Search Results (281)

Hyperbranched poly(β-amino ester) (HPAE) is identified as a unique non-viral carrier capable of sustaining high-efficiency transfection under elevated plasmid concentrations, overcoming the aggregation and toxicity limitations of conventional lipid and PEI reagents. We demonstrate that transfection enhancement is driven by concentration-dependent synergism between membrane accumulation and endosomal escape. Guided by this mechanism, a half-volume transfection strategy was established to transiently elevate plasmid concentration without compromising cell viability, enabling superior lentivirus yield and purity. These findings define plasmid concentration as a previously overlooked regulatory axis in nanoparticle-mediated gene delivery and position HPAE as a high-performance platform for scalable therapeutic vector production. Full article

Gut dysbiosis, defined as a disruption in the structure or function of the intestinal microbiota, is increasingly recognized as a key contributor to inflammatory, metabolic, and neuropsychiatric diseases. Conventional interventions such as broad-spectrum antibiotics, generic probiotics, and fecal microbiota transplantation (FMT) often show limited and inconsistent efficacy because they lack specificity, durability, and robust safety controls. In contrast, recent advances in DNA-based technologies are reshaping the therapeutic landscape by enabling targeted, programmable, and mechanistically informed modulation of the gut ecosystem. This review presents an integrated overview of three major domains driving this shift: CRISPR-based systems that selectively delete, silence, or reprogram microbial genes; synthetic biology-driven live therapeutics engineered to sense disease-associated cues and execute controlled responses; and metagenomics-informed strategies that tailor interventions to patient-specific microbial gene profiles and functional deficits. Additionally, we examine the continued evolution of FMT toward DNA-optimized workflows and defined microbial consortia that offer safer, more standardized alternatives to crude donor material. Across these domains, we discuss delivery platforms (including bacteriophages, conjugative plasmids, extracellular vesicles, and synthetic nanoparticles), and compare their efficiency, specificity, and scalability. We further highlight how DNA-guided interventions interface with host immunity—shaping Treg/Th17 balance, mucosal barrier function, and inflammatory signaling—while also analyzing ecological and evolutionary risks, biocontainment strategies, and regulatory classification gaps that will govern clinical translation. Together, these developments signal a transition from empirical microbiome manipulation to rational ecosystem engineering. DNA-guided therapies hold strong promise for precise and personalized management of gut-related diseases, but their success will depend on rigorous ecological risk assessment, long-term monitoring, and adaptive regulatory frameworks alongside continued technological innovation. Full article

Biologically relevant primary cell samples are inherently heterogeneous and often require selective enrichment prior to genetic manipulation. We previously demonstrated a vortex-assisted microfluidic platform that integrates size-selective cell trapping with electroporation; however, its limited processing capacity constrained applications requiring larger sample volumes. Here, we present a scaled version of this integrated system achieved through electrode array redesign and electrical optimization. The updated architecture increases processing capacity while preserving size-selective trapping behavior, electric field uniformity, and device stability. Systematic optimization of electrical and buffer conditions enables efficient delivery of plasmid DNA and in vitro-transcribed mRNA into primary human cells, with performance approaching benchmark chemical transfection methods. By scaling an integrated trapping–electroporation workflow without compromising delivery performance, this platform advances microfluidic cell engineering toward practical processing of heterogeneous primary cell samples. Full article

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species. 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

Background/Objectives: Antigen presenting cells (APCs) and immune cells have unique properties to drive or suppress immune responses. They are therefore key targets for the expression of vaccine antigens or transgene proteins. To better determine the utility of different molecular therapies to modify these cells, mRNA and DNA-based molecular therapy vectors were compared for their ability to genetically modify immune cells after intradermal injections in mice. DNA-based vectors included naked plasmid DNA, plasmid packaged in lipid nanoparticles (LNPs), and replication-defective adenovirus (Ad) vectors. mRNA delivery was mediated by packaging into LNPs like those used in COVID-19 vaccines. Methods: Each vector was used to deliver Cre recombinase into Cre reporter mice whose cells were activated to express green fluorescent protein (GFP) and firefly luciferase after Cre recombination. The mice were injected intradermally (ID) near the base of their tail at a site that drains into the inguinal lymph node. Luciferase activity was imaged in the living mice 1 or 4 days after vector injection. The animals were then euthanized, and luciferase activity was imaged in the draining inguinal lymph node. Cells were prepared from the intradermal injection site and from the draining lymph node to determine which immune cells were genetically modified by phenotyping CD45, CD3, and CD11b GFP-positive cells by flow cytometry. Given that the skin uniquely contains Langerhans dendritic cells, these CD207⁺ cells were also phenotyped in skin samples and in the draining lymph node. Results: In both the skin and in the draining lymph node, the rank order of luciferase and GFP activation by the vectors were: (1) Ad; (2) mRNA-LNP; (3) DNA-LNP; and (4) naked DNA. Only mRNA-LNP and Ad vectors mediated obvious luciferase activity in the living animals and in the draining lymph nodes by imaging. Notably, both vectors appeared to leak from the ID injection site and not only modify the draining lymph node but also strongly modify the livers of the mice. Naked DNA and DNA-LNP mediated detectable GFP activation in the skin and draining lymph node in some mice, but this activity was low and did not reach statistical significance when compared to PBS-treated animals. mRNA-LNPs and Ad both mediated significant Cre delivery in CD45⁺, CD3⁺, CD11b⁺, and CD207⁺ immune cells in the skin and in the lymph node, with adenovirus mediating consistently higher levels of expression in all of the tested cells. Conclusions: These data indicate that mRNA-LNP and Ad vectors mediate stronger modification of skin and lymph node immune cells after intradermal injections. Naked DNA and DNA-LNPs were markedly less potent at this activity than the other vectors. These data are consistent with the higher vaccine potency of mRNA-LNP and Ad vectors and suggest that approaches that increase targeting of immune cell subsets may have utility to increase efficacy while also reducing off-target modification of tissues like the liver. Full article

The GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technology) system enables efficient gene stacking within an Agrobacterium T-DNA. Using unidirectional site-specific recombinases and alternating selection markers, it allows precise, sequential assembly of multiple genes directly within an Agrobacterium virulence plasmid. Here, we modified Agrobacterium tumefaciens strain EHA105 to create JGT105 as a GAANTRY recipient and constructed a 15.8 kb T-DNA containing five cargo sequences. We compared the performance of the JGT105 5-stack strain against a conventional binary vector carrying the same cargo sequences in Arabidopsis and potato transformation. The transformation efficiencies were comparable for the GAANTRY strain and the binary vector (potato: 83% vs. 82%; Arabidopsis: 1.73% vs. 1.95%). Single T-DNA insertion frequencies were also similar between the two systems (17.6% for GAANTRY vs. 24.5% for the binary vector construct in potato; 10.3% vs. 18.2% in Arabidopsis, respectively). Notably, the GAANTRY construct had significantly reduced vector backbone transfer in potato (10.0% vs. 26.5%) for the binary vector, whereas rates were higher in Arabidopsis (37.5% vs. 48.9%). These results show that the JGT105 GAANTRY strain is an effective T-DNA delivery system, matching binary vector transformation efficiency while offering lower backbone integration frequency, facilitating the generation of high-quality, multi-gene transgenic plants. Full article

Background/Objectives: Prostate cancer is the most commonly diagnosed cancer in men and a leading cause of cancer-related mortality, highlighting the need for delivery systems capable of efficiently transporting both chemotherapeutic drugs and therapeutic genes to tumor cells. Generation-3 diaminobutyric polypropylenimine (DAB) dendrimers display low toxicity, high drug loading capacity and efficient gene delivery, and can be engineered as camptothecin-bearing PEGylated carriers complexed with plasmid DNA. The aim of this study was to compare microfluidic processing with conventional hand mixing for the preparation of camptothecin-bearing PEGylated DAB dendriplexes and to evaluate the impact of formulation methods and microfluidic parameters on their physicochemical properties, cellular uptake and gene expression in prostate cancer cells. Methods: Camptothecin-bearing PEGylated DAB dendrimers were synthesized and complexed with plasmid DNA to form dendriplexes. Formulations were prepared either by microfluidics, using different total flow rates and aqueous: organic flow rate ratios, or by conventional hand mixing. The resulting dendriplexes were characterized for DNA condensation, particle size, polydispersity index and zeta potential. Morphology was assessed by transmission electron microscopy. Cellular uptake of fluorescein-labelled DNA and β-galactosidase reporter gene expression were evaluated in PC3-Luc and DU145 prostate cancer cells. Results: Both microfluidic and hand-mixed methods produced stable, nanosized, positively charged dendriplexes with efficient and sustained DNA condensation (more than 99% over 24 h). Microfluidic processing, particularly at an aqueous: organic flow rate ratio of 3:1, yielded dendriplexes with hydrodynamic diameters and zeta potentials comparable to or slightly improved over hand-mixed formulations. These microfluidic conditions significantly enhanced cellular uptake in both PC3-Luc and DU145 cells. In PC3-Luc cells, this translated into β-galactosidase expression levels comparable to hand-mixed dendriplexes and higher than naked DNA, whereas in DU145 cells, transfection efficiencies remained modest for all formulations despite increased uptake. Conclusions: Microfluidic processing enables the reproducible and scalable preparation of camptothecin-bearing PEGylated DAB dendriplexes with tunable physicochemical properties. Under selected conditions, in vitro cellular uptake and gene expression were comparable to conventional hand mixing, supporting microfluidics as a robust alternative platform for the manufacture of dendrimer-based systems for combined chemo–gene delivery in prostate cancer. Full article

Background/Objectives: The emergence of immune-evasive SARS-CoV-2 variants highlights the need for adaptable vaccine strategies. Trimeric receptor-binding domain (tRBD) antigens offer structural and immunological advantages over monomeric RBDs, but DNA vaccine efficacy has been limited by inefficient antigen expression, particularly in non-dividing antigen-presenting cells. Although cytoplasmic transcription–based DNA platforms have been developed to overcome nuclear entry barriers, their utility for antigen structure–function optimization remains underexplored. This study evaluated whether integrating a rationally designed trimeric RBD with a T7-driven cytoplasmic transcription system could enhance immunogenic performance. Methods: A DNA vaccine encoding a tandem trimeric SARS-CoV-2 RBD was delivered using a T7 RNA polymerase-driven cytoplasmic transcription system. In vitro antigen expression was assessed following Lipofectamine 3000-mediated transfection. In vivo, mice were immunized with the SM-102-based Rpol/tRBD/LNP formulation, and immunogenicity was assessed by antigen-specific antibody titers, serum neutralizing activity, and T-cell response profiling, together with basic safety/tolerability evaluations. Results: The T7-driven cytoplasmic transcription system markedly increased antigen mRNA and protein expression compared with conventional plasmid delivery. Rpol/tRBD vaccination induced higher anti-RBD IgG titers, enhanced neutralizing antibody activity, and robust CD8⁺ T cell responses relative to monomeric RBD and plasmid-based trimeric RBD vaccines. Immune responses were Th1-skewed and accompanied by germinal center activation without excessive inflammatory cytokine induction, body-weight loss, or hepatic and renal toxicity. Conclusions: This study demonstrates that integrating rational trimeric antigen engineering with direct cytoplasmic transcription enables balanced and well-tolerated immune activation in a DNA vaccine context. The T7 autogene-based platform provides a flexible framework for antigen structure–function optimization and supports the development of next-generation DNA vaccines targeting rapidly evolving viral pathogens. Full article

Background/Objectives: A key disadvantage of DNA vaccines is ineffective uptake of plasmid DNA, resulting in low immunogenicity. A way to overcome it is forced DNA delivery, which requires specialized equipment and/or reagents. Effective delivery of plasmids without specialized devices or using commonly available ones would significantly increase DNA vaccine applicability. Here, we delivered DNA by intradermal injections, facilitating them by optimized sonoporation (SP) or electroporation (EP), and we compared these methods by their capacity to support the production of foreign proteins in mice. Methods: DNA delivery was optimized using the plasmid encoding firefly luciferase (Luc) (pVaxLuc). Luc production was assessed by bioluminescence imaging (BLI) (IVIS, PerkinElmer, Shelton, CT, USA; LumoTrace Fluo, Abisense, Dolgoprudny, Russia). Female BALB/c mice were injected intradermally (id) with pVaxLuc in phosphate buffers of varying ionic strengths. Injection sites were subjected to SP (Intelect Mobile, Chattanooga, UK) or EP (CUY21EDITII, BEX Co., Tokyo, Japan) or left untreated. Optimal delivery protocols were selected based on the highest in vivo levels of photon flux according to BLI. Optimal protocols for id injections with/without EP were applied to DNA-immunized mice with HIV-1 clade A reverse transcriptase. Antibody response induced by DNA immunization was assessed by ELISA. Results: The optimal phosphate buffers for id delivery had ionic strengths from 81 to 163 mmol/L. The optimal SP regimen included an acoustic pressure of 2.4 W/cm² applied in a duty cycle of 2%. The optimal EP regimen included bipolar driving pulses of 100 V, a pulse duration of 10 ms, and an interval between the pulses of 20 ms. Optimized DNA delivery by id/SP injection was inferior to both id/EP and id alone. DNA immunization with HIV-1 RT by id injections induced anti-RT antibodies in a titer of 10⁴ and by id/EP in a titer of 10⁵. Conclusions: Electroporation of the sites of id DNA injection provided the highest levels of production of luciferase reporters and induced a strong antibody response against HIV-1 RT. Full article

Cancer immunotherapy increasingly relies on nucleic acid-based vaccines, yet achieving efficient and safe delivery remains a critical limitation. Polyplexes—electrostatic complexes of cationic polymers and nucleic acids—have emerged as versatile carriers offering greater chemical tunability and multivalent targeting capacity compared to lipid nanoparticles, with lower immunogenicity than viral vectors. This review summarizes key design principles governing polyplex performance, including polymer chemistry, architecture, and assembly route—emphasizing microfluidic fabrication for improved size control and reproducibility. Mechanistically, effective systems support stepwise delivery: tumor targeting, cellular uptake, endosomal escape (via proton-sponge, membrane fusion, or photochemical disruption), and compartment-specific cargo release. We discuss therapeutic applications spanning plasmid DNA, siRNA, miRNA, mRNA, and CRISPR-based editing, highlighting preclinical data across multiple tumor types and early clinical evidence of on-target knockdown in human cancers. Particular attention is given to physiological barriers and engineering strategies—including size-switching systems, charge-reversal polymers, and tumor-penetrating peptides—that improve intratumoral distribution. However, significant challenges persist, including cationic toxicity, protein corona formation, manufacturing variability, and limited clinical responses to date. Current evidence supports polyplexes as a modular platform complementary to lipid nanoparticles in selected oncology indications, though realizing this potential requires continued optimization alongside rigorous translational development. Full article

The mammary gland is a valuable model in cancer research and developmental biology. Gene delivery techniques are crucial for mammary tissue research to understand how genes function and study on diseases such as cancer. Viral vector-based approaches provide a high degree of transduction efficiency, but they raise safety and immunogenicity concerns, whereas non-viral vector-based approaches are considered safer and have lower immunogenicity than viral methods. Unfortunately, non-viral gene delivery has rarely been applied to the mammary glands because it is technically challenging. Here, we developed a novel method for in vivo transfection of epithelial cells lining murine mammary glands via intraductal injection of plasmid DNA using a breath-controlled glass capillary and subsequent electroporation (EP) of the injected area. Female mice were transfected with plasmids harboring the enhanced green fluorescent protein (EGFP) gene. Widespread EGFP fluorescence was observed in the mammary epithelial cells of the ducts and adipocytes adjacent to the ducts. As this in vivo gene delivery method is simple, safe, and efficient for gene transfer to the mammary glands, we named this technique “Mammary Intraductal Gene Electroporation” (MIGE). The MIGE method is a useful experimental tool for studies on mammary gland development and differentiation as well as breast cancer research. Full article

Coconut genetic improvement remains challenging due to low regeneration efficiency and limited transformation success. We optimized major components of a gene gun-mediated transient transformation system and evaluated explant types to support future establishment of a stable transformation pipeline. Three reporter genes (eGFP, GUS, and RUBY) were compared in coconut callus, and eGFP was selected as the most suitable due to its strong and non-destructive fluorescence. Background interference in GUS staining was reduced by adjusting the methanol–GUS ratio to 4:10. Single-factor optimization using callus tissue identified 0.4 M mannitol, 300–500 μg gold particles, 1.5 μg plasmid DNA, a 6.5 cm target distance, and 7 MPa pressure as effective parameters for biolistic delivery. Among the callus types, spongy callus showed strong transient eGFP expression but displayed loose and watery morphology consistent with non-embryogenic callus. In contrast, crumbly and smooth callus exhibited compact structures previously associated with embryogenic competence, although transient expression levels were lower. Among differentiated tissues, germinated zygotic embryo plumules and distal young leaflets exhibited moderate transient expression, supporting their suitability as transformation targets. These findings provide practical guidance on reporter selection, parameter refinement, and explant choice for future establishment of an efficient genetic transformation system in coconut. Full article

Background/Objectives: Cervical cancer is a leading cause of cancer-related mortality among women, primarily driven by persistent infections with high-risk human papillomavirus (HPV), particularly HPV-16. Vaccines based on plasmid DNA encoding the viral oncogenes E6 and E7 represent a promising immunotherapeutic strategy, but their efficacy remains limited due to poor cellular uptake. Cell-penetrating peptides such as RALA improve intracellular delivery, and functionalization with octa-arginine peptide conjugated to mannose (R8M) further enhances targeting of antigen-presenting cells (APCs). This study aimed to obtain the minicircle DNA (mcDNA) encoding mutant HPV-16 E6 and/or E7 antigens, and optimize its complexation with mannosylated RALA-based nanoparticles to improve vector delivery and consequently antigen presentation. Methods: Nanoparticles were formulated at different concentrations of RALA, with and without R8M functionalization. Their characterization included hydrodynamic diameter, polydispersity index, zeta potential, complexation efficiency (CE), stability, morphology, and Fourier-Transform Infrared Spectroscopy. In vitro assays in JAWS II dendritic cells (DCs) assessed biocompatibility, transfection efficiency and target gene expression. Results: Optimal conditions were obtained at 72.5 µg/mL of RALA, producing nanoparticles smaller than 150 nm with high CE (>97%) and uniform size distribution. Functionalization with R8M at 58 µg/mL preserved these characteristics when complexed with all mcDNA vectors. The formulations were biocompatible and effectively transfected DCs. Mannosylated formulations enhanced antigenic expression compared to non-mannosylated counterparts, evidencing a mannose-receptor-mediated uptake, while increasing the production of pro-inflammatory cytokines. Conclusions: Nanoparticles based on the RALA peptide and functionalized with R8M significantly improved mcDNA transfection and gene expression in APCs. These findings support further investigation of this system as a targeted DNA vector delivery platform against HPV-16. Full article