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.

Ion channel proteins (e.g., K+ channels) actively participate in external field-induced electroporation (EP), yet the mechanism by which open/conductive (O/O) state K+ channels influence bilayer EP remains unclear. Here, we employ molecular dynamics (MD) simulations to investigate the impact of the O/O-state KcsA channels on membrane EP. Two systems─pure POPC bilayers and bilayers embedded with the O/O-state KcsA─were subjected to nanosecond pulsed electric fields (nsPEF) of varying amplitudes and polarities. In addition to lipid pore formation, nsPEF directly triggers pore generation within the selectivity filter (SF) of KcsA, which evolves into complex pores accompanied by protein conformational changes. Negative pulses preferentially initiate SF pore formation, while positive pulses at lower amplitudes favor complex pore development. Poration time (tep) analysis demonstrates that O/O-state KcsA significantly accelerates membrane poration, particularly under negative pulses. To quantitatively characterize the poration, umbrella sampling (US) was applied to compute free energy profiles along reaction coordinates (RC). Biased simulations show that pure membranes exhibit similar free energy barriers for poration under positive and negative pulses, whereas KcsA-containing membranes display lower free energy under negative pulses, corroborating unbiased MD findings. These differences arise from asymmetric channel geometries, enhanced membrane potential heterogeneity, and altered water dipole distributions relative to those of pure membranes. This study advances understanding of field-protein interactions with the O/O-state KcsA channel-containing membranes and offers novel insights into its EP mechanisms.

Chemokine CCL5 overexpression combined with radiotherapy modulates Th1-mediated immune response and leads to significant tumor growth delay in mouse tumor models.

Mechanical disruption of cell membranes to enable intracellular delivery has been gaining traction as a methodology. Here, we show that a mechanical disruption-based strategy, filtroporation (FP), can be applied to edit the beta globin gene efficiently in human hematopoietic stem and progenitor cells. Gene expression analyses from RNA-Seq datasets demonstrate that electroporation (EP) yields greater transcriptional changes compared to FP globally, and gene pathway enrichment analyses suggest EP promotes stem cell differentiation while FP promotes cell division. Membrane repair occurs within 30 s of disruption by FP, and calcium signaling plays a key role in membrane repair in this context. Studies with fluorescently tagged membrane repair proteins, GRAF1, SNAP23, and CHMP4B, implicate the involvement of three mechanisms to reconstitute the cell barrier following FP. This work supports the evidence that the choice of intracellular delivery method affects transcriptional stem cell profile, and that membrane repair after mechanical disruption is a fast, multi-pathway process.

Over the past decade, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, traditional CAR-T cell engineering employs viral vectors, which has several limitations. Additionally, the immunosuppressive tumor microenvironment, particularly mediated by the PD-1/PD-L1 pathway, significantly restricts CAR-T cell efficacy. CRISPR/Cas9-mediated PD-1 knockout can enhance CAR-T cell anti-tumor activity, but traditional electroporation (EP) method often damages T cells. Herein, a novel lipid nanoparticles (LNPs)-mediated delivery technology are introduced to engineer CAR-T cells. The LNPs platform enables the simultaneous expression of CAR cassette and CRISPR/Cas9 gene editor in T cells via co-delivery of multiplex RNAs (CD19 CAR mRNA+Cas9 mRNA+sgRNA targeting PD-1). Importantly, LNPs exhibit higher transfection efficiency and superior cell viability compared to traditional electroporation method. The engineered CAR-T cells with PD-1 knockout, which express anti-CD19 CAR, can specifically kill CD19+ Nalm-6 tumor cells in vitro and display enhanced anti-tumor activity in vivo. Furthermore, LNPs-mediated co-delivery of Cas9 mRNA and sgRNAs targeting PD-1, TRAC, and B2M enables triple-knockout of T cells with high editing efficiencies (76% for PD-1, 86% for TRAC, and 80% for B2M), highlighting the ability for multiplex gene editing. This LNP-mediated delivery strategy has great potentials for the development of safer and more efficacious CAR-T cells.

The purpose of this study is to investigate the phenolic substance contents of Hypericum perforatum and Rheum ribes plants and their cytotoxicity on lung cancer cells (A549) and to reveal the effects of electroporation (EP) on the antiproliferative activities of these plants. Antiproliferative activities of plant extracts were determined in A549 cells, and their biocompatibility was evaluated in L-929 fibroblast cells through MTT assay. In electrochemotherapy (extracts+EP) applications of A549 cancer cells, eight square wave electrical pulse sequences with an intensity of 800V/cm were used with various doses of plant extracts. Both plant extracts were observed to be abundant in phenolic compounds and exhibited almost no cytotoxic effects, with IC50 values higher than 1000µg/mL for L-929 fibroblast cells. However, A549 cancer cells showed very good sensitivity to the cytotoxic activities of Rheum ribes and Hypericum perforatum extracts, with IC50 values of 297.32 and 241.10 µg/mL, respectively. The cytotoxic activity of both plant extracts increased considerably with EP, and cell viability percentages of extract+EP groups were observed to decrease significantly compared to extract-only groups (p

The action mechanism of bipolar pulse (BP) on biological plasma membranes─which boast complex compositions and asymmetric lipid distributions between the inner and outer leaflets─remains incompletely elucidated. In this study, coarse-grained molecular dynamics (MD) simulation techniques were used to conduct a systematic investigation of the mammalian average plasma membrane model, aiming to analyze the effects of BP and its application sequence on the electroporation process of the plasma membrane. During the experiments, two BP modes were used: depolarization-hyperpolarization bipolar pulses (D-H BP) and hyperpolarization-depolarization bipolar pulses (H-D BP). In both BP modes, we found that the plasma membrane exhibited two distinct electroporation states at the end of the pulse interval, which were defined as the Retaining state and the Resealing state. As the interval lengthens, the frequency of the Retaining state progressively declines, while that of the Resealing state steadily rises. Under H-D BP, the plasma membrane exhibits a higher frequency of the Retaining state and a notably larger perforation area compared to D-H BP. Analyzed within the Martini coarse-grained framework, our results primarily reveal a qualitative trend regarding the application sequence of BP. This trend suggests that the sequence may be a relevant factor for the electroporation (EP) process, potentially affecting pore expansion and localization, a finding that warrants further investigation.

Background and Aim: Genetically engineered pigs are invaluable biomedical models for xenotransplantation and the study of human diseases. Although electroporation (EP) and lipofection are individually effective for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) delivery, their combined application in porcine embryos has not been systematically evaluated. This study aimed to determine whether packaging Cas9-guided RNA complexes in cationic lipids enhances EP-mediated gene editing efficiency without compromising embryonic development. Materials and Methods: Porcine zygotes with their zona pellucida removed were edited using RNPs targeting beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) or growth hormone receptor (GHR) genes. Four treatment groups were tested: (1) EP with RNPs (EP), (2) EP with lipofectamine-packaged RNPs (EPL), (3) transfection with lipofectamine-packaged RNPs before EP (TL + EPL), and (4) EP followed by lipofection (EPL + TL). Blastocyst formation was evaluated morphologically, and mutation rates were assessed by Sanger sequencing followed by tracking of indels by decomposition (TIDE) analysis. Results: Blastocyst formation rates were comparable across all treatments, indicating that lipofectamine packaging and EP caused no detectable cytotoxicity. For B4GALNT2, no mutations were induced by EP alone, whereas TL + EPL treatment significantly increased total and mosaic mutation rates (p < 0.05). For GHR, the total mutation and mosaic mutation rates were likewise higher in TL + EPL compared with EP, although mutation efficiency (indel percentage per edited embryo) remained unchanged. These results suggest that pre-EP lipofection promotes RNP uptake by facilitating lipid-membrane interactions that are potentiated by subsequent membrane destabilization through EP. Conclusion: Packaging RNPs in cationic lipids and applying sequential lipofection followed by EP significantly enhances CRISPR/Cas9-mediated mutagenesis in porcine zygotes without affecting developmental competence. This dual-delivery approach provides a simple, reproducible, and low-toxicity workflow for generating gene-edited embryos, with potential applicability to large-animal biomedical models. Keywords: cationic lipid, CRISPR/Cas9, electroporation, genome-editing efficiency, lipofectamine, porcine zygote, xenotransplantation.

BackgroundGene therapy has emerged as a transformative biomedical approach, offering new therapeutic possibilities from many so far uncurable diseases through the introduction of recombinant nucleic acids into target cells. Among non-viral delivery techniques, gene electrotransfer (GET) has become one of the frequently applied methods in clinical trials. It is based on the application of short, high-intensity electric pulses that transiently permeabilize cell membranes and enable the efficient transfer of plasmid DNA or other types of recombinant nucleic acids into various cell types. Beyond its role in gene delivery, GET can trigger complex cellular responses, as the introduced DNA interacts with intracellular DNA sensing pathways involved in innate immunity and inflammation. These responses can influence the therapeutic outcome – either by enhancing antitumour and vaccine-related immune activation or by reducing transfection efficiency when excessive inflammation or cell death occur. Our experimental findings in tumour, muscle, and skin models have shown that even non-coding plasmid DNA delivered by GET can induce local immune stimulation and tissue-specific inflammatory signaling, suggesting that the delivered DNA itself contributes to therapeutic efficacy.ConclusionsThe dual nature of cellular responses following plasmid DNA GET represents both an opportunity and a challenge. Controlled activation of innate immunity can be harnessed to amplify antitumour or vaccine efficacy, while excessive responses may hinder applications requiring cell survival and sustained expression. Understanding these mechanisms enables the rational optimization of GET parameters and plasmid vector design to fully exploit the adjuvant effect or reduce the off-target effect of DNA sensing after GET, based on the desired application.

Highly pathogenic avian influenzas (HPAIs) is a continuing public health threat. Here, we describe the development of plasmid-encoded H5N1 hemagglutinin antigens representing clades that have caused human zoonoses. Electroporation (EP) delivery of clade 2.3.2.1c HA (pCamb) DNA was immunogenic in mice but only partially protective against clade 2.3.4.b challenge. Homologous challenge resulted in complete protection, suggesting that matched clade antigens are important for protection. Contemporary clade 2.3.4.4b HA (pMich) DNA plasmids supported robust cellular and humoral responses when delivered via EP. Co-immunization with plasmids representing both H5 clades supported high titers of binding and neutralizing antibodies against both antigens and complete protection from clade 2.3.4.4b challenge. We formulated the pMich plasmid DNA in lipid nanoparticles (LNPs). A single dose of pMich DNA-LNP supported long-lived immunity in mice that was protective against challenge at both acute and memory timepoints. These data demonstrate that DNA can support robust anti-HPAI immunity and protection.

Abstract Hereditary arrhythmogenic cardiomyopathy (ACM) and hypertrophic cardiomyopathy (HCM) are autosomal dominant inherited heart diseases responsible for most cases of sudden cardiac death in healthy people aged younger than 35, specially in young athletes. Modelling these disorders in large animals is expected to serve as an invaluable resource for investigating disease-specific pathways and mutation impacts. Moreover, by facilitating rigorous preclinical testing, it opens avenues for developing targeted gene therapies. We have established a working pipeline for generating genetically modified pig models using CRISPR/Cas9 technology to investigate key cardiac proteins involved in hereditary heart conditions. Specifically, this initiative aims to create large animal models with targeted mutations in the TMEM43, PKP2 and MYBPC3 genes. Our methodology involves maturing oocytes from pig ovaries, followed by in vitro fertilization with Yucatan pig semen. Prior fertilization, oocytes undergo CRISPR/Cas9-mediated genetic modification via electroporation (EP) or cytoplasmic microinjection (CMI) with Cas9 proteins and specific guide RNAs (gRNAs). Furthermore, we have established the surgical procedures required for embryo transfer. Surrogate sows, prepared through hormonally induced heat cycles, undergo an abdominal laparotomy to insert CRISPR-modified embryos containing PKP2 mutations directly into the oviduct. Post-fertilization viability rates are 33-36%, and development to the blastocyst stage is achieved in 11-12% of cases for EP and 18% for CMI. Among these, CMI has proven the most efficient, particularly for the PKP2 model, where an impressive 80% of blastocysts exhibit successful gene editing. This high success rate marks a significant advance in generating precise and robust large-animal disease models. In our first trial, ultrasound monitoring of the gilt at day 35 post-surgery revealed successful implantation of seven embryonic vesicles in the uterine wall. Although the development of these fetuses was naturally interrupted before they reached term, the implantation of the embryos constituted the first major challenge initially presented by the transfer process. Consequently, we are currently working on improving the pregnancy diagnostics and monitoring of the pregnant sow in future transfers to obtain our first generation of genetically modified piglets after a gestation period of 115 days. By applying cutting-edge gene-editing techniques on pig oocytes, we aim to produce animal models that replicate human cardiac disease pathology with high fidelity, enhancing their translational potential for understanding disease mechanisms and testing new therapies. As such, this work represents a major step toward the implementation of personalized therapeutic strategies in cardiology, bridging the gap between molecular insights and clinical application.Distribution of mutations in blastocysts Ultrasound of developing embryo at 35d

Lipid nanoparticle-mediated epitope editing in human HSPCs enables resistance to CD45-directed CAR-T cell therapy

BackgroundLassa fever (LF) is an acute viral hemorrhagic illness endemic to West Africa, with no licensed vaccines or targeted treatments available, highlighting a critical gap in global health preparedness. T cell-mediated immunity plays a central role in viral control and survival. Synthetic DNA vaccines offer a promising strategy to induce both humoral and cellular immunity against LF.MethodsA Phase 1b, randomized, double-blind, placebo-controlled trial was conducted to assess the safety, tolerability, and immunogenicity of INO-4500, a DNA vaccine encoding the Lassa virus (Josiah strain) glycoprotein precursor (GPC). A total of 220 healthy adults were randomized to receive either 1 mg or 2 mg of INO-4500 (intervention), or placebo, administered intradermally (ID) followed by electroporation (EP) at Day 0 and Week 4. Safety was evaluated through Week 48. Primary immunogenicity endpoints included humoral and cellular immune responses at multiple timepoints post-vaccination.ResultsINO-4500 was well tolerated, with no Grade 3 or higher treatment-emergent adverse events (TEAEs) deemed to be related to the intervention; 88.6% of all TEAEs were Grade 1. No cases of attributable hearing loss were reported. INO-4500 groups demonstrated statistically significant increases in Lassa virus GPC-specific binding antibodies at Weeks 6 and 12 compared to placebo, with the 2 mg group eliciting the strongest responses. T cell responses remained elevated above baseline through Week 48 in both INO-4500 groups, indicating durable cellular immunity.ConclusionsDNA vaccine INO-4500 was well tolerated and elicited durable humoral and cellular immune responses in healthy adults. These findings support further clinical development of INO-4500 as a potential preventive vaccine to reduce LF-associated morbidity and mortality in endemic regions.Clinical Trial Registrationhttps://clinicaltrials.gov, identifier NCT04093076

Effective targeting of E2F1 transcription factor via siRNA gene electrotransfer in HT-29 colorectal carcinoma xenografts.

Gene Electrotransfer and DNA Sensing

Revolutionizing pediatric gene and cell therapy: The hope for lipid-based nanoparticles in blood disorders.

Improving bleomycin electrochemotherapy with gold nanoparticles: first in vivo study on intra-tumoral field amplification.

Induced pluripotent stem cells (iPSCs) are useful for studying genetic and rare diseases and can be generated by reprogramming immortalized lymphoblastoid cell lines (LCLs) stored in global repositories with detailed genotype and phenotype data. Traditional bulk-type electroporators are commonly used for gene electrotransfer in reprogramming, but they have major drawbacks, including high costs associated with electric pulse generators and the requirement for fixed volumes for costly reprogramming factors. These limitations hinder cost-effective and scalable iPSC generation, particularly when working with large numbers of LCLs with diverse genotypes. We aimed to develop a flow-through-type electroporator utilizing microchannels for the generation of iPSCs from LCLs, to reduce the costs associated with traditional bulk-type electroporators and enable parallel processing for LCLs with various genotypes. We applied a continuous wave of biphasic alternating voltage (~10 V one-sided amplitude) to micro-scaled electrodes within the microchannel to develop a flow-through electroporator. Numerical simulations were conducted to assess the electric field distribution and its applicability to pore formation in the plasma membrane. To optimize electroporation and flow conditions, we used plasmid pCXLE-EGFP (encoding Green Fluorescent Protein, GFP) for gene electrotransfer to LCLs. Reprogramming factors (pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pCXLE-hUL) were also delivered to the cells via the same system. The flow-through electroporator achieved 31% transfection efficiency with 78% cell viability, 2 d post-electroporation. In each condition, only 3 µL of cell suspension was used with 107 cells/mL of cells and 500 ng/µL plasmid vector. A reprogramming efficiency of 0.048% was obtained, which is comparable to that achieved using bulk-type electroporators. This developed flow-through electroporator with microchannel technology offers significant advantages over traditional methods, including the potential to reduce costs and the ability to process small volumes of cell suspension, making it suitable for parallel processing of LCLs with diverse genotypes. The system provides a promising approach for scalable and potentially cost-effective iPSC generation.

Expression of inducible damage-associated molecular patterns after interleukin-12 gene electrotransfer in mouse melanoma and colorectal cell lines.

Synchronous nanowire-assisted electroporation and peracetic acid oxidation to inhibit VBNC cells formation: Reversible electroporation pores reinforce permeation of peracetic acid for cellular destruction.