Improved Transfection Efficiency Research Articles

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes.

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

Poly(2-methyl-2-oxazoline) as a polyethylene glycol alternative for lipid nanoparticle formulation

Lipid nanoparticles (LNPs) have emerged as the platform of choice for mRNA delivery. Polyethylene glycol (PEG) is considered a key component of currently approved LNP-based delivery systems as it ensures particle stability and shapes various facets of LNP behavior in biological systems. Whilst PEG has numerous characteristics that are favorable for delivery systems, there is a growing body of evidence that suggests that it is immunogenic. Thus, next-generation mRNA therapeutics are likely to benefit from the identification of PEG alternatives. Towards this end, we have assessed the suitability of poly(2-methyl-2-oxazoline) (PMOZ) for LNP-based mRNA delivery. We compared the properties and bioactivities of PMOZ-containing LNPs to that of a standard composition that includes PEG. Decreasing the percentage of PMOZ in formulations improved transfection efficiency and enhanced the immunostimulatory potential. Reducing the PMOZ density was shown to enhanced antigen-specific T-cell responses in vivo. Interestingly, we found that this was not the case for antibody responses. A direct comparison between LNPs that contain the same amount of PEG or PMOZ strongly suggests that the former induces stronger CD8+ T-cell responses while the latter induces superior neutralizing titers. These findings augur well for the further development of PMOZ as a PEG replacement for LNP-based mRNA delivery approaches.

Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells.

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines' culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

Abstract 7247: Exosome-mediated delivery of RNA and DNA: Enhanced efficiency with polycationic PEI-PEG

Abstract Emerging advances in gene therapy over the past few decades have revolutionized biomedical research. Our lab has pioneered bovine milk and colostrum-derived exosomes (Exo) and devised a nanoplatform for nucleic acid delivery achieving targetability with folic acid (FA) functionalization. We showed that bovine colostrum exosomes complexed with high MW polyethyleneimine (PEI-60K) can efficiently delivery siRNA and plasmid DNA (pDNA) in vitro and in vivo. We hypothesize that lower MW PEI may improve transfection efficiency and enhance oral uptake of EPM-siRNA, in addition to presumably higher degradability in a cellular system. We now report that exosomes and PEI of lower MW (25K)-PEG copolymer is as effective or more effective for delivery of siRNA and pDNA than PEI-60K. Incubation of exosomes, PEI-25K and siRNA in the presence of 5’-32P-labeled siRNA as a tracer showed high siRNA entrapment (>90%) based on the distribution of the radioactivity in EPM-siRNA precipitate compared with the supernatant. The efficacy of the EPM matrix is gene specific. As treatment of A549 lung cancer cells with EPM-(PEI-60K)-siKRAS showed 60% knockdown of target KRAS gene, which was enhanced to 70% by EPM-(PEI-25K)-siKRAS. Moreover, p53-pDNA delivered by EPM-25K resulted in nearly 2-fold higher expression of p53 in p53-null H1299 lung cancer cells compared with EPM-60K. However, still lower MW PEI (PEI-15K) diminished the transfection efficiency (36% KRAS knockdown). EPM formulated with PEI-60K and 25K had similar effect on cell viability of HEK293 cells. Upon in vivo testing, EPM-25K loaded with siKRAS and functionalized with folic acid (FA) showed time-dependent growth inhibition of A549-innoculated orthotopic lung tumors in NOD Scid mice eliciting 76% (p<0.01) tumor growth inhibition. The tumor inhibition was accompanied with 50% (p<0.05) knockdown of the target gene in the tumor tissue, while the adjacent lung tissue showed no modulation. The anti-tumor efficacy of siKRAS delivered by FA-functionalized EPM-25K is the same or somewhat higher than the efficacy reported previously with FA-functionalized EPM-60K.In conclusion, based on our in vitro and in vivo results, lower MW PEI (PEI-25K) is a suitable alternative for PEI-60K with boosted efficacy in terms of knockdown of KRAS. Our proposed technology offers a platform to advance and overcome the obstacles in the clinical translation of gene therapy. Funding: Work supported from funds provided by 3P Biotechnologies and in part Agnes Brown Duggan Funds. Disha Nagesh Moholkar is supported by IPIBS Fellowship, University of Louisville. Citation Format: Disha Nagesh Moholkar, Raghuram Kandimalla, Margaret Wallen, Jeyaprakash Jeyabalan, Wendy Spencer, Farrukh Aqil, Ramesh C. Gupta. Exosome-mediated delivery of RNA and DNA: Enhanced efficiency with polycationic PEI-PEG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7247.

Abstract 7249: Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy

Abstract Cancer poses a global health challenge, driving the quest for innovative therapies. Messenger ribonucleic acid (mRNA) lipid nanoparticles (LNPs) are a promising strategy in cancer immunotherapy. However, a major obstacle lies in efficiently transfecting immune cells like macrophages and dendritic cells (DCs) with LNPs. This study seeks to improve cancer immunotherapy by optimizing mRNA-LNP formulations. Therefore, we systematically evaluated three components of LNPs, namely ionizable lipids, phospholipids, and sterols, and fine-tuned the components and their molar ratios. A comprehensive screening process was conducted to determine the efficiency of LNPs, involving in vitro experiments on macrophages and DCs, as well as in vivo assessments. Among six ionizable lipids assessed in different cell lines (RAW264.7 and DC2.4), SM-102 significantly enhanced the transfection efficiency in macrophages and DCs. Subsequently, we evaluated these LNPs in an in vivo setting. We quantified the bioluminescence signal after administering LNPs loaded with luciferase mRNA. The results demonstrated a significant increase in luciferase expression when using SM-102 and ALC-0315 lipids. To validate this finding, we conducted a T cell response study, which revealed an increased percentage of CD8+ T cells with SM-102-based LNPs compared to other formulations. Having identified SM-102 as a potent ionizable lipid, we proceeded to evaluate three phospholipids in vitro. The results indicated a significant enhancement in transfection efficiency with DOPE lipids compared to DSPC. Consequently, we selected DOPE as the phospholipid for subsequent screening. Lastly, we explored different types of sterols with varying molar ratios in macrophages and DC cells. Our findings unveiled a notable improvement in mRNA delivery, particularly in DCs, when the molar ratio of β-sitosterol was increased to 19.5%, while concurrently decreasing the molar ratio of cholesterol to 19% within the same formulation. It is noteworthy that the addition of β-sitosterol alone led to reduced transfection efficiency in DCs but resulted in a significant enhancement in macrophages. Lastly, we evaluated the top four formulations in vivo, and the results demonstrated a significant increase when β-sitosterol was added to the formulation with cholesterol, at molar ratios of 19.5% and 19%, respectively. Overall, altering lipid composition and their molar ratios profoundly impact mRNA-LNP transfection. The optimized formulations, comprising SM-102, DOPE, and cholesterol with or without β-sitosterol, outperformed the alternatives. These refined formulations hold promise for clinical applications, potentially enhancing passive therapeutic agent delivery into macrophages and DCs, offering promising prospects for advancing cancer immunotherapy. Citation Format: Yasir Alshehry, Matthew Fernandez, Raneem Aldaqqa, Asma Al-Terawi, Douglas Sweet, Sandro da Rocha. Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7249.

Recent Progress in Nucleic Acid Pulmonary Delivery toward Overcoming Physiological Barriers and Improving Transfection Efficiency.

Pulmonary delivery of therapeutic agents has been considered the desirable administration route for local lung disease treatment. As the latest generation of therapeutic agents, nucleic acid has been gradually developed as gene therapy for local diseases such as asthma, chronic obstructive pulmonary diseases, and lung fibrosis. The features of nucleic acid, specific physiological structure, and pathophysiological barriers of the respiratory tract have strongly affected the delivery efficiency and pulmonary bioavailability of nucleic acid, directly related to the treatment outcomes. The development of pharmaceutics and material science provides the potential for highly effective pulmonary medicine delivery. In this review, the key factors and barriers are first introduced that affect the pulmonary delivery and bioavailability of nucleic acids. The advanced inhaled materials for nucleic acid delivery are further summarized. The recent progress of platform designs for improving the pulmonary delivery efficiency of nucleic acids and their therapeutic outcomes have been systematically analyzed, with the application and the perspectives of advanced vectors for pulmonary gene delivery.

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x106 cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Advancing in vivo modelling and delivery systems for triplet repeat expansion‐mediated corneal endothelial dystrophy: Implications in Anti‐miR therapy

Aims/Purpose: Triplet repeat expansion‐mediated endothelial corneal dystrophy, including Fuchs endothelial corneal dystrophy (FECD), is characterized by abnormal expansions of trinucleotide repeats, resulting in corneal endothelium dysfunction [1,2]. This study aims to characterize a reliable in vivo model, that it lacks, and explore the therapeutic potential of anti‐miRs, which have shown efficacy in other disorders involving triplet repeat expansions [3,4,5]. Current invasive delivery methods for eye‐based treatments present challenges, so our focus is on optimizing non‐invasive delivery methods for anti‐miR‐based therapy.Methods: A transgenic mouse model with a significant CTG repeat expansion (>1000), established in DM1, was investigated as an in vivo model for corneal endothelial dystrophy. The Fuchs‐like phenotype of the disease was characterized using immunohistochemistry and FISH techniques. Delivery methods were explored to enhance the targeting of corneal endothelial cells (CEC). Delivery efficacy and marker expression were assessed using fluorescence, RT‐qPCR, PCR, western blotting, and immunocytochemistry techniques. FECD cell cultures were established to evaluate the challenging FECD‐specific delivery systems.Results: A novel in vivo model has been characterized for studying corneal endothelial dystrophy caused by triplet repeat expansion, exhibiting similarities to the Fuchs‐like phenotype. Enhanced delivery methods have been identified to improve transfection efficiency in endothelial (HUVEC) and CEC. Successful establishment of FECD cell cultures has allowed the evaluation of these enhanced delivery systems.Conclusions: The first successful characterization of an in vivo model for triplet repeat expansion‐mediated corneal endothelial dystrophy has been achieved. This model shows potential for assessing the reversal of these phenotypes in the ocular region. Moreover, the successful delivery of new systems across CEC suggests the possibility of non‐invasive administration of anti‐miR directly into the eye. This breakthrough opens the door for future research on anti‐miRs in other diseases caused by triplet repeat expansion, such as FECD.ReferenceIn PMID: [1] 28886202, [2] 34130750, [3] 29946070, [4] 32805487, [5] 22764244.

Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma

MicroRNA (miR)–200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c–based gene therapy to inhibit OSCC growth has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA (pDNA) encoding miR-200c delivered via nonviral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were used to transfect pDNA encoding miR-200c into OSCC cells, and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c, were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line–derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and improve clinical outcomes in OSCC treatment, while more comprehensive examinations to confirm the safety and efficacy of the CaCO3/pDNA miR-200c system using various preclinical models are needed.

A poly(amidoamine)-based polymeric nanoparticle platform for efficient in vivo delivery of mRNA

The successful use of mRNA vaccines enabled and accelerated the development of several new vaccine candidates and therapeutics based on the delivery of mRNA. In this study, we developed bioreducible poly(amidoamine)-based polymeric nanoparticles (PAA PNPs) for the delivery of mRNA with improved transfection efficiency. The polymers were functionalized with chloroquinoline (Q) moieties for improved endosomal escape and further stabilization of the mRNA-polymer construct. Moreover, these PAAQ polymers were covalently assembled around a core of multi-armed ethylenediamine (Mw 800, 2 % w/w) to form a pre-organized polymeric scaffolded PAAQ (ps-PAAQ) as a precursor for the formation of the mRNA-loaded nanoparticles. Transfection of mammalian cell lines with EGFP mRNA loaded into these PNPs showed a favorable effect of the Q incorporation on GFP protein expression. Additionally, these ps-PAAQ NPs were co-formulated with PEG-polymer coatings to shield the positive surface charge for increased stability and better in vivo applicability. The ps-PAAQ NPs coated with PEG-polymer displayed smaller particle size, electroneutral surface charge, and higher thermal stability. Importantly, these nanoparticles with both Q and PEG-polymer coating induced significantly higher luciferase activity in mice muscle than uncoated ps-PAAQ NPs, following intramuscular injection of PNPs loaded with luciferase mRNA. The developed technology is broadly applicable and holds promise for the development of new nucleotide-based vaccines and therapeutics in a range of infectious and chronic diseases.

Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages.

mRNA lipid nanoparticle (LNP) technology presents enormous opportunities to prevent and treat various diseases. Here, we developed a novel series of LNPs containing ionizable amino-lipids showing a remarkable array of tunable and pH-sensitive lyotropic liquid crystalline mesophases including the inverse bicontinuous cubic and hexagonal phases characterized by high-throughput synchrotron radiation X-ray scattering. Furthermore, with an interest in developing mRNA therapeutics for lung macrophage targeting, we discovered that there is a strong correlation between the mesophase transition of the LNPs during acidification and the macrophage association/transfection efficiency of mRNAs. The slight molecular structural differences between the SM-102 and ALC-0315 ionizable lipids are linked to the LNP's ability to transform their internal structures from an amorphous state to the inverse micellar, hexagonal, and finally cubic structures during endosomal maturation. SM-102 LNPs showed exceptionally improved transfection efficiency due to their ability to form a cubic structure at a lower pH than the ALC-0315 analogues, which remained within the hexagonal structure, previously attributed to promoting endosomal escape of the ionizable LNPs. Overall, the new knowledge draws our attention to the important role of mesophase transition in endosomal escape, and the novel LNP libraries reported herein have broad prospects for advancing mRNA therapeutics.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

HTERT Gene Modification Using CRISPR-dCas9-dnmt3a System as a Therapeutic Approach Against Glioma.

Abnormal DNA methylation patterns have been reported in various diseases, including different cancers. CRISPR/Cas9 is a low-cost and highly effective gene editing tool that has lately revolutionized biotechnology. Studies have shown that the CRISPR/Cas9 system can effectively target and correct methylation. Telomerase plays a survival role for cancer cells. It is encoded by the hTERT gene. The effectiveness of CRISPR/Cas9 in targeting hTERT to treat glioma cancer cells was assessed in this study. EF1a-hsaCas9-U6-gRNA vector carrying sgRNA and Cas9 hybrids were used to transfect U87 glioma cells. Four and eight μg/mL polybrene concentrations were investigated to improve transfection efficiency. The expression level of hTERT that has undergone metabisulfite modification was assessed using real-time PCR. Flow cytometry and Western blotting were also used to determine whether telomerase was present in the cells. High-resolution melting analysis (HRM) was used to examine the hTERT promoter's methylation. Finally, flow cytometry was used to measure the apoptotic rate of transfected U87 cells. The findings demonstrated that gRNA significantly boosted transfection effectiveness. Significant variations were seen in the expression of hTERT in U87 cells at 4 μg/mL polybrene and 80 μg/mL transfection compared to transfection without gRNA and basal cells. Flow cytometry showed a decrease in hTERT levels in transfected cells. Furthermore, transfection with gRNA increased U87 cell apoptosis compared to transfection without gRNA. It appears that the designed CRISPR/Cas9 system can reduce hTERT expression and telomerase activity and thus inhibit glioma cell growth.

Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.

Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p<0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine™ 2000 and our PGC medium was the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them. This article is protected by copyright. All rights reserved.

Improved biocompatibility of dendrimer-based gene delivery by histidine-modified nuclear localization signals

Polyamidoamine (PAMAM) dendrimers have been explored as an alternative to polyethylenimine (PEI) as a gene delivery carrier because of their relatively low cytotoxicity and excellent biocompatibility. The transfection efficiency of PAMAM dendrimers can be improved by the addition of nuclear localization signal (NLS), a positively charged peptide sequence recognized by cargo proteins in the cytoplasm for nuclear transport. However, increased positive charges from NLS can cause damage to the cytoplasmic and mitochondrial membranes and lead to reactive oxygen species (ROS)-induced cytotoxicity. This negative effect of NLS can be negated without a significant reduction in transfection efficiency by adding histidine, an essential amino acid known as a natural antioxidant, to NLS. However, little is known about the exact mechanism by which histidine reduces cytotoxicity of NLS-modified dendrimers. In this study, we selected cystamine core PAMAM dendrimer generation 2 (cPG2) and conjugated it with NLS derived from Merkel cell polyomavirus large T antigen and histidine (n = 0–3) to improve transfection efficiency and reduce cytoxicity. NLS-modified cPG2 derivatives showed similar or higher transfection efficiency than PEI 25 kDa in NIH3T3 and human mesenchymal stem cells (hMSC). The cytotoxicity of NLS-modified cPG2 derivatives was substantially lower than PEI 25 kDa and was further reduced as the number of histidine in NLS increased. To understand the mechanism of cytoprotective effect of histidine-conjugated NLS, we examined ROS scavenging, hydroxyl radical generation and mitochondrial membrane potential as a function of the number of histidine in NLS. As the number of hisidine increased, cPG2 scavenged ROS more effectively as evidenced by the hydroxyl radical antioxidant capacity (HORAC) assay. This was consistent with the reduced intracellular hydroxyl radical concentration measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) assay in NIH3T3. Finally, fluorescence imaging with JC-1 confirmed that the mitochondrial membranes of NIH 3T3 were well-protected during the transfection when NLS contained histidine. These experimental results confirm the hypothesis that histidine residues scavenge ROS that is generated during the transfection process, preventing the excessive damage to mitochondrial membranes, leading to reduced cytotoxicity.

Peptide-Based Vectors for Gene Delivery

Gene therapy is the ultimate therapeutic technology for diseases related to gene abnormality. However, the use of DNA alone has serious problems, such as poor stability and difficulty in entering target cells. The development of a safe and efficient gene delivery system is the cornerstone of gene therapy. Of particular interest, multifunctional peptides are rationally designed as non-viral vectors for efficient gene delivery. As components of gene delivery vectors, these peptides play critically important roles in skeleton construction, the implementation of targeting strategies, cell membrane penetration, endosome rupture, and nuclear transport. In recent years, the research of functional peptide-based gene delivery vectors has made important progress in improving transfection efficiency. The latest research progress and future development direction of peptide-based gene delivery vectors are reviewed in this paper.

Poly-L-lysine as a crosslinker in bile acid and alginate nanoaggregates for gene delivery in auditory cells.

Background: Hearing loss is a condition that may affect a wide array of patients from various backgrounds. There are no cures for sensorineural hearing loss. Gene therapy is one possible method of improving hearing status; however, gene delivery remains challenging. Materials & methods: Polymer nanoaggregates of alginate and poly-L-lysine were prepared with and without bile acid. The nanoaggregates had physical properties, cytotoxicity, gene release and gene expression analyzed. Results & discussion: The nanoparticles produced had appropriate size and charge, low cytotoxicity between 0.5 and 1.0mg/ml and linear gene release but poor transfection efficiency. Conclusion: The present study provides preliminary evidence for the efficacy of polymer nanotechnology with bile acids for inner ear gene delivery; optimization is required to improve transfection efficiency.

Blended Block Polycation Micelles Enhance Antisense Oligonucleotide Delivery.

Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an n-butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.