Showing papers on "Electroporation published in 2000"

Increased DNA vaccine delivery and immunogenicity by electroporation in vivo.

Abstract: DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.

In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves.

Abstract: A convenient, Agrobacterium-mediated transient expression assay has been evaluated for rapid analysis of plant promoters and transcription factors in vivo. By simple infiltration of Agrobacterium cells carrying appropriate plasmid constructs into tobacco leaves in planta, reproducible expression assays could be conducted in as little as 2-3 days without using expensive equipment (e.g. biolistic gun or electroporation apparatus) or complicated procedures (e.g. preparation of protoplasts). Biotic and abiotic treatments could be applied to the intact plant to examine their influence on promoter activity and gene expression. Using this method, we have tested the stress-responsive as-1 and heat shock elements, yeast GAL4 transactivation system, two promoters of pathogenesis-related (PR) genes as well as a heat shock promoter. Through deletion analyses of tobacco PR1a promoter and a novel myb1 promoter, we have also successfully identified the cis-regulatory regions in these promoters that are responsive to salicylic acid treatment or tobacco mosaic virus infection. Together, our results demonstrate that Agrobacterium-mediated transient expression is a simple and efficient method for in vivo assays of plant promoters and transcription factors.

Electroporation of cells and tissues

Abstract: Electrical pulses that cause the transmembrane voltage of fluid lipid bilayer membranes to reach at least U/sub m//spl ap/0.2 V, usually 0.5-1 V, are hypothesized to create primary membrane "pores" with a minimum radius of -1 nm. Transport of small ions such as Na/sup +/ and Cl/sup -/ through a dynamic pore population discharges the membrane even while an external pulse tends to increase U/sub m/, leading to dramatic electrical behavior. Molecular transport through primary pores and pores enlarged by secondary processes provides the basis for transporting molecules into and out of biological cells. Cell electroporation in vitro is used mainly for transfection by DNA introduction, but many other interventions are possible, including microbial killing. Ex vivo electroporation provides manipulation of cells that are reintroduced into the body to provide therapy. In vivo electroporation of tissues enhances molecular transport through tissues and into their constitutive cells. Tissue electroporation, by longer, large pulses, is involved in electrocution injury. Tissue electroporation by shorter, smaller pulses is under investigation for biomedical engineering applications of medical therapy aimed at cancer treatment, gene therapy, and transdermal drug delivery. The latter involves a complex barrier containing both high electrical resistance, multilamellar lipid bilayer membranes and a tough, electrically invisible protein matrix.

Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice.

Abstract: Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

Medical applications of electroporation

Abstract: In vivo electroporation, first reported in 1987, makes it possible to render cell membranes temporarily permeable to substances that otherwise would not be able to effectively enter the cell interior. Micro- or millisecond pulses of electrical field strengths exceeding the natural cellular transmembrane potential difference of approximately I V results in permeabilization ("poration") of cell membranes. This phenomenon opens up numerous applications in the medical field. Electroporative delivery of chemotherapeutic drugs into tumor cells has proven successful in clinical studies to treat malignant tumors and is nearing market Introduction in Europe. For gene therapy applications, delivery of DNA by electroporation into a variety of tissues has been shown to consistently result in a 100-1000-fold enhancement of gene expression. Other applications of electroporation discussed in this paper include intravascular delivery of drugs and genes with electroporation catheters, electroinsertion of molecules into membranes, intraocular delivery of drugs and genes, and transdermal drug delivery. The use of electroporation for drug and gene delivery in vivo is clearly gaining momentum, and new medical applications are emerging at an increasing rate.

Controlled electroporation and mass transfer across cell membranes

Abstract: Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought. Electroporation is achieved by applying a voltage between the two chambers, and diffusive transport is achieved either by a difference in solute concentration between the liquids surrounding the cell and the cell interior or by a differential in concentration between the two chambers themselves. Electric current and diffusive transport are restricted to a flow path that passes through the opening.

Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes

Abstract: The use of tumor antigen loaded dendritic cells (DC) is one of the most promising approaches to induce a tumor specific immune response in vivo. Several strategies have been designed to load DC with tumor antigens. In this study, we investigated the delivery of in vitro transcribed RNA and plasmid DNA into monocyte-derived, ie non-proliferating human DC, using several nonviral transfection methods including electroporation and lipofection. Green fluorescent protein (GFP) was used as a reporter gene and influenza matrix protein 1 (M1) as a model antigen for HLA class I restricted antigen presentation. Using electroporation in combination with DNA or with RNA, up to 11% of DC were GFP-positive. Using liposomes as a vehicle for DNA transport up to 10% of the DC were GFP-positive. A significant increase in transfection efficacy, of up to 20%, was observed when GFP RNA was used in combination with liposomes. Importantly, the RNA transfected DC retained their typical morphological and immunophenotypical characteristics. In addition, DC transfected with M1 RNA were able to stimulate autologous peripheral M1-specific memory cytotoxic T lymphocytes (CTL), as well as M1-specific CTL clones. Furthermore, comparison of DNA-transfected DC with RNA-transfected DC revealed the latter to be far better stimulators of antigen-specific T cells. This RNA transfection technique consequently represents a very promising tool for future immunotherapy strategies.

The Role of Electroporation in Defibrillation

Abstract: Electric shock is the only effective therapy against ventricular fibrillation. However, shocks are also known to cause electroporation of cell membranes. We sought to determine the impact of electroporation on ventricular conduction and defibrillation. We optically mapped electrical activity in coronary-perfused rabbit hearts during electric shocks (50 to 500 V). Electroporation was evident from transient depolarization, reduction of action potential amplitude, and upstroke dV/dt. Electroporation was voltage dependent and significantly more pronounced at the endocardium versus the epicardium, with thresholds of 229+/-81 versus 318+/-84 V, respectively (P=0.01, n=10), both being above the defibrillation threshold of 181.3+/-45.8 V. Epicardial electroporation was localized to a small area near the electrode, whereas endocardial electroporation was observed at the bundles and trabeculas throughout the entire endocardium. Higher-resolution imaging revealed that papillary muscles (n=10) were most affected. Electroporation and conduction block thresholds in papillary muscles were 281+/-64 V and 380+/-79 V, respectively. We observed no arrhythmia in association with electroporation. Further, preconditioning with high-energy shocks prevented reinduction of fibrillation by 50-V shocks, which were otherwise proarrhythmic. Endocardial bundles are the most susceptible to electroporation and the resulting conduction impairment. Electroporation is not associated with proarrhythmic effects and is associated with a reduction of vulnerability.

Cell/tissue analysis via controlled electroporation

Abstract: An electrical current is created across an electrically conductive medium comprising a cell which may be part of a tissue of a living organism. A first electrical parameter which may be current, voltage, or electrical impedance is measured. A second electrical parameter which may be current, voltage or a combination of both is then adjusted and/or analyzed. Adjustments are carried out to facilitate analysis and/or obtain a desired degree of electroporation. Analysis is carried out to determine characteristics of the cell membrane and/or tissue.

Toxicity of anticancer agents mediated by electroporation in vitro.

Abstract: Electroporation is a physical event that temporarily reduces cell membrane barrier properties. Diminished membrane barrier properties are achieved by exposing cells to pulsed electric fields. When a cell has been treated with electric fields it is possible for extracellular agents to gain access to

Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo.

Abstract: Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Electromanipulation of mammalian cells: fundamentals and application

Abstract: Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian cells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of cells. Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermabilization in response to a breakdown pulse. Transient stretching force assumes a maximum value in low-conductivities pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible fields of (genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed shout 100 /spl mu/s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivities. Such media are also beneficial because field-inducted apoptosis does not occur under these conditions (in contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: 1) to high incorporation rates of plasmids (DNA) or artificial chromesomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising gents for cancer treatment, as shown by first clinical trials.

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene.

Abstract: We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells.

Abstract: . Purpose: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. Methods: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. Results: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. Conclusions: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.

Brief Report: Muscle Transfection by Electroporation with High-Voltage and Short-Pulse Currents Provides High-Level and Long-Lasting Gene Expression

Abstract: Gene transfer into muscle by electroporation with low-voltage and long-pulse (LV/LP, 100 V/50 msec) currents was shown to be more efficient than simple intramuscular DNA injection. Nevertheless, transgene expression declined from day 7 and only reached 10% of the maximum 3 weeks after electroporation. We have optimized electroporation conditions including voltage, pulse number, and the amount of injected luciferaseencoding plasmid DNA in the tibialis anterior muscle. Using high-voltage and short-pulse (HV/SP, 900 V/100 mu sec) currents, we observed an average 500-fold increase in luciferase expression, in comparison with nonelectroporated muscle. Moreover, sustained and long-lasting gene expression was observed for at least 6 months. When we compared HV/SP currents with LV/LP currents, luciferase expression was similar 24 hr after electroporation. One month later, whereas luciferase expression was stable in muscle electroporated with HV/SP currents, it decreased 600-fold in muscle electroporated ...

Electrical impedance tomography to control electroporation

Abstract: Images created by electrical impedance tomography (EIT) are used to adjust one or more electrical parameters and obtain a desired degree of electroporation of cells in tissue The parameters include current, voltage and a combination thereof The cells are subjected to conditions such that they become permeabilized but are preferably not subjected to conditions which result in irreversible pore formation and cell death The electroporation can analyze cell membranes, diagnose tissues and the patient as well as to move materials into and out of cells in a controlled manner

Electrochemotherapy, electrogenetherapy, and transdermal drug delivery : electrically mediated delivery of molecules to cells

Abstract: Reviews. Principles of Membrane Electroporation and Transport of Macromolecules, Eberhard Neumann, Sergej Kakorin, and Katja Toensing. Instrumentation and Electrodes for In Vivo Electroporation, Gunter A. Hofmann. Numerical Modeling for In Vivo Electroporation, Dejan Semrov and Damijan Miklavcic. In Vitro Delivery of Drugs and Other Molecules to Cells, Marie-Pierre Rols, Muriel Golzio, Christine Delteil, and Justin Teissie. The Basis of Electrochemotherapy, Lluis M. Mir and Stephane Orlowski. Electrochemotherapy: Animal Model Work Review, Gregor Sersa. Clinical Trials for Solid Tumors Using Electrochemotherapy, Richard Heller, Richard Gilbert, and Mark J. Jaroszeski. In Vitro and Ex Vivo Gene Delivery to Cells by Electroporation, Sek Wen Hui and Lin Hong Li. Delivery of Genes In Vivo Using Pulsed Electric Fields, Mark J. Jaroszeski, Richard Gilbert, Claude Nicolau, and Richard Heller. Mechanism of Transdermal Drug Delivery by Electroporation, Timothy E. Vaughan and James C. Weaver. Mechanistic Studies of Skin Electroporation Using Biophysical Methods, Mark R. Prausnitz, Uwe Pliquett, and Rita Vanbever. Electrochemotherapy Protocols. Treatment of Murine Transplanted Subcutaneous Tumors Using Systemic Drug Administration, Stephane Orlowski and Lluis M. Mir. Electrochemotherapy of Murine Melanoma Using Intratumor Drug Administration, Richard Heller, Richard Gilbert, and Mark J. Jaroszeski. Treatment of a Tumor Model with ECT Using 4+4 Electrode Configuration, Maja Cemazar. Treatment of Multiple Spontaneous Breast Tumors in Mice Using Electrochemotherapy, Stephane Orlowski and Lluis M. Mir. Electroporation of Muscle Tissue In Vivo, Julie Gehl and Lluis M. Mir. Treatment of Human Pancreatic Tumors Xenografted in Nude Mice by Chemotherapy Combined with Pulsed Electric Fields, Sukhendu B. Dev, Gunter A. Hofmann, and Gurvinder S. Nanda. Distribution of Bleomycin in a Rat Model, Per Engstrom, Leif G. Salford, and Bertil R. R. Persson. Treatment of Rat Bladder Cancerwith Electrochemotherapy In Vivo, Yoko Kubota, Teruhiro Nakada, and Isoji Sasagawa. Electrochemotherapy for the Treatment of Soft Tissue Sarcoma in a Rat Mode, Richard Gilbert, Mark J. Jaroszeski, and Richard Heller. Treatment of Spontaneous Soft Tissue Sarcomas in Cat, Stephane Orlowski and Lluis M. Mir. Treatment of Rat Glioma with Electrochemotherapy, Leif G. Salford, Per Engstrom, and Bertil R. R. Persson. Treatment of Liver Malignancies with Electrochemotherapy in a Rat Model, Mark J. Jaroszeski, Richard Gilbert, and Richard Heller. Treatment of Liver Tumors in Rabbit, Stephane Orlowski and Lluis M. Mir. Electrogenetherapy Protocols. Electrically Mediated Reporter Gene Transfer into Normal Rat Liver Tissue, Mark J. Jaroszeski, Richard Gilbert, Claude Nicolau, and Richard Heller. Reporter/Functional Gene Transfer in Rat Brain, Toru Nishi, Kimio Yoshizato, Tomoaki Goto, Hideo Takeshima, Shigeo Yamashiro, Jun-ichi Kuratsu, Hideyuki Saya, and Yukitaka Ushio. In Vivo Gene Electroporation in the Mouse Testis, Tatsuo Muramatsu. Ex Vivo Stromal Cell Electroporation of Factor IX cDNA for Treatment of Hemophilia B, Armand Keating, Edward Nolan, Robin Filshie, and Sukhendu B. Dev. In Ovo Gene Electroporation into Early Chicken Embryos, Tatsuo Muramatsu. Transdermal Delivery Protocols. Electrical Impedance Spectroscopy for Rapid and Noninvasive Analysis of Skin Electroporation, Uwe Pliquett and Mark R. Prausnitz. An In Vitro System for Measuring the Transdermal Voltage and Molecular Flux Across the Skin in Real Time, Tani Chen, Robert Langer, and James C. Weaver. Using Surface Electrodes to Monitor the Electric-Pulse-Induced Permeabilization of Porcine Skin, Stephen A. Gallo, Patricia G. Johnson, and Sek Wen Hui. Transdermal Delivery Using Surface Electrodes in Porcine Skin, Patricia G. Johnson, Stephen A. Gallo, and Sek Wen Hui. Transdermal Drug Delivery by Skin Electroporation in the Rat, Rita Vanbever and Veronique Preat. In Vivo Skin-Targeted Gene Delivery by

In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity.

Abstract: When cancer cells, including melanoma cells, are genetically altered to secrete cytokines, irradiated and injected into subjects, long-term antitumour immunity is induced. Optimally, existing melanomas induced to produce cytokines in vivo could stimulate this same immune response. Although in vivo electroporation enhances plasmid expression, electroporation of plasmids encoding granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-2 (IL2) into B16 mouse melanomas did not significantly alter tumour growth at the concentration tested. Electrochemotherapy, which causes short-term, complete regressions of treated tumour but no resistance to challenge, was combined with plasmid delivery. The combination treatment resulted in the induction of long-term immunity to recurrence and resistance to challenge in up to 25% of mice.

Electroporation-enhanced gene delivery in mammary tumors.

Abstract: Electroporation was applied to enhance gene transfer into subcutaneous MC2 murine breast tumors. Cultured MC2 cells were also transfected by electroporation or by cationic liposomes in the presence of serum using pSV-luc plasmids. Electroporation parameters and liposome formulation were optimized to achieve the highest relative levels of transfection. An electric field threshold for successful electrotransfection in cultured cells appeared around 800-900 V/cm. The liposomes used contained the cationic lipid dioleoyl-3-trimethylammonium propane (DOTAP). Multilamellar vesicles (MLV) had a 10-fold advantage over small unilamellar vesicles (SUV) in cell culture transfection. For in vivo gene delivery, the plasmids were injected either alone, or in complex with MLV or SUV DOTAP liposomes. A series of six electric pulses 1 ms long were applied across tumors, using caliper electrodes on the skin surface. Electric field strengths ranged from 400-2300 V/cm. Luciferase expression was approximately two orders of magnitude higher than controls in tumors treated with pulses > or =800 V/cm. Differences between enhanced relative levels of transfection using uncomplexed plasmid and lipoplexes were not statistically significant. Distribution of DNA into tumor tissues was monitored by fluorescence in situ PCR. The highest numbers of fluorescent cells were found in tumors electroporated following the injection of plasmid. The significant transfection improvement shows that in vivo electroporation is a powerful tool for local gene delivery to tumors.

Continuous Erythropoietin Delivery by Muscle-Targeted Gene Transfer Using in Vivo Electroporation

Abstract: It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGSEpo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 mug of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 mug of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 ± 33.0 mU/ml on day 7 and gradually decreased to 35.9 ± 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 ± 1.1 to 67.8 ± 2.2% on day 32 (p<0.001). In pCAGGS-Epo t...

Misexpression of genes in brain vesicles by in ovo electroporation.

Abstract: Transfection to living chick embryos in ovo by electroporation has been recently developed. In this mini-review, misexpression in brain vesicles is introduced. To transfect, expression plasmid is inserted in the brain vesicle, and the square pulse of 25 V, 50 ms was charged five times. The translation product of the transfected gene is detected 2 h after electroporation, and reaches the peak at 24 h after electroporation. Transfection is so effective that this method is contributing greatly to the study of the molecular mechanisms of morphogenesis.

High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34 + cells via electroporation-mediated gene delivery

Abstract: The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and adult bone marrow (BM) CD34+ cells. We describe optimal culture and electroporation parameters for efficient gene delivery in prestimulated T lymphocytes (16.3 +/-1.3%), as well as 2-day cultured adult BM CD34+ cells (29.6+/-4.6%). PHA-stimulated T cells were most receptive for transfection after 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of prestimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolonged period. In addition, fresh unstimulated BM CD34+ cells were consistently transfected (5.2+/-0.4%) with minimal cytotoxicity (<5%), even without preliminary CD34+ cell purification. Both T cells and CD34+ cells retained their phenotype and functional capacity after electroporation. These results demonstrate that electroporation is a suitable nonviral transfection technique that may serve applications in gene therapy protocols using T lymphocytes or CD34+ cells.