Search Results (175)

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

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

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

Background/Objectives: Nanoparticles (NPs) were previously explored as enhancers in electroporation due to their potential to locally amplify electric fields near cell membranes, with gold nanoparticles (AuNPs) in particular showing promise in improving membrane permeability and gene electrotransfer (GET). In this study, we systematically investigated the influence of NP properties—including size, shape, surface functionalization, and material—on electroporation efficacy. Methods: A combined approach using theoretical modeling and experimental validation was employed, encompassing numerical simulations, membrane permeabilization assays, transmission electron microscopy, and GET efficiency measurements. Results: Numerical results revealed that the presence of NPs alters local electric field distributions, but the amplification is highly localized, regardless of NP conductivity or geometry. Experimentally, only two out of six tested NP types produced a statistically significant, yet modest, increase in membrane permeability at one electric field intensity. Similarly, GET improvement was observed with only one NP type, with no dependence on concentration or functionalization. Conclusions: Overall, our findings demonstrate that NPs, under tested conditions, do not substantially enhance cell membrane permeability or GET efficacy. These conclusions are supported by both computational modeling and in vitro experiments. Full article

Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME’s core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform’s ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME’s potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck. Full article

Background/Objectives: Co-transfection of multiple DNAs is important to many research and therapeutic applications. While the optimization of single plasmid transfection is common, multiple plasmid co-transfection analyses are limited. Here we provide empirical data regarding multiple plasmid co-transfection while altering the number of species of plasmids transfected (up to four different plasmids) and the amount of plasmids/cell using the two most common non-viral techniques, electroporation and lipofection. Methods: A549 human lung epithelial cells were transfected using lipofectamine 2000 or electroporation with combinations of plasmids, each expressing one of four different fluorescent proteins from the CAGG promoter. Twenty-four hours later, cells were analyzed by spectral flow cytometry to determine the number of cells expressing each fluorescent protein and the amount of fluorescent signal of each protein in a cell. Results and Conclusions: For electroporation, while the fraction of cells expressing plasmids increased with increasing amounts of DNA, increasing the number of plasmid species did not alter the fraction of expressing cells and had no effect on levels of expression in individual cells. By contrast, for lipofection, the fraction of cells expressing plasmids was not affected by the amount of DNA added but both the fraction of cells expressing and the level of protein produced in these cells decreased for each plasmid species as the number of delivered species increased. For both lipofection and electroporation after single plasmid transfection, the expressing cells had greater numbers of plasmid copies/cell than non-expressing cells. Multiple plasmid lipofection resulted in more plasmid copies/cell in co-expressing than non-expressing cells. Multiple plasmid electroporation was the inverse of this with fewer plasmid copies/cell in co-expressing than non-expressing cells. 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

The liver plays a pivotal role in metabolism, detoxification, and protein synthesis and comprises several cell types, including hepatocytes and cholangiocytes. Primary human hepatocytes in 2D cultures rapidly dedifferentiate and lose their function, making their use as a reliable cell model challenging. Therefore, developing robust three-dimensional cell culture models is crucial, especially for diseases lacking reliable animal models. The aim of this study was to optimize a protocol for the directed differentiation of induced pluripotent stem cells into liver progenitor cells, achieving the high-level expression of specific markers. As a result, we established a 2D culture of liver progenitor cells capable of differentiating into three cell types: a 3D organoid culture containing hepatocyte- and cholangiocyte-like cells and a 2D cell culture comprising stellate-like cells. To evaluate gene delivery efficiency, liver progenitor cells were transduced with various rAAV serotypes carrying an eGFP reporter cassette at different multiplicities of infection (MOIs). Our results revealed that rAAV serotype 2/2 at MOI of 100,000 achieved the highest transduction efficiency of 93.6%, while electroporation demonstrated a plasmid delivery efficiency of 54.3%. These findings suggest that liver progenitor cells are a promising tissue-like cell model for regenerative medicine and demonstrate high amenability to genetic manipulation, underscoring their potential in gene therapy and genome editing studies. 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

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. Full article

This study designed three sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting the prolactin gene receptor (PRLR) in fetal cattle, utilized Cas9 to cleave endogenous DNA, and screened stable cell lines for somatic cell nuclear transfer experiments to investigate the impact of different editing sites on embryonic development. The results showed that sgRNA139 had the highest cleavage efficiency (Fcut = 0.65, Indels = 42.19%), while sgRNA109 had the lowest (Fcut = 0.45, Indels = 35.31%). No significant differences were observed in cell growth status after electroporation (p > 0.05), and the transfection efficiency exceeded 90% after five days of culture. In the evaluation of key embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate (p < 0.01), whereas sgRNA139 showed no significant effect on the cleavage rate (p > 0.05), but its blastocyst rate was slightly lower than that of the control group (p > 0.05). This study demonstrates that highly specific sgRNAs and stable edited cell lines used as donor cells can significantly regulate the later stages of embryonic development. This study not only provides new experimental evidence for the functional study of the PRLR but also lays an important theoretical foundation for the innovation of molecular breeding technologies in dairy cattle. Full article

RyR1-related myopathies (RyR1-RMs) include a wide range of genetic disorders that result from mutations in the RYR1 gene. Pathogenic variants lead to defective intracellular calcium homeostasis and muscle dysfunction. Fixing intracellular calcium leaks by stabilizing the RyR1 calcium channel has been identified as a promising therapeutic target. Gene therapy via prime editing also holds great promise as it can cure diseases by correcting genetic mutations. However, as more than 700 variants have been identified in the RYR1 gene, a universal treatment would be a more suitable solution for patients. Our investigation into the RyR1-S2843A mutation has yielded promising results. Using a calcium leak assay, we determined that the S2843A mutation was protective when combined with pathogenic mutations and significantly reduced the Ca²⁺ leak of the RyR1 channel. Our study demonstrated that prime editing can efficiently introduce the protective S2843A mutation. In vitro experiments using the RNA electroporation of the prime editing components in human myoblasts achieved a 31% introduction of this mutation. This article lays the foundation for a new therapeutic approach for RyR1-RM, where a unique once-in-a-lifetime prime editing treatment could potentially be universally applied to all patients with a leaky RyR1 channel. Full article

Background: The majority of antigen-based SARS-CoV-2 (SCV2) vaccines utilized in the clinic have had the Spike protein or domains thereof as the immunogen. While the Spike protein is highly immunogenic, it is also subject to genetic drift over time, which has led to a series of variants of concern that continue to evolve, requiring yearly updates to the vaccine formulations. In this study, we investigate the potential of the N-terminal ectodomain of the ORF3a protein encoded by the orf3a gene of SCV2 to be an evolution-resistant vaccine antigen. This domain is highly conserved over time, and, unlike many other SCV2 conserved proteins, it is present on the exterior of the virion, making it accessible to antibodies. ORF3a is also important for eliciting robust anti-SARS-CoV-2 T-cell responses. Methods: We designed a DNA vaccine by fusing the N-terminal ectodomain of orf3a to macrophage-inflammatory protein 3α (MIP3α), which is a chemokine utilized in our laboratory that enhances vaccine immunogenicity by targeting an antigen to its receptor CCR6 present on immature dendritic cells. The DNA vaccine was tested in mouse immunogenicity studies, vaccinating by intramuscular (IM) electroporation and by intranasal (IN) with CpG adjuvant administrations. We also tested a peptide vaccine fusing amino acids 15–28 of the ectodomain to immunogenic carrier protein KLH, adjuvanted with Addavax. Results: The DNA IM route was able to induce 3a-specific splenic T-cell responses, showing proof of principle that the region can be immunogenic. The DNA IN route further showed that we could induce ORF3a-specific T-cell responses in the lung, which are critical for potential disease mitigation. The peptide vaccine elicited a robust anti-ORF3a antibody response systemically, as well as in the mucosa of the lungs and sinus cavity. Conclusions: These studies collectively show that this evolutionarily stable region can be targeted by vaccination strategies, and future work will test if these vaccines, alone or in combination, can result in reduced disease burden in animal challenge models. Full article

The successful application of primordial germ cells (PGCs) is an ideal method for generating gene-edited birds. However, barriers to efficient DNA transfection in PGCs lead to low transfection efficiency, limiting the generation of genetically modified chickens. The current study utilized chemical transfection and electroporation methods to determine the optimal transfection conditions for the PGC line under feeder- and serum-free medium. Among the tested methods, the Lonza electroporation system exhibited the highest transduction efficiency, with a previously unreported rate of 71.13 ± 1.26%. Optimal transfection conditions were achieved using 4 µg of DNA and 100 µL of Entranster^TM-E in 1 × 10⁶ PGCs. Furthermore, the optimal electroporation conditions resulted in low cell death and normal expression of pluripotency-related genes, highlighting the low cytotoxicity. The resulting electroporation models were then used to deliver the enhanced green fluorescent protein (EGFP) gene to the Z chromosome with a Cas9-gRNA plasmid, achieving a 7-day insertion efficiency of 14.63 ± 1.07%. Our study highlights the vast potential of electroporation technology for the transfection of PGCs. Full article

Electroporation is an efficient method for nucleotide and protein transfer, and is used for clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9)-mediated genome editing. In this study, we investigated the effects of electroporation on platelet-derived growth factor receptor alpha (PDGFRA) and receptor tyrosine kinase (RTK) expression in U-251 and U-87 MG cells. PDGFRA mRNA and protein expression decreased 2 days after electroporation in both cell lines, with recovery observed after 13 days in U-87 MG cells. However, in U-251 MG cells, PDGFRα expression remained suppressed, despite mRNA recovery after 13 days. Similar expression profiles were observed for lipofection in the U-251 MG cells. Comprehensive RNA sequencing confirmed electroporation-induced up- and down-regulation of RTK mRNA in U-251 MG cells 2 days post-electroporation. In contrast, recombinant adeno-associated virus (rAAV) transfected with mNeonGreen fluorescent protein or Cas9 did not affect PDGFRA, RTKs, or inflammatory cytokine expression, suggesting fewer adverse effects of rAAV on U-251 MG cells. These findings emphasize the need for adequate recovery periods following electroporation or the adoption of alternative methods, such as rAAV transfection, to ensure the accurate assessment of CRISPR-mediated gene editing outcomes. Full article