CAR-T cell therapies have emerged as a revolutionary approach in the field of haematological malignancies, offering unprecedented clinical efficacy when patients have exhausted any other treatment ...option. Six CAR-T products are available worldwide, but many of the manufacturing challenges are yet to be addressed. A significant fraction of the cost is related to manufacturing the viral vector required to introduce the clinically relevant CAR construct. As such, non-viral gene delivery platforms such as electroporation or mechanoporation can potentially decrease the costs of manufacturing associated with CAR-T products. This study focused on establishing a scalable manufacturing method for CAR-T cell therapies using electroporation and a transposon-transposase system employed to ensure stable integration of the CAR transgene.
Several stages were undertaken to establish the scalable manufacturing process: (1) electroporation optimisation, (2) identification of suitable cell viability recovery strategy, (3) small-scale expansion studies followed by (4) scalable expansion (ongoing). The workflow used featured a screening across electroporation conditions, followed by a study focused on changing the feeding strategy to maximize cell viability recovery post-electroporation. Once these were identified, the non-viral CAR-T product was characterized using growth rate, metabolic profile, CD4:CD8 ratios, CD8 subsets and CAR expression. This work was conducted using cryopreserved CD3+ cells, RPMI supplemented with 10% FBS, 2 mM of L-glutamine and 50 IU/mL of IL-2 as expansion medium and TransAct as activation method.
Here, suitable electroporation programs that minimised cell viability loss after electroporation were identified. In addition, a DNA-to-cell ratio study demonstrated that 1 µg of each minicircle per million cells transfected is sufficient to generate CD3+ populations with viability levels above 90% with CAR expression levels reaching 30%, 48 hours past electroporation. On harvesting day, our optimized process led to a CAR-T product with a CD4:CD8 ratio of 3, with 95% of the CD8 in the central memory phenotype. CAR expression levels were shown to be above 25% on harvesting day. A total of 20-fold increase was reported during the 7-day expansion period. The next stage of this study will include the integration of the electroporation with stirred tank bioreactors to demonstrate the feasibility of modular manufacturing in the non-viral CAR-T space.
Nonthermal irreversible electroporation is a new tissue ablation technique that consists of applying pulsed electric fields across cells to induce cell death by creating permanent defects in the cell ...membrane. Nonthermal irreversible electroporation is of interest because it allows treatment near sensitive tissue structures such as blood vessels and nerves. Two recent articles report that electrolytic reaction products at electrodes can be combined with electroporation pulses to augment and optimize tissue ablation. Those articles triggered a concern that the results of earlier studies on nonthermal irreversible electroporation may have been tainted by unaccounted for electrolytic effects. The goal of this study was to reexamine previous studies on nonthermal irreversible electroporation in the context of these articles. The study shows that the results from some of the earlier studies on nonthermal irreversible electroporation were affected by unaccounted for electrolysis, in particular the research with cells in cuvettes. It also shows that tissue ablation ascribed in the past to irreversible electroporation is actually caused by at least 3 different cytotoxic effects: irreversible electroporation without electrolysis, irreversible electroporation combined with electrolysis, and reversible electroporation combined with electrolysis. These different mechanisms may affect cell and tissue ablation in different ways, and the effects may depend on various clinical parameters such as the polarity of the electrodes, the charge delivered (voltage, number, and length of pulses), and the distance of the target tissue from the electrodes. Current clinical protocols employ ever-increasing numbers of electroporation pulses to values that are now an order of magnitude larger than those used in our first fundamental nonthermal irreversible electroporation studies in tissues. The different mechanisms of cell death, and the effect of the clinical parameters on the mechanisms may explain discrepancies between results of different clinical studies and should be taken into consideration in the design of optimal electroporation ablation protocols.
We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells ...with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.
Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies ...is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.
In this article, the authors present a deep learning‐assisted nanofountain probe electroporation system in combination with microconfinement arrays to trap and transfect single cells. Using the combined platform, the authors demonstrate automated intracellular delivery and genetic perturbation in hard‐to‐transfect cells followed by temporal tracking of the perturbed cell colonies.
•E2 is the combination of reversible electroporation and electrolysis.•E2’s cell killing mechanism is ascribed to a synergistic effect of both components.•Cell death is initiated within the first two ...minutes after E2 application.•However, cell death occurs significantly delayed.•There is significant but innocuous temperature increase during E2 application.
Electrolytic Electroporation (E2) is the combination of reversible electroporation and electrolysis. It has been proposed as a novel treatment option to ablate tissue percutaneously. The present in vitro study in cells in suspension was performed to investigate the underlying mechanisms of action of E2. Different types of experiments were performed to isolate the effects of the electrolysis and the electroporation components of the treatment. Additionally, thermal simulations were performed to determine whether significant temperature increase contributes to the effect. The results indicate that E2’s cell killing efficacy is due to a combinational effect of electrolysis and reversible electroporation that takes place within the first two minutes after E2 application. The results further show that cell death after E2 treatment is significantly delayed. These observations suggest that cell death is induced in permeabilized cells due to the uptake of electrolysis species. Thermal simulations revealed a significant but innocuous temperature increase.
Electroporation based therapies and treatments (e.g. electrochemotherapy, gene electrotransfer for gene therapy and DNA vaccination, tissue ablation with irreversible electroporation and transdermal ...drug delivery) require a precise prediction of the therapy or treatment outcome by a personalized treatment planning procedure. Numerical modeling of local electric field distribution within electroporated tissues has become an important tool in treatment planning procedure in both clinical and experimental settings. Recent studies have reported that the uncertainties in electrical properties (i.e. electric conductivity of the treated tissues and the rate of increase in electric conductivity due to electroporation) predefined in numerical models have large effect on electroporation based therapy and treatment effectiveness. The aim of our study was to investigate whether the increase in electric conductivity of tissues needs to be taken into account when modeling tissue response to the electroporation pulses and how it affects the local electric distribution within electroporated tissues.
We built 3D numerical models for single tissue (one type of tissue, e.g. liver) and composite tissue (several types of tissues, e.g. subcutaneous tumor). Our computer simulations were performed by using three different modeling approaches that are based on finite element method: inverse analysis, nonlinear parametric and sequential analysis. We compared linear (i.e. tissue conductivity is constant) model and non-linear (i.e. tissue conductivity is electric field dependent) model. By calculating goodness of fit measure we compared the results of our numerical simulations to the results of in vivo measurements.
The results of our study show that the nonlinear models (i.e. tissue conductivity is electric field dependent: σ(E)) fit experimental data better than linear models (i.e. tissue conductivity is constant). This was found for both single tissue and composite tissue. Our results of electric field distribution modeling in linear model of composite tissue (i.e. in the subcutaneous tumor model that do not take into account the relationship σ(E)) showed that a very high electric field (above irreversible threshold value) was concentrated only in the stratum corneum while the target tumor tissue was not successfully treated. Furthermore, the calculated volume of the target tumor tissue exposed to the electric field above reversible threshold in the subcutaneous model was zero assuming constant conductivities of each tissue.Our results also show that the inverse analysis allows for identification of both baseline tissue conductivity (i.e. conductivity of non-electroporated tissue) and tissue conductivity vs. electric field (σ(E)) of electroporated tissue.
Our results of modeling of electric field distribution in tissues during electroporation show that the changes in electrical conductivity due to electroporation need to be taken into account when an electroporation based treatment is planned or investigated. We concluded that the model of electric field distribution that takes into account the increase in electric conductivity due to electroporation yields more precise prediction of successfully electroporated target tissue volume. The findings of our study can significantly contribute to the current development of individualized patient-specific electroporation based treatment planning.
Objective: The goal of our study was to determine the importance of electric field orientation in an anisotropic muscle tissue for the extent of irreversible electroporation damage by means of an ...experimentally validated mathematical model. Methods: Electrical pulses were delivered to porcine skeletal muscle in vivo by inserting needle electrodes so that the electric field was applied in direction either parallel or perpendicular to the direction of the muscle fibres. Triphenyl tetrazolium chloride staining was used to determine the shape of the lesions. Next, we used a single cell model to determine the cell-level conductivity during electroporation, and then generalised the calculated conductivity changes to the bulk tissue. Finally, we compared the experimental lesions with the calculated field strength distributions using the Sørensen-Dice similarity coefficient to find the contours of the electric field strength threshold beyond which irreversible damage is thought to occur. Results: Lesions in the parallel group were consistently smaller and narrower than lesions in the perpendicular group. The determined irreversible threshold of electroporation for the selected pulse protocol was 193.4 V/cm with a standard deviation of 42.1 V/cm, and was not dependent on field orientation. Conclusion: Muscle anisotropy is of significant importance when considering electric field distribution in electroporation applications. Significance: The paper presents an important advancement in building up from the current understanding of single cell electroporation to an in silico multiscale model of bulk muscle tissue. The model accounts for anisotropic electrical conductivity and has been validated through experiments in vivo .
CAR‐T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR ...enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+ B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non‐Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR‐T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time‐consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non‐viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non‐viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.
In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium ...chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.
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