Search Results (120)

High-frequency irreversible electroporation (H-FIRE) is a nonthermal tumor ablation technique that disrupts the blood–brain barrier (BBB) in a focal and reversible manner. However, the mechanisms underlying this disruption remain poorly understood, particularly the role of small tumor-derived extracellular vesicles (sTDEVs) released from ablated tumor cells. In this study, we investigate the proteomic and functional alterations of sTDEVs released from F98 glioma and LL/2 Lewis lung carcinoma cells following H-FIRE ablation. Mass spectrometry analysis revealed 108 unique proteins in sTDEVs derived from ablative doses of H-FIRE, which are capable of disrupting the BBB in an in vitro model. Proteomic analysis of TDEVs highlights key changes in pathways related to integrin signaling, Platelet-derived growth factor receptor (PDGFR) signaling, and ubiquitination, which may underline their interactions with brain endothelial cells. These “disruptive” sTDEVs exhibit enhanced tropism for cerebral endothelial cells both in vitro and in vivo, where they persist in the brain longer than sTDEVs released after non-ablative H-FIRE doses. Notably, when introduced into a healthy Fischer rat model, disruptive sTDEVs are associated with increased recruitment of Iba1+ immune cells, suggesting a potential role in modulating post-ablation immune responses. However, despite their altered protein composition, these vesicles do not directly increase BBB permeability in vivo. This study is the first to demonstrate that electroporation-based tumor ablation significantly alters the composition and functionality of tumor-derived extracellular vesicles, potentially influencing the tumor microenvironment post-ablation. These findings have important implications for developing multimodal treatment strategies that combine H-FIRE with systemic therapies to enhance efficacy while managing the peritumoral microenvironment. Full article

Newborn mice (up to 6 d after birth) are suitable for genetic manipulations, such as facial vein-mediated injection, owing to their hairless and thin skin. Their small body volumes also facilitate the rapid dissemination of injected solutions, supporting gene engineering-related experiments. However, anesthesia in newborns is challenging because of the potential risks associated with anesthetic agents. Isoflurane inhalation anesthesia is an option, although its effects on brain development remain under investigation. In this study, we established a reproducible protocol for delivering nucleic acids to juvenile mouse testes using a simple isoflurane-based anesthetic system prepared from common laboratory equipment. Using this system, nucleic acids were successfully delivered to juvenile mouse testes via intra-testicular injection, followed by in vivo electroporation. The present isoflurane-based method achieved >90% postoperative survival with normal maternal nursing observations. Gene delivery resulted in limited transfection of seminiferous tubules but efficient interstitial Leydig cell transfection. Thus, gene engineering in somatic and germ cells in neonatal mice will be facilitated using the anesthetic protocol established in this study. Full article

RAC3 encodes a small Rho-family GTPase essential for cytoskeletal regulation and neurodevelopment, and de novo RAC3 variants typically act as gain-of-function alleles that cause severe neurodevelopmental disorders. In this study, we analyzed a fetus with multisystem congenital anomalies and identified a de novo RAC3 p.(T17R) variant by genome sequencing. To elucidate the pathogenicity of this variant, we combined in silico variant prioritization, structural and energetic modeling, and pathogenicity prediction with in vitro biochemical assays, including GDP/GTP exchange, GTP hydrolysis, effector pull-down, and luciferase reporter analyses in COS7 cells, as well as morphological analysis of primary hippocampal neurons. Furthermore, we performed in vivo analyses using a mouse in utero electroporation to assess cortical neuron migration, axon extension, and dendritic development. Our biochemical results suggest that RAC3-T17R exhibits markedly increased GDP/GTP exchange, with a preference for GDP binding, and undetectable GTP hydrolysis. The mutant displayed minimal binding to canonical RAC effectors (PAK1, MLK2, and N-WASP) and failed to activate SRF-, NFκB-, or AP1-dependent transcription. Neuronal overexpression of RAC3-T17R impaired axon formation in vitro, while in vivo expression delayed cortical neuron migration and axon extension and reduced dendritic arborization. Clinically, the fetus exhibited corpus callosum agenesis, microcephaly, organomegaly, and limb contractures. Collectively, these findings indicate that the RAC3 p.(T17R) variant may represent a signaling-deficient allele with pleiotropic, variant-specific mechanisms that disrupt corticogenesis and broader organogenesis. Our multi-tiered in silico–in vitro–in vivo approach demonstrates that noncanonical RAC3 variants can produce complex, multisystem developmental phenotypes beyond previously recognized RAC3-related neurodevelopmental disorders. Full article

Objectives: There is a critical need for effective focal therapies for patients with inoperable or anatomically complex tumors where conventional ablation techniques pose high risk or are ineffective. Integrated Nanosecond Pulsed Irreversible Electroporation (INSPIRE) is a novel non-thermal ablation modality which uses real time temperature feedback during pulse delivery to safely treat tumors near critical structures. This study evaluated the impact of exposed electrode length on ablation zone size, reproducibility, and cardiac safety in a large animal model. Methods: INSPIRE treatments were performed in an in vivo healthy porcine liver model. All treatments administered 6000 V 1000 ns pulses with a 45 °C temperature set point. Treatments were administered percutaneously via an electrode and grounding pad approach using an internally cooled electrode applicator. The exposed electrode region at the distal end of the applicator was set to either 0.5, 1.0, 1.5, or 2.0 cm. Ablation zones were assessed via ultrasound, contrast-enhanced CT, and gross pathology one week post-treatment. Cardiac safety was evaluated by measuring pre- and post-treatment serum Troponin levels. Results: All treatments were completed without adverse events. Troponin levels remained stable (pre: 0.249 ng/mL; post: 0.224 ng/mL), indicating no measurable cardiac injury. The 1.5 cm exposure length produced the largest and most consistent ablation volumes, with a mean volume of 12.8 ± 2.6 cm³ and average dimensions of 3.7 × 2.7 cm in under 6 min. Increasing exposure length beyond 1.5 cm introduced greater variability and reduced treatment volumes. Conclusions: INSPIRE enables safe, large-volume, single-applicator ablation without a need for electrical pulse synchronization with R wave in cardiac rhythm. The 1.5 cm exposure length offers optimal balance between energy delivery and treatment consistency. These findings support further clinical investigation of INSPIRE for non-thermal ablation of inoperable tumors. Full article

We previously demonstrated lipid nanoparticle-mediated CRISPR-Cas9 gene editing to disrupt the gene encoding cytochrome P450 oxidoreductase (Cypor), combined with transient administration of acetaminophen (APAP), to repopulate the liver with healthy hepatocytes and rescue a phenylketonuria mouse model. This study aimed to investigate electroporation-mediated delivery of Cypor-targeting CRISPR-Cas9 ribonucleoproteins into wild-type hepatocytes, combined with liver engraftment under APAP treatment, as an in vivo selection approach in a mouse model of homozygous familial hypercholesterolemia (Ldlr^−/−). Electroporation provides higher delivery efficiency compared to lipid nanoparticles. We observed engraftment levels up to 13% engraftment of electroporated Cypor-deficient hepatocytes with indels in the liver of Ldlr^−/− mice after transient APAP administration, while negligible engraftment was observed in no-APAP controls (mean 9% and 2%, respectively, p = 0.0121). The engraftment of Cypor-deficient Ldlr^+/+ hepatocytes was associated with reductions in LDL-cholesterol (18%) and triglycerides (52%) compared to the untransplanted control Ldlr^−/− mice fed a Western diet for 5 weeks, but offered no protection from the development of diet-induced aortic root atherosclerosis or liver steatosis. While biochemical markers for liver damage normalized after discontinuation of APAP, we observed persistent lipid accumulation in the liver of Ldlr^−/− mice grafted with Cypor-deficient Ldlr^+/+ hepatocytes, likely stemming from the impact of Cypor deficiency on cholesterol clearance. Therefore, the combination of CRISPR-Cas9-mediated Cypor knockdown to induce clonal expansion of gene-edited hepatocytes using transient APAP administration is not a viable therapeutic strategy for familial hypercholesterolemia due to the essential role of Cypor in cholesterol metabolism. Unlike findings from phenylketonuria mouse model studies, the loss of Cypor function could not be compensated by unedited native hepatocytes in Ldlr^−/− mice. Collectively, our results demonstrate that electroporation is a viable and informative approach for evaluating gene editing strategies for the treatment of inherited metabolic diseases that affect the liver. Our electroporation procedure revealed that a one-size-fits-all gene editing strategy may not be universally applicable for treating inherited metabolic liver disorders. Tailored gene editing and selection strategies may be needed for different liver disorders. Full article

Recent studies have demonstrated the clinical potential of nucleic acid therapeutics (NATs). However, their efficient and scalable delivery remains a major challenge for both ex vivo and in vivo gene therapy. Microfluidic platforms have emerged as a powerful tool for overcoming these limitations by enabling precise intracellular delivery and consistent therapeutic carrier fabrication. This review examines microfluidic strategies for gene delivery at the cellular level. These strategies include mechanoporation, electroporation, and sonoporation. We also discuss the synthesis of lipid nanoparticles, polymeric particles, and extracellular vesicles for systemic administration. Unlike conventional approaches, which treat ex vivo and in vivo delivery as separate processes, this review focuses on integrated microfluidic systems that unify these functions. For example, genetic materials can be delivered to cells that secrete therapeutic extracellular vesicles (EVs), or engineered cells can be encapsulated within hydrogels for implantation. These strategies exemplify the convergence of gene delivery and carrier engineering. They create a single workflow that bridges cell-level manipulation and tissue-level targeting. By synthesizing recent technological advances, this review establishes integrated microfluidic platforms as being fundamental to the development of next-generation NAT systems that are scalable, programmable, and clinically translatable. Full article

Pancreatic cancer is among the deadliest malignancies, with limited treatment options and poor prognosis. Novel strategies are therefore urgently needed. Sotorasib, a KRAS G12C-specific inhibitor, offers targeted treatment for a small subset of patients with this mutation. Electrochemotherapy (ECT), which enhances the cytotoxicity of chemotherapeutic agents through electroporation-induced membrane permeabilization, has shown promise in various tumor types, including deep-seated malignancies such as pancreatic cancer. Combining ECT with sotorasib may potentiate antitumor effects in KRAS G12C-mutated pancreatic cancer; however, preclinical data on such combinations are lacking. This proof-of-concept study evaluated the cytotoxic effects of ECT using bleomycin (BLM) or cisplatin (CDDP) in combination with sotorasib in KRAS G12C-mutated MIA PaCa-2 and KRAS G12D-mutated PANC-1 pancreatic cancer cell lines. ECT alone significantly reduced cell viability, particularly in MIA PaCa-2 cells, where electric pulses induced approximately 75% cell death. Combining ECT with sotorasib resulted in an additive effect on KRAS G12C-mutated MIA PaCa-2 cells, though no synergy was observed, likely due to the high intrinsic sensitivity to electric pulses. These results support the potential of combining physical and molecular therapies in a subset of pancreatic cancer patients and lay the groundwork for further in vivo studies to optimize treatment parameters and explore clinical translatability. Full article

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6–8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron–glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation. Full article

Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah^-/- graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver. Full article

Background/Objectives: The AXL receptor tyrosine kinase is a promising therapeutic target in solid tumors, yet conventional viral vector-engineered CAR-T cells face critical limitations, including risks of insertional mutagenesis and immunogenicity from murine-derived single-chain variable fragments (scFvs). This study aimed to develop and evaluate mRNA-engineered fully human AXL CAR-T (^mfhAXL CAR-T) cells as a safer, scalable alternative for solid tumor immunotherapy. Methods:^mfhAXL CAR-T cells were generated via electroporation-mediated delivery of in vitro transcribed mRNA encoding a fully human AXL-specific CAR. CAR expression kinetics and T-cell viability were quantified by flow cytometry. Antitumor activity was assessed through in vitro co-cultures with AXL-positive lung and pancreatic cancer cells, measuring cytotoxicity, cytokine secretion, and specificity. In vivo efficacy was evaluated in a lung cancer xenograft mouse model, with tumor volume and body weight monitored over 14 days. Results: Flow cytometry confirmed transient but high CAR expression (>90% at 24 h) with preserved T-cell viability (>90%). In vitro, ^mfhAXL CAR-T cells exhibited dose-dependent cytotoxicity and antigen-specific cytokine secretion. In vivo, four administrations of ^mfhAXL CAR-T cells suppressed tumor growth without body weight loss. Conclusions: The mRNA-electroporated ^mfhAXL CAR-T platform enables cost-effective, large-scale production, offering a safer alternative to viral vector-based approaches by eliminating risks of insertional mutagenesis and immunogenicity. Full article

Skeletal muscle plays a pivotal role in physical activity, protein storage and energy utilization. Skeletal muscle wasting due to immobilization, aging, muscular dystrophy and cancer cachexia has negative impacts on the quality of life. The deletion of myostatin, a growth and differentiation factor-8 (GDF-8) augments muscle mass through hyperplasia and hypertrophy of muscle fibers. The present study examines the impact of myostatin deletion using CRISPR/Cas9 editing on the myogenic differentiation (MD) of C2C12 muscle stem cells. A total of five myostatin loci were targeted using guided RNAs that had been previously cloned into a vector. The clones were transfected in C2C12 cells via electroporation. The cell viability and MD of myostatin-edited clones (Mstn^−/−) were compared with C2C12 (Mstn^+/+) using a series of assays, including MTT, sulforhodamine B, immunocytochemistry, morphometric analysis and RT-qPCR. The clones sequenced showed evidence of nucleotides deletion in Mstn^−/− cells. Mstn^−/− cells demonstrated a normal physiological performance and lack of cytotoxicity. Myostatin depletion promoted the myogenic commitment as evidenced by upregulated MyoD and myogenin expression. The number of MyoD-positive cells was increased in the differentiated Mstn^−/− clones. The Mstn^−/− editing upregulates both mTOR and MyH expression, as well as increasing the size of myotubes. The differentiation of Mstn^−/− cells upregulates ActRIIb; in contrast, it downregulates decorin expression. The data provide evidence of successful CRISPR/Cas9-mediated myostatin deletion. In addition, targeting myostatin could be a beneficial therapeutic strategy to promote MD and to restore muscle loss. In conclusion, the data suggest that myostatin editing using CRISPR/Cas9 could be a potential therapeutic manipulation to improve the regenerative capacity of muscle stem cells before in vivo application. Full article

The primary method of treatment for patients suffering from drug-resistant focal-onset epilepsy is resective surgery, which adversely impacts neurocognitive function. Radio frequency (RF) ablation and laser ablation are the methods with the most promise, achieving seizure-free rates similar to resection but with less negative impact on neurocognitive function. However, there remains a number of concerns and open technical questions about these two methods of thermal ablation, with the primary ones: (1) heating; (2) hemorrhage and bleeding; and (3) poor directionality. Irreversible electroporation (IRE) is a proven method of focal ablation, which circumvents all three of the primary concerns regarding focal RF and laser ablation. Here, we demonstrate the in vivo application of a flexible implant with organic electrodes for focal ablation of epilepsy foci using high-frequency IRE (H-FIRE) in mice. Our results show that local, targeted ablation is possible in the close neighborhood of the electrode, paving the way for the clinical application in the treatment of focal epilepsy. Full article

Gene electrotransfer (GET) is a physical method of gene delivery to various tissues utilizing pulsed electric fields to transiently permeabilize cell membranes to allow for genetic material transfer and expression. Optimal pulsing parameters dictate gene transfer efficiency and cell survival, which are critical for the wide adaptation of GET as a gene therapy technique. Tissue heterogeneity complicates the delivery process, requiring the extensive optimization of pulsing protocols currently empirically optimized. These experiments are time-consuming and resource-intensive, requiring large numbers of animals for in vivo optimization. Advances in machine learning (ML) and computing power, data analysis, and model generation using ML techniques, such as neural networks, enable predictive modeling for GET. ML models have been used previously to predict ablation performance in irreversible electroporation procedures and single-cell electroporation platforms. In this work, we present ML predictive models that could be used to optimize pulsing parameters based on already completed experiments. The models were trained on 132 data points from 19 papers with the Matlab Statistics and Machine Learning Toolbox. An artificial neural network (ANN) was generated that could predict binary treatment outcomes with an accuracy of 71.8%. Support vector machines (SVMs) using selected features based on ${\chi }^2$ tests were also explored. All models used a maximum of 24 features as input, spread across target species, needle configuration, pulsing parameters, and plasmid parameters. Pulse voltage and pulse width dominated as the critical parameters, followed by field strength, dose, and electrode with the greatest impact on GET efficiency. This study elucidates areas where predictive ML algorithms may ideally inform GET study design to accelerate optimization and improve efficiencies upon the further training of these models. Full article

Background and Aims: Cancer cachexia is a complex syndrome affecting most cancer patients and is directly responsible for about 20% of cancer-related deaths. Previous studies showed muscle proteolysis hyper-activation and mitophagy induction in tumor-bearing animals. While basal mitophagy is required for maintaining muscle mass and quality, excessive mitophagy promotes uncontrolled protein degradation, muscle loss and impaired function. BNIP3, a key mitophagy-related protein, is significantly increased in the muscles of both mice and human cancer hosts. This study aimed to define the potential of mitigating mitophagy via BNIP3 downregulation in preserving mitochondrial integrity, counteracting skeletal muscle loss in experimental cancer cachexia. Methods: Two in vivo gene delivery methods were performed to knock down muscle BNIP3: electroporation of a BNIP3-specific shRNA expression vector or adenovirus injection. Results: The electroporation effectively reduced muscle BNIP3 in healthy mice but was ineffective in C26 tumor-bearing mice. In contrast, adenovirus-mediated BNIP3 knockdown successfully decreased BNIP3 levels also in tumor hosts. Although BNIP3 knockdown did not impact overall on body or muscle mass, it improved muscle fiber size in C26-bearing miceh2, suggesting partial prevention of muscle atrophy. Mitochondrial respiratory chain complexes (OxPhos) and TOM20 protein levels were consistently rescued, indicating improvements in mitochondrial mass, while H₂O₂ levels were unchanged among the groups, suggesting that BNIP3 downregulation does not impair the endogenous control of oxidative balance. Conclusions: These findings suggest that a fine balance between mitochondrial disposal and biogenesis is fundamental for preserving muscle homeostasis and highlight a potential role for BNIP3 modulation against cancer-induced muscle wasting. Full article

RAC3 encodes a small GTPase of the Rho family that plays a critical role in actin cytoskeleton remodeling and intracellular signaling regulation. Pathogenic variants in RAC3, all of which reported thus far affect conserved residues within its functional domains, have been linked to neurodevelopmental disorders characterized by diverse phenotypic features, including structural brain anomalies and facial dysmorphism (NEDBAF). Recently, a novel de novo RAC3 variant (NM_005052.3): c.196C>T, p.R66W was identified in a prenatal case with fetal akinesia deformation sequence (a spectrum of conditions that interfere with the fetus’s ability to move), and complex brain malformations featuring corpus callosum agenesis, diencephalosynapsis, kinked brainstem, and vermian hypoplasia. To investigate the mechanisms underlying the association between RAC3 deficiency and this unique, distinct clinical phenotype, we explored the pathophysiological significance of the p.R66W variant in brain development. Biochemical assays revealed a modest enhancement in intrinsic GDP/GTP exchange activity and an inhibitory effect on GTP hydrolysis. Transient expression studies in COS7 cells demonstrated that RAC3-R66W interacts with the downstream effectors PAK1, MLK2, and N-WASP but fails to activate SRF-, AP1-, and NFkB-mediated transcription. Additionally, overexpression of RAC3-R66W significantly impaired differentiation in primary cultured hippocampal neurons. Acute expression of RAC3-R66W in vivo by in utero electroporation resulted in impairments in cortical neuron migration and axonal elongation during corticogenesis. Collectively, these findings suggest that the p.R66W variant may function as an activated version in specific signaling pathways, leading to a distinctive and severe prenatal phenotype through variant-specific mechanisms. Full article