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High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator

Laboratoire de Physiologie, Groupe Physiologie et Physiopathologie de l'Exercice et du Handicap, Groupement d'Intérêt Public-Exercice Sport Santé, Faculté de Médecine J. Lisfranc, 15 rue Ambroise Paré, Saint-Etienne Cedex, France.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 09/2002; 296(2):443-50. DOI: 10.1016/S0006-291X(02)00901-4
Source: PubMed

ABSTRACT Efficiency and reproducibility of gene electrotransfer depend on the electrical specifications provided by the pulse generator, such as pulse duration, pulse number, pulse frequency, pulse combination, and current intensity. Here, we describe the performances of GET42, a pulse generator specifically designed for gene electrotransfer into skeletal muscle. Expression of beta-galactosidase in the Tibialis anterior muscle of Sprague-Dawley male rats was increased 250-fold by GET42 compared to DNA injection alone. Combination of high and low current intensity pulses further increased transfection efficiency (400-fold compared to DNA injection without electrotransfer). Varying degrees of muscle necrosis were observed after gene electrotransfer. Nevertheless, muscle necrosis was dramatically reduced after optimization of cumulated pulse duration without significant reduction in transfection efficiency. Physiological applicability was illustrated by the analysis of cytochrome c promoter transactivation. In conclusion, GET42 has proven to be a reliable and efficient pulse generator for gene electrotransfer experiments, and provides a powerful mean to study in vivo the regulation of gene expression.

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Available from: Anne-Cécile Durieux, Jun 11, 2014
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    • "Indeed, an efficient transfection into skeletal muscles and tumors required the application of limited number of long pulses (several ms) at a low frequency (1 Hz). But, a very high number of repetitive short but stronger pulses (several μs) at a high frequency (kHz) give a high level of gene expression into the same tissues (Lucas et al. 2002; Vicat et al. 2000; Rizzuto et al. 1999; Mir et al. 1999; Durieux et al. 2002). A suitable protocol using a short high voltage pulse (kV/cm, μs) followed by several longer low voltage pulses (V/cm, ms) at a low frequency (1 Hz) has been proposed to aid gene delivery into skeletal muscle (Bureau et al. 2000). "
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    • "Transfection efficiency was calculated as the percentage of the area stained in blue over the whole muscle cross-sectional area [7] [30]. Muscle damage was assessed from Hemalun –Eosin – Safran (HES) staining and expressed as the percentage of damaged area over the whole crosssectional area [7] [30]. 2.6. "
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    ABSTRACT: We determined over a 3-week period some of the factors that may influence the kinetic of gene expression following in vivo gene electrotransfer. Histochemical analysis of beta-galactosidase and biochemical analysis of luciferase expressions were used to determine reporter gene activity in the Tibialis anterior muscles of young Sprague-Dawley male rats. Transfection efficiency peaked 5 days after gene electrotransfer and then exponentially decreased to reach non-detectable levels at day 28. Reduction of muscle damage by decreasing the amount of DNA injected or the cumulated pulse duration did not improve the kinetic of gene expression. Electrotransfer of luciferase expression plasmids driven either by viral or mammalian promoters rather show that most of the decrease in transgene expression was related to promoter origin/strength. By regulating the amount of transgene expression, the promoter origin/strength could modulate the immune response triggered against the foreign protein and ultimately the kinetic of transgene expression.
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    ABSTRACT: The advantages of non-viral gene transfer systems are safety and low immunogenicity, therefore they are well suited for use as vectors in gene therapy. The main disadvantage, namely their low gene-transfer-efficiency, can be improved through the development of systemic gene transfer systems using targeted vectors with high specificity and gene transfer efficiency. The intravenous application of PEI22lin/DNS complexes leads to a high gene expression in the lung, but with high toxicity. This observation can be explained by the positive surface charge of the DNS complexes and the uncomplexed free PEI, which leads to aggregation of erythrocytes. DNS complexes can be isolated from free uncomplexed PEI by gel filtration. The systemic application of gel filtrated PEI22lin complexes to non-tumor bearing mice resulted in reduced toxicity however there was a decreased in gene expression compared to non-filtrated complexes. The same experiment was performed on tumor bearing mice and again reduced toxicity was observed and interestingly slightly higher gene expression found in the tumor compared to the non-filtrated complexes. Shielding the positive surface charge of the PEI22lin complexes by transferrin led to increased gene expression in the tumor with reduced expression in the lung and other organs. The improved tumor targeted gene expression was associated with reduced systemic toxicity. Tumor targeted gene expression appears to be dependent on the tumor model as this observation was only found in neuro2A neuroblastoma tumor model in A/J mice and not in B16F10 melanoma tumor models of C57BL/6 mice and CT26 colon carcinoma tumor models of BALB/c mice. To enhance the intracellular efficiency of the vectors, the endosomolytic active peptide melittin was incorporated into the transferrin targeted complexes. This led to a further increase in gene expression in the Neuro2A tumor models in A/J mice. For the local gene transfer, electroporation proved to be an easy to handle method to obtain a high gene expression in tissue. The non-invasive kaliper electrode was suitable for gene transfer to both muscle and tumor. The applied voltage showed to be the most important parameter in expression. The use of electroporation for intratumoral transfer of the therapeutic gene encoding for the cytokine TNF was unsuccessful. However, systemic application of the TNF-α gene in transferrin targeted complexes in combination with the intraperitoneal application of the chemotherapeutic Doxil® showed a clear synergistic effect. A significant delay in the tumor growth and in some cases a complete regression of the tumor was observed. The enzyme cytochrome P450 metabolizes the non toxic prodrug cyclophosphamide (CPA) into the cytotoxic drug. Electroporation of the cytochrome P450 gene into the tumor lead to its localized protein expression. When followed by the intraperitoneal application of CPA, a significant delay in the tumor growth of the human hepatocellular carcinoma Huh7 was observed in SCID mice. When applied to the Neuro2A tumor model in A/J mice, this application scheme showed a complete tumor regression in two animals. Furthermore the systemic application of the P450 gene in transferrin targeted complexes containing melittin in combination with CPA led to a strong delay in the tumor growth. In summary, this work describes a new anti-cancer strategy using the combination of chemotherapeutics and non-viral gene delivery resulting in a synergistic therapeutic effect in vivo. This promising strategy will be more effective with the improvement of non-viral gene delivery systems which have better targeted gene expression with lower toxicity.
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