Showing papers on "Electroporation published in 2023"

A comparative analysis of microglial inducible Cre lines

Abstract: Cre/LoxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. The field of microglial biology has particularly benefited from this technology as microglia have historically been difficult to transduce with virus or electroporation methods for gene delivery. Here, we interrogate four of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency and spontaneous recombination, depending on the Cre line and loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency in microglia, which could be extended to other cell types. There is increasing evidence that microglia are key regulators of neural circuit structure and function. Microglia are also major drivers of a broad range of neurological diseases. Thus, reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field of microglial biology and the development of microglia-based therapeutics.

Doxorubicin Loading into Milk and Mesenchymal Stem Cells’ Extracellular Vesicles as Drug Delivery Vehicles

Abstract: Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs’ CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

Abstract: CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells. The yields of edited primary human lymphocytes can be increased substantially, with respect to those obtained via electroporation, by delivering a CRISPR ribonucleoprotein alongside an amphiphilic peptide identified via screening.

Magnetic–Acoustic Sequentially Actuated CAR T Cell Microrobots for Precision Navigation and In Situ Antitumor Immunoactivation

Abstract: Despite its clinical success, chimeric antigen receptor T (CAR T)‐cell immunotherapy remains limited in solid tumors, owing to the harsh physical barriers and immunosuppressive microenvironment. Here a CAR‐T‐cell‐based live microrobot (M‐CAR T) is created by decorating CAR T with immunomagnetic beads using click conjugation. M‐CAR Ts are capable of magnetic–acoustic actuation for precision targeting and in situ activation of antitumor immune responses. Sequential actuation endows M‐CAR Ts with magnetically actuated anti‐flow and obstacle avoidance as well as tissue penetration driven by acoustic propulsion, enabling efficient migration and accumulation in artificial tumor models. In vivo, sequentially actuated M‐CAR Ts achieves long‐distance targeting and accumulate at the peritumoural area under programmable magnetic guidance, and subsequently acoustic tweezers actuate M‐CAR Ts to migrate into deep tumor tissues, resulting in a 6.6‐fold increase in accumulated exogenous CD8+ CAR T cells compared with that without actuation. Anti‐CD3/CD28 immunomagnetic beads stimulate infiltrated CAR T proliferation and activation in situ, significantly enhancing their antitumor efficacy. Thus, this sequential‐actuation‐guided cell microrobot combines the merits of autonomous targeting and penetration of intelligent robots with in situ T‐cell immunoactivation, and holds considerable promise for precision navigation and cancer immunotherapies.

Qualitative Investigation of Experience and Quality of Life in Patients Treated with Calcium Electroporation for Cutaneous Metastases

Abstract: Simple Summary Some cancer patients develop cutaneous metastases at late stage of disease, and the tumours may be present for months or years. Some cancers are more prone to disseminating to the skin, such as breast and lung cancer. When cancer manifests on the body’s surface or on the face, symptoms and distress can have a significant impact on the quality of life of patients. Surgical and medical treatment is difficult, and local treatments are important. Calcium electroporation is a novel cancer treatment. It includes injecting calcium-solution and applying electric pulses to tumour tissue. There is a scarcity of literature on patient experience of living with cutaneous metastases. Our study’s objective was to examine the patient’s perspective on CaEP treatment of skin tumours, as well as the treatment’s impact on health-related quality of life (QoL). We evaluated quality of life in patients with skin metastases treated with calcium electroporation using qualitative interviews. Calcium electroporation enhanced health-related quality of life by reducing symptoms and increasing social inclination. Peer accounts are important in preparation for treatment. Abstract (1) Background: Calcium electroporation is a novel cancer treatment. It includes injecting calcium-solution and applying electric pulses to tumour tissue. Data on quality of life for patients with cutaneous metastases treated with calcium electroporation is limited. We evaluated quality of life in patients with skin metastases treated with calcium electroporation using qualitative interviews. (2) Methods: This investigation featured a subgroup from a non-randomised phase II study (CaEP-R) at Zealand University Hospital, Denmark, studying response to calcium electroporation in cutaneous metastasis (ClinicalTrials no. NCT04225767). Participants were interviewed at baseline before calcium electroporation treatment and after two months. Data was analysed phenomenologically; (3) Results: Interviews were conducted February 2020–November 2021. Nine patients were included, of which seven participated in both interviews. All seven patients expected treated tumours to disappear, symptom relief and minimal side effects. Most patients requested peer accounts. All patients found the treatment uncomfortable but acceptable; all thought their fears of electric pulses exceeded their experience. All would repeat the treatment if effective. Successful treatment had a positive effect on pain, symptomatic wounds, sleep, vigour and social inclination; (4) Conclusions: Calcium electroporation enhanced health-related quality of life by reducing symptoms and increasing social inclination. Peer accounts provide patients with a shortcut to confidence in treatment on top of doctors’ recommendations.

Improved DNA Vaccine Delivery with Needle-Free Injection Systems

Abstract: DNA vaccines have inherent advantages compared to other vaccine types, including safety, rapid design and construction, ease and speed to manufacture, and thermostability. However, a major drawback of candidate DNA vaccines delivered by needle and syringe is the poor immunogenicity associated with inefficient cellular uptake of the DNA. This uptake is essential because the target vaccine antigen is produced within cells and then presented to the immune system. Multiple techniques have been employed to boost the immunogenicity and protective efficacy of DNA vaccines, including physical delivery methods, molecular and traditional adjuvants, and genetic sequence enhancements. Needle-free injection systems (NFIS) are an attractive alternative due to the induction of potent immunogenicity, enhanced protective efficacy, and elimination of needles. These advantages led to a milestone achievement in the field with the approval for Restricted Use in Emergency Situation of a DNA vaccine against COVID-19, delivered exclusively with NFIS. In this review, we discuss physical delivery methods for DNA vaccines with an emphasis on commercially available NFIS and their resulting safety, immunogenic effectiveness, and protective efficacy. As is discussed, prophylactic DNA vaccines delivered by NFIS tend to induce non-inferior immunogenicity to electroporation and enhanced responses compared to needle and syringe.

Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation.

Abstract: Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.

PIRET — A Platform for Treatment Planning in Electroporation-Based Therapies

Abstract: Tissue electroporation is the basis of several therapies. Electroporation is performed by briefly exposing tissues to high electric fields. It is generally accepted that electroporation is effective where an electric field magnitude threshold is overreached. However, it is difficult to preoperatively estimate the field distribution because it is highly dependent on anatomy and treatment parameters. Objective: We developed PIRET, a platform to predict the treatment volume in electroporation-based therapies. Methods: The platform seamlessly integrates tools to build patient-specific models where the electric field is simulated to predict the treatment volume. Patient anatomy is segmented from medical images and 3D reconstruction aids in placing the electrodes and setting up treatment parameters. Results: Four canine patients that had been treated with high-frequency irreversible electroporation were retrospectively planned with PIRET and with a workflow commonly used in previous studies, which uses different general-purpose segmentation (3D Slicer) and modeling software (3Matic and COMSOL Multiphysics). PIRET outperformed the other workflow by 65 minutes (× 1.7 faster), thanks to the improved user experience during treatment setup and model building. Both approaches computed similarly accurate electric field distributions, with average Dice scores higher than 0.93. Conclusion: A platform which integrates all the required tools for electroporation treatment planning is presented. Treatment plan can be performed rapidly with minimal user interaction in a stand-alone platform. Significance: This platform is, to the best of our knowledge, the most complete software for treatment planning of irreversible electroporation. It can potentially be used for other electroporation applications.

Irreversible Electroporation in Pancreatic Cancer—An Evolving Experimental and Clinical Method

Abstract: Pancreatic cancer has no symptoms until the disease has advanced and is aggressive cancer with early metastasis. Up to now, the only curative treatment is surgical resection, which is possible in the early stages of the disease. Irreversible electroporation treatment offers new hope for patients with unresectable tumors. Irreversible electroporation (IRE) is a type of ablation therapy that has been explored as a potential treatment for pancreatic cancer. Ablation therapies involve the use of energy to destroy or damage cancer cells. IRE involves using high-voltage, low-energy electrical pulses to create resealing in the cell membrane, causing the cell to die. This review summarizes experiential and clinical findings in terms of the IRE applications. As was described, IRE can be a non-pharmacological approach (electroporation) or combined with anticancer drugs or standard treatment methods. The efficacy of irreversible electroporation (IRE) in eliminating pancreatic cancer cells has been demonstrated through both in vitro and in vivo studies, and it has been shown to induce an immune response. Nevertheless, further investigation is required to assess its effectiveness in human subjects and to comprehensively understand IRE’s potential as a treatment option for pancreatic cancer.

Augmenting Intracellular Cargo Delivery of Extracellular Vesicles in Hypoxic Tissues through Inhibiting Hypoxia-Induced Endocytic Recycling.

Abstract: As mesenchymal stem-cell-derived small extracellular vesicles (MSC-sEVs) have been widely applied in treatment of degenerative diseases, it is essential to improve their cargo delivery efficiency in specific microenvironments of lesions. However, the interaction between the microenvironment of recipient cells and MSC-sEVs remains poorly understood. Herein, we find that the cargo delivery efficiency of MSC-sEVs was significantly reduced under hypoxia in inflammaging nucleus pulposus cells due to activated endocytic recycling of MSC-sEVs. Hypoxia-inducible factor-1 (HIF-1)-induced upregulated RCP (also known as RAB11FIP1) is shown to promote the Rab11a-dependent recycling of internalized MSC-sEVs under hypoxia via enhancing the interaction between Rab11a and MSC-sEV. Based on this finding, si-RCP is loaded into MSC-sEVs using electroporation to overcome the hypoxic microenvironment of intervertebral disks. The engineered MSC-sEVs significantly inhibit the endocytic recycling process and exhibit higher delivery efficiency under hypoxia. In a rat model of intervertebral disk degeneration (IDD), the si-RCP-loaded MSC-sEVs successfully treat IDD with improved regenerative capacity compared with natural MSC-sEV. Collectively, the findings illustrate the intracellular traffic mechanism of MSC-sEVs under hypoxia and demonstrate that the therapeutic capacity of MSC-sEVs can be improved via inhibiting endocytic recycling. This modifying strategy may further facilitate the application of extracellular vesicles in hypoxic tissues.

Characterization of Experimentally Observed Complex Interplay between Pulse Duration, Electrical Field Strength, and Cell Orientation on Electroporation Outcome Using a Time-Dependent Nonlinear Numerical Model

Abstract: Electroporation is a biophysical phenomenon involving an increase in cell membrane permeability to molecules after a high-pulsed electric field is applied to the tissue. Currently, electroporation is being developed for non-thermal ablation of cardiac tissue to treat arrhythmias. Cardiomyocytes have been shown to be more affected by electroporation when oriented with their long axis parallel to the applied electric field. However, recent studies demonstrate that the preferentially affected orientation depends on the pulse parameters. To gain better insight into the influence of cell orientation on electroporation with different pulse parameters, we developed a time-dependent nonlinear numerical model where we calculated the induced transmembrane voltage and pores creation in the membrane due to electroporation. The numerical results show that the onset of electroporation is observed at lower electric field strengths for cells oriented parallel to the electric field for pulse durations ≥10 µs, and cells oriented perpendicular for pulse durations ~100 ns. For pulses of ~1 µs duration, electroporation is not very sensitive to cell orientation. Interestingly, as the electric field strength increases beyond the onset of electroporation, perpendicular cells become more affected irrespective of pulse duration. The results obtained using the developed time-dependent nonlinear model are corroborated by in vitro experimental measurements. Our study will contribute to the process of further development and optimization of pulsed-field ablation and gene therapy in cardiac treatments.

Highly efficient mRNA delivery with nonlinear microfluidic cell stretching for cellular engineering.

Abstract: In the past few years, messenger RNA (mRNA) has emerged as a promising therapeutic agent for the treatment and prevention of various diseases. Clinically, mRNA-based drugs have been used for cancer immunotherapy, infectious diseases, and genomic disorders. To maximize the therapeutic efficacy of mRNA, the exact amount of mRNAs must be delivered to the target locations without degradation; however, traditional delivery modalities, such as lipid carriers and electroporation, are suboptimal because of their high cost, cell-type sensitivity, low scalability, transfection/delivery inconsistency, and/or loss of cell functionality. Therefore, new effective and stable delivery methods are required. Accordingly, we present a novel nonlinear microfluidic cell stretching (μ-cell stretcher) platform that leverages viscoelastic fluids, i.e., methylcellulose (MC) solutions, and cell mechanoporation for highly efficient and robust intracellular mRNA delivery. In the proposed platform, cells suspended in MC solutions with mRNAs were injected into a microchannel where they rapidly passed through a single constriction. Owing to the use of viscoelastic MC solutions, a high shear force was applied to the cells, effectively creating transient nanopores. This feature allows mRNAs to be effectively internalized through generated membrane discontinuities. Using this platform, high delivery efficiency (∼97%), high throughput (∼3.5 × 105 cells per min), cell-type-/cargo-size-insensitive delivery, simple operation (single-step), low analyte consumption, low-cost operation (<$1), and nearly clogging-free operation were demonstrated, demonstrating the high potential of the proposed platform for application in mRNA-based cellular engineering research.

Comprehensive review on thermal aspects of nonthermal irreversible electroporation

Abstract: Irreversible electroporation (IRE) is an innovative cell ablation method based on the concept that the application of excessive electric pulses induces a lethal increase in the permeability of the cell membrane owing to nanoscale defects, resulting in a gentle form of necrotic cell death. Although the mechanism of cell death by IRE is primarily nonthermal, thermal effects are inevitable because electric pulses inherently generate Joule heat. The larger the applied voltage to treat a large target, the greater the Joule heating and the consequent temperature rise. Therefore, the temperature increase due to Joule heating during pulse application should be carefully controlled to minimize thermal damage. Research on IRE is an interdisciplinary endeavor incorporating health science for humanitarian relief and engineering. Therefore, this study provides a comprehensive review of the thermal aspects of IRE based on existing in vitro and in vivo experimental and numerical studies. The paper begins with an overview of IRE treatment covering the geometry and arrangement of electrodes, pulse parameters, and cell death mechanisms, followed by sections on thermal damage evaluation that summarize the significant work of experiments, analysis, and comparisons. Finally, thermal mitigation strategies, including electrode modification, lowering the IRE threshold, and modified pulsing protocols, are discussed.

mRNAs encoding IL-12 and a decoy-resistant variant of IL-18 synergize to engineer T cells for efficacious intratumoral adoptive immunotherapy

Abstract: Interleukin-12 (IL-12) gene transfer enhances the therapeutic potency of adoptive T cell therapies. We previously reported that transient engineering of tumor-specific CD8 T cells with IL-12 mRNA enhanced their systemic therapeutic efficacy when delivered intratumorally. Here, we mix T cells engineered with mRNAs to express either single-chain IL-12 (scIL-12) or an IL-18 decoy-resistant variant (DRIL18) that is not functionally hampered by IL-18 binding protein (IL-18BP). These mRNA-engineered T cell mixtures are repeatedly injected into mouse tumors. Pmel-1 T cell receptor (TCR)-transgenic T cells electroporated with scIL-12 or DRIL18 mRNAs exert powerful therapeutic effects in local and distant melanoma lesions. These effects are associated with T cell metabolic fitness, enhanced miR-155 control on immunosuppressive target genes, enhanced expression of various cytokines, and changes in the glycosylation profile of surface proteins, enabling adhesiveness to E-selectin. Efficacy of this intratumoral immunotherapeutic strategy is recapitulated in cultures of tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells on IL-12 and DRIL18 mRNA electroporation.

Targeting the Inside of Cells with Biologicals: Toxin Routes in a Therapeutic Context

Abstract: Numerous toxins translocate to the cytosol in order to fulfil their function. This demonstrates the existence of routes for proteins from the extracellular space to the cytosol. Understanding these routes is relevant to multiple aspects related to therapeutic applications. These include the development of anti-toxin treatments, the potential use of toxins as shuttles for delivering macromolecular cargo to the cytosol or the use of drugs based on toxins. Compared with other strategies for delivery, such as chemicals as carriers for macromolecular delivery or physical methods like electroporation, toxin routes present paths into the cell that potentially cause less damage and can be specifically targeted. The efficiency of delivery via toxin routes is limited. However, low-delivery efficiencies can be entirely sufficient, if delivered cargoes possess an amplification effect or if very few molecules are sufficient for inducing the desired effects. This is known for example from RNA-based vaccines that have been developed during the coronavirus disease 2019 pandemic as well as for other approved RNA-based drugs, which elicited the desired effect despite their typically low delivery efficiencies. The different mechanisms by which toxins enter cells may have implications for their technological utility. We review the mechanistic principles of the translocation pathway of toxins from the extracellular space to the cytosol, the delivery efficiencies, and therapeutic strategies or applications that exploit toxin routes for intracellular delivery.

Engineered Cancer-Derived Small Extracellular Vesicle-Liposome Hybrid Delivery System for Targeted Treatment of Breast Cancer.

Abstract: Cancer-derived small extracellular vesicles (sEVs) may be a promising drug delivery system that targets cancer cells due to their unique features, such as native homing ability, biological barrier crossing capability, and low immune response. However, the oncogenic cargos within them pose safety concerns, hence limiting their application thus far. We proposed using an electroporation-based strategy to extract the endogenous cargos from cancer-derived sEVs and demonstrated that their homing ability was still retained. A membrane fusion technique was used to fuse these sEVs with liposomes to form hybrid particles, which possessed both benefits of sEVs and liposomes. Anti-EGFR monoclonal antibodies were modified on the hybrid particles to improve their targeting ability further. The engineered hybrid particles showed higher drug loading ability that is 33.75 and 43.88% higher than that of liposomes and sEVs, respectively, and improved targeting ability by 52.23% higher than hybrid particles without modification. This delivery system showed >90% cell viability and enhanced treatment efficiency with 91.58 and 79.26% cell migration inhibition rates for the miR-21 inhibitor and gemcitabine, respectively.

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beauty composite system

Abstract: Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo. This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Abstract: Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Exploring the Fate of Antibody-Encoding pDNA after Intramuscular Electroporation in Mice

Abstract: DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This study reports the quantitative evaluation and localization of the administered pDNA over time and its association with corresponding mRNA levels and systemic protein concentrations. pDNA encoding the murine anti-HER2 4D5 mAb was administered to BALB/c mice via intramuscular injection followed by electroporation. Muscle biopsies and blood samples were taken at different time points (up to 3 months). In muscle, pDNA levels decreased 90% between 24 h and one week post treatment (p < 0.0001). In contrast, mRNA levels remained stable over time. The 4D5 antibody plasma concentrations reached peak levels at week two followed by a slow decrease (50% after 12 weeks, p < 0.0001). Evaluation of pDNA localization revealed that extranuclear pDNA was cleared fast, whereas the nuclear fraction remained relatively stable. This is in line with the observed mRNA and protein levels over time and indicates that only a minor fraction of the administered pDNA is ultimately responsible for the observed systemic mAb levels. In conclusion, this study demonstrates that durable expression is dependent on the nuclear uptake of the pDNA. Therefore, efforts to increase the protein levels upon pDNA-based gene therapy should focus on strategies to increase both cellular entry and migration of the pDNA into the nucleus. The currently applied methodology can be used to guide the design and evaluation of novel plasmid-based vectors or alternative delivery methods in order to achieve a robust and prolonged protein expression.

Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.

Abstract: Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

Electric-field-induced topological changes in multilamellar and in confined lipid membranes

Abstract: It is well known that lipid membranes respond to a threshold transmembrane electric field through a reversible mechanism called electroporation, where hydrophilic water pores form across the membrane, an effect widely used in biological systems. The effect of such fields on interfacially-confined (stacked or supported) lipid membranes, on the other hand, which may strongly modulate interfacial properties, has not to our knowledge been previously studied. Motivated by recent surface forces experiments showing a striking effect of electric fields on lubrication by confined lipid bilayers, we carried out all-atom molecular dynamics simulations of such membranes under transverse electric fields. We find that in addition to electroporation, a new feature emerges of locally merged bilayers which act to bridge the confining interfaces. These features shed light on the remodelling of confined lipid membrane stacks by electric fields, and provides insight into how such fields may modulate frictional and more generally surface interactions in the presence of lipid-based boundary layers.

A simple, rapid, and efficient method for generating multigene‐knockout culture cells by the CRISPR/Cas9 system

Abstract: We evaluated the efficacy of simultaneous multiple‐gene knockout in human culture cells. By simple co‐transfection of HeLa cells with a mixture of pX330‐based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin‐resistant cells, Cas9/single‐guide RNA (sgRNA)‐transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co‐transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA‐induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co‐transfection‐based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene‐knockout cell lines.