Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

Abstract

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 g of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.Genes can be transferred into skeletal muscle cells of rodents and primates by intramuscular injection of plasmid DNA, and the resulting gene expression has been reported to last as long as several months (1, 2). Similarly, various viral vectors such as adenoviral, retroviral, and AAV-based vectors (3), have been used to transduce myofibers in vivo. The i.m. injection of plasmid DNA, however, has several advantages over viral vectors. First, plasmid DNA vectors are easier to construct and can be prepared as pharmaceutical-grade solutions (4) without the risk of contamination with wild-type infectious particles. Second, previous infection by wild-type adenovirus or AAV may induce a neutralizing antibody response that could preclude administration of the recombinant virus. In contrast, anti-DNA antibodies have never been detected in experiments of muscle DNA injection (2), therefore it is possible to readminister plasmid DNA by i.m. injection if repeated therapy or escalation is required.Despite the promise of i.m. injection of plasmid vectors for treating serum protein deficiencies, several important issues remain to be addressed before this approach becomes feasible for human gene therapy. The potential clinical usefulness of direct gene transfer to muscle of plasmid DNA is in fact limited by the low and highly variable level of gene expression (1, 2, 5, 6). Therefore, although DNA injection is potentially very powerful as a vaccination method because a low level of gene expression is sufficient to trigger immunoresponses, it is necessary to increase the efficiency of DNA uptake after i.m. injection of plasmid vectors before using this technique as a standard gene correction procedure.One of the most efficient methods implemented to achieve gene transfer and expression in mammalian cells is based on electric pulses (7). Electroporation has been used to introduce foreign DNA in different cell types (7), but it has also recently met with some success in in vivo applications. Gene transfer by electrical permeabilization has been obtained in skin (8, 9), corneal endothelium (10), melanoma (11), brain (12), liver, (13) and muscle (14) of experimental animals.We have shown previously that electropermeabilization can increase severalfold the uptake by rat muscle of a plasmid encoding the Escherichia coli lacZ gene (15). In this study...

Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo

“…Erythropoietin cDNA expression after muscle injection was increased to levels adequate to induce an increase in hematocrit in mice. 19 An increase in IL-5 protein expression was observed after muscle electroporation. 17 It is apparent that protein expression observed after in vivo electroporation is ample to induce quantifiable responses in vivo.…”

“…14 Longer length (ms) pulses also result in increased expression of injected plasmids to mouse testes, 15 rat hepatocytes, 13 mouse melanoma cells 16 and mouse skeletal muscle, [17][18][19] but tissue damage is observed at field strengths greater than 100 V/cm. 13,15 The results presented here demonstrate that in vivo electroporation enhances delivery of plasmid DNA to rat hepatocellular carcinomas.…”

“…11 This method has recently been used to deliver reporter genes in vivo to normal rat hepatocytes, 12,13 rat brain tumor cells, 14 mouse testes, 15 mouse melanoma cells, 16 and skeletal muscle. [17][18][19] A previous study by this laboratory demonstrated in vivo gene delivery to normal liver tissue using electroporation. 12 In the present study, the delivery of plasmids encoding reporter genes to a solid visceral tumor is demonstrated.…”

Genetic vaccines against Ep‐CAM break tolerance to self in a limited subset of subjects: Initial identification of predictive biomarkers

“…DNA vaccination in mice was performed via both electroporation according to Rizzuto et al [32] and adenovirus vaccination according to Mennuni et al [17]. Monkey immunization studies were performed at The Biomedical Primate Research Center (BPRC, Rijswijk, The Netherlands) using a group of four rhesus macaques (Macaca mulatta).…”

Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at distant site

“…Most of the data related to muscle electroporation delivery are based on the investigation of reporter genes, 10 except for one report in which erythropoietin was delivered to muscle using electroporation to generate a therapeutic effect. 11 To date, muscle electroporation gene delivery has not been tested in any disease model. We present data to demonstrate that electrotransfection of the IL-12 gene into muscle induced significant and durable expression of IFN -, increased CD8 + T-cell population in peripheral blood, and inhibition of tumor establishment and growth.…”