation. By reversing this polarization, a therapeutic activity can be induced. However, the direct delivery of these nucleotides has been challenging due to limitations such as rapid clearance, degradation, and reduction in half-life. Therefore, safe and efficient carrier vehicles are fundamental for microRNA delivery. Here, we present a comprehensive analysis of miRNA-based nano-systems for chronic neuropathic pain, focusing on their impact in dorsal root ganglia. This review provides a critical evaluation of various delivery platforms, including viral, polymeric, lipid-based, and inorganic nanocarriers, emphasizing their therapeutic potential as well as their limitations in the treatment of chronic neuropathic pain. Innovative strategies such as hybrid nanocarriers and stimulus-responsive systems are also proposed to enhance the prospects for clinical translation. Serving as a roadmap for future research, this review aims to guide the development and optimization of miRNA-based therapies for effective and sustained neuropathic pain management. Full article

PAMAM dendrimers are distinguished by their capacity for functionalization, which enhances the properties of the compounds they transport, rendering them highly versatile nanoparticles with extensive applications in the biomedical domain, including drug, vaccine, and gene delivery. These dendrimers can be internalized into cells through various endocytic mechanisms, such as passive diffusion, clathrin-mediated endocytosis, and caveolae-mediated endocytosis, allowing them to traverse the cytoplasm and reach intracellular targets, such as the mitochondria or nucleus. Despite the significant challenge posed by the cytotoxicity of these nanoparticles, which is contingent upon the dendrimer size, surface charge, and generation, numerous strategies have been documented to modify the dendrimer surface using polyethylene glycol and other chemical groups to temporarily mitigate their cytotoxic effects. The potential of PAMAM dendrimers in cancer therapy and other biomedical applications is substantial, owing to their ability to enhance bioavailability, pharmacokinetics, and pharmacodynamics of active ingredients within the body. This underscores the necessity for further investigation into the optimization of internalization pathways and cytotoxicity of these nanoparticles. This review offers a comprehensive synthesis of the current literature on the diverse cellular internalization pathways of PAMAM dendrimers and their cargo molecules, emphasizing the mechanisms of entry, intracellular trafficking, and factors influencing these processes. Full article

Glioblastoma multiforme (GBM), the most aggressive and therapy-resistant glioma subtype, remains an urgent clinical challenge due to its invasive nature, molecular heterogeneity, and the protective constraints of the blood–brain barrier (BBB). Liposomes and extracellular vesicles (EVs) have emerged as two of the most promising nanocarrier systems capable of overcoming these limitations through improved drug delivery and cellular targeting. Their applications in glioma therapy span chemotherapy, immunotherapy, and gene therapy, each presenting distinct advantages and mechanisms of action. Liposomes offer structural flexibility, controlled release, and a well-established clinical framework, while EVs provide innate biocompatibility, low immunogenicity, and the ability to mimic natural intercellular communication. Both systems demonstrate the capacity to traverse the BBB and selectively accumulate in tumor tissue, yet they differ in scalability, cargo loading efficiency, and translational readiness. Comparative evaluation of their functions across therapeutic modalities reveals complementary strengths that may be leveraged in the development of more effective, targeted strategies for glioma treatment. Full article

Exogenous RNA application, also known as spray-induced gene silencing (SIGS), is a new approach in plant biotechnology that utilizes RNA interference (RNAi) to modify plant traits. This technique involves applying RNA solutions of double-stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), or microRNA (miRNA) directly onto plant surfaces. This triggers RNAi-mediated silencing of specific genes within the plant or invading pathogens. While extensively studied for enhancing resistance to pathogens, the application of exogenous RNA to regulate plant endogenous genes remains less explored, creating a rich area for further research. This review summarizes and analyzes the studies reporting on the exogenously induced silencing of plant endogenes and transgenes using various RNA types. We also discuss the RNA production and delivery approaches, analyze the uptake and transport of exogenous RNAs, and the mechanism of action. The analysis revealed that SIGS/exoRNAi affects the expression of plant genes, which may contribute to crop improvement and plant gene functional studies. Full article

Current osteoarthritis (OA) therapies fail to yield long-term clinical benefits, due in part to the lack of a mechanism for the targeted and confined delivery of therapeutics to OA joints. This study evaluates if M2 macrophages are effective cell vehicles for the targeted and confined delivery of therapeutic genes to OA joints. CT bioluminescence in vivo cell tracing and fluorescent microscopy reveal that intraarticularly injected M2 macrophages were recruited to and retained at inflamed synovia. The feasibility of an M2 macrophage-based adoptive gene transfer strategy for OA was assessed using IL-1Ra as the therapeutic gene in a mouse tibial plateau injury model. Mouse M2 macrophages were transduced with lentiviral vectors expressing IL-1Ra or GFP. The transduced macrophages were intraarticularly injected into injured joints at 7 days post-injury and OA progression was monitored with plasma COMP and histology at 4 weeks. The IL-1Ra-expressing M2 macrophage treatment reduced plasma COMP, increased the area and width of the articular cartilage layer, decreased synovium thickness, and reduced the OARSI OA score without affecting the osteophyte maturity and meniscus scores when compared to the GFP-expressing M2 macrophage-treated or PBS-treated controls. When the treatment was given at 5 weeks post-injury, at which time OA should have developed, the IL-1Ra-M2 macrophage treatment also reduced plasma COMP, had a greater articular cartilage area and width, decreased synovial thickness, and reduced the OARSI OA score without an effect on the meniscus and osteophyte maturity scores at 8 weeks post-injury. In conclusion, the IL-1Ra-M2 macrophage treatment, given before or after OA was developed, delayed OA progression, indicating that the M2 macrophage-based adoptive gene transfer strategy for OA is tenable. Full article

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease (ESKD) globally. Despite advances in our understanding of its pathophysiology, current therapies are often insufficient to stop its progression. In recent years, microRNAs (miRNAs)—small, non-coding RNA molecules involved in post-transcriptional gene regulation—have emerged as critical modulators of key pathogenic mechanisms in DKD, including fibrosis, inflammation, oxidative stress, and apoptosis. Numerous studies have identified specific miRNAs that either exacerbate or mitigate renal injury in DKD. Among them, miR-21, miR-192, miR-155, and miR-34a are associated with disease progression, while miR-126-3p, miR-29, miR-146a, and miR-215 demonstrate protective effects. These molecules are also detectable in plasma, urine, and renal tissue, making them attractive candidates for diagnostic and prognostic biomarkers. Advances in therapeutic technologies such as antagomiRs, mimics, locked nucleic acids, and nanoparticle-based delivery systems have opened new possibilities for targeting miRNAs in DKD. Additionally, conventional drugs, including SGLT2 inhibitors, metformin, and GLP-1 receptor agonists, as well as dietary compounds like polyphenols and sulforaphane, may exert nephroprotective effects by modulating miRNA expression. Recent evidence also highlights the role of gut microbiota in regulating miRNA activity, linking metabolic and immune pathways relevant to DKD progression. Further research is needed to define stage-specific miRNA signatures, improve delivery systems, and develop personalized therapeutic approaches. Modulation of miRNA expression represents a promising strategy to slow DKD progression and improve patient outcomes. Full article

Polycationic gene vectors have been studied extensively for gene delivery, and the charge density of polycations plays a pivotal role in condensing nucleic acids. Recently, we have synthesized two kinds of polycations with varied charge densities: poly(2-(dimethylamino)ethyl methacrylate) (denoted as A100) and a copolymer of 2-(tetramethyleneimino)ethyl methacrylate and 2-(diisopropyl-amino)ethyl methacrylate with a 3:1 feed ratio (denoted as B75D25). Despite its lower charge density, B75D25-based vectors exhibit higher transfection efficiency than A100-based vectors, prompting the hypothesis that hydrophobic interactions, rather than solely high charge density, enhance DNA complexation and gene delivery. This study aims to investigate the molecular mechanisms underlying these differences using molecular dynamics (MD) simulations to study the complexation of DNA with B75D25s and A100s. Our simulations reveal that DNA is quite uniformly covered by B75D25s, and the complexation is not only driven by the electrostatic attraction with DNA but more importantly by the hydrophobic interactions among B75D25s. In contrast, only a small fraction of A100s bind to DNA, which is due to the strong electrostatic repulsion among A100s. Our results reveal the contribution of hydrophobic interactions to the complexation of low-charge-density B75D25s with DNA. These results suggest that high charge density may not be essential for DNA condensation and efficient gene delivery. Full article