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Delivery of RNAi-Based Oligonucleotides by Electropermeabilization

Centre National de la Recherche Scientifique (CNRS), Institut de Pharmacologie et de Biologie Structurale (IPBS) BP 64182, 205 route de Narbonne, Toulouse F-31077, France
Université Paul Sabatier de Toulouse, IPBS, Toulouse F-31077, France
Author to whom correspondence should be addressed.
Pharmaceuticals 2013, 6(4), 510-521;
Received: 31 December 2012 / Revised: 19 March 2013 / Accepted: 27 March 2013 / Published: 10 April 2013
(This article belongs to the Special Issue RNAi-Based Therapeutics)


For more than a decade, understanding of RNA interference (RNAi) has been a growing field of interest. The potent gene silencing ability that small oligonucleotides have offers new perspectives for cancer therapeutics. One of the present limits is that many biological barriers exist for their efficient delivery into target cells or tissues. Electropermeabilization (EP) is one of the physical methods successfully used to transfer small oligonucleotides into cells or tissues. EP consists in the direct application of calibrated electric pulses to cells or tissues that transiently permeabilize the plasma membranes, allowing efficient in vitro and in vivo cytoplasmic delivery of exogenous molecules. The present review reports on the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred, the mechanism(s) of their electrotransfer and the technical settings for pre-clinical purposes.

1. Introduction

RNA interference (RNAi) is a natural process allowing gene silencing at post-transcriptional level [1]. It offers the possibility of targeting and silencing any pathological protein in a specific way [2]. RNAi is mediated endogenously by microRNAs (miRNAs) [3] and experimentally by small interfering RNAs (siRNAs) or miRNA mimics [4]. Both are small (~22 nt) noncoding RNAs that, once loaded into the RNA-induced silencing complex (RISC), bind to their target messenger RNA (mRNA) impairing its translation. As a result, gene expression is suppressed [5,6].
However, clinical success of RNAi has been hampered by its poor cellular uptake and stability. To overcome these problems, progress has been made to develop new technologies to optimize the chemistry of siRNAs on the one hand, and achieve their effective delivery on the other hand [7]. In fact, their physicochemical characteristics (i.e., large molecular weight and anionic charges) prevent passive diffusion across the plasma membrane of most cell types. Thus, delivery methods are required to allow oligonucleotides to enter cells while being biocompatible, safe, and targeted. Delivery is therefore one of the major challenges for the development of RNAi-based therapeutics [8].
Electropermeabilization (EP) is a promising non-viral biophysical method for in vitro and in vivo delivery of various molecules such as drugs [9] and nucleic acids [10,11]. EP was introduced in 1960s [12] and consists in the direct application of external electric field pulses to permeabilize target cells or tissues. Under calibrated electric conditions, these pulses transiently destabilize the plasma membrane, causing its permeabilization [13]. Since the first report in 2002 [14], numerous publications have demonstrated potency of this technique for siRNA delivery [15,16,17,18,19,20]. Efficiency and convenience of this technique (i.e., simplicity of the procedure, low cost and speed) led to its extensive use for both external and internal tissues [21,22]. Moreover, very few side-effects have been reported (mostly superficial burns under poorly controlled conditions), emphasizing the innocuousness of this method for clinical use. To date, several preclinical and clinical studies have shown encouraging results by using hydrophilic cytotoxic drugs or plasmid DNA combined with EP demonstrating antitumor effectiveness [23,24,25,26,27,28].
This article reviews the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred and the associated mechanism(s) of their electrotransfer. Elucidation of the mechanisms involved in RNAi-based oligonucleotides electro-delivery will lead to a better optimization of future treatments and will allow the development of new approaches to EP-based therapy.

2. How to Perform Electrotransfer of RNAi-Based Oligonucleotides

The basic instrumentation for EP comprises a pulse generator and specific electrodes. However, the definition of the electrical parameters and the design of the electrodes are crucial steps for efficient and safe electrotransfer into the target cells and tissues.
Electrical parameters. Electrical conditions are characterized by physical parameters: electric field intensity (E) and number of electric pulses (N), their duration (T) and frequency (F). The definition of these parameters are essential to achieve effective transfer while preserving cell viability and avoiding unwanted effects on the patient (essentially superficial burns and muscle contractions). Depending on the nature of the molecule to be transferred, there are two types of electrical treatment: electrochemotherapy (ECT) and electrogenetherapy (EGT).
ECT is the combination of a cytotoxic (low molecular weight) drug, such as cisplatin or bleomycin, with electric pulses applied to the tumor. This method uses high electric field intensity (kV/cm) of short duration (microseconds). ECT protocols (8 pulses of 100 µs at 1,300 V/cm; 1 Hz) have been approved in human clinics to treat malignant cutaneous and subcutaneous melanoma [29]. ECT has also been used successfully in veterinary medicine for treatment of feline sarcoma [30], perianal and mast cell tumors in dogs [31,32] and equine sarcoids [33].
The EGT parameters allow the electrotransfer of macromolecules (e.g., nucleic acids) for gene therapy purposes. Compared to ECT, this procedure uses lower field intensity (V/cm) with longer duration (milliseconds) to increase the electrophoretic movement of the electrotransferred macromolecule during the electric pulse. In animal models, EGT has been performed in many different tissues: skeletal [34,35], cardiac muscle [36], liver [37,38], skin [39,40], spleen [41], kidney [42], brain [43], joints [44] and tumor [20]. EGT has also been used successfully in veterinary medicine for the treatment of mast cell tumors in dogs [45]. This procedure is a simple way to obtain an efficient transfer of siRNA both in vitro and in vivo [20].
Other electrical settings are reported. They consist of combinations of pulses of high voltage and short duration (HV, permeabilizing pulse) [46] followed by low voltage and long duration non-permeabilizing pulses (LV, electrophoretic pulse). Studies performed on mice skeletal muscle showed that the HV-LV pulse sequence leads to an efficient gene transfer, rather similar to what was obtained with the EGT parameters [47].
It is of note that the definition of the electrical parameters is depending on the tissue treated. In fact, tissue electrical response depends on its origin, shape and environment. The type of electrodes used also modify the tissue electrical response.
Electrodes. Indeed, the success of the EP technique is linked to the proper distribution of the electric field in the tissue, that is dependent on the type of electrodes used. Numerous electrode configurations have been developed for therapeutic purposes: parallel plate, needle, contact wire, etc. [48].
The parallel plate electrodes are the most frequently used for electrotransfer. This consists of placing the electrodes on both sides of the tissue prior to electric pulse delivery [11,49]. This simple design has produced high response rates in animal studies [17,50]. Their limitation is that tissue should fit into the inter-electrode space and that the high field at the point of contact of the electrode with the skin can induce superficial burns if sharp angles are present in their design.
If the parallel plate electrodes have been shown to be well suited for treatment of cutaneous tumors, needle electrodes are more efficient in intraoperative settings, for treating the deepest regions or in large animals [51,52]. Needle electrodes are inserted through the skin allowing deeper penetration of the electric field into the tissue. However, with these electrodes, the electric field is heterogeneous as it is confined to the immediate proximity of the needles. A strong burning of the tissues in contact with the needles was reported. Several configurations are in development, such as linear and circular arrays [51].
The contact wire electrodes have been shown to be very efficient and convenient when large tissue surfaces (several square centimeters) must be treated. They are easy to use at the cutaneous level. Crossed configurations in the field distribution can be easily obtained by changing their orientation on the skin surface [48].
New electrode designs are under development in order to adapt field distribution to the geometry of the tumor, enabling cancer cell permeabilization with minimum tissue damage. These improvements are based on numerical modeling, but the irregular shape of the tumor and the heterogeneity of the surrounding layers render this numerical modeling difficult [53].

3. Electrotransfer of RNAi-Based Oligonucleotides and Mechanisms

EP represents a very attractive delivery method that has led to abundant literature, but only a few reports concern the mechanism of delivery [13]. We showed that electrotransfer of large molecules such as plasmid DNA is a multistep process: electrophoretic migration towards the permeabilized membrane, insertion into the membrane, all within the pulse delivery, followed by a slow translocation across the membrane and migration towards the nucleus [54,55] (Figure 1). Electrotransferred plasmid DNA is not injected into the cytoplasm as observed for small molecules, such as anticancer drugs. Small molecules enter into cells across permeabilized zones of the membrane facing both electrodes. This fast entry occurred mostly by post pulse diffusion process [56] (Figure 1). EP appears also to be well adapted for all kinds of nucleic acids including RNAi-based oligonucleotides [2].
Figure 1. Mechanisms of electrotransfer of molecules. Propidium iodide (PI) or small molecules mostly enter the cells by diffusion through both sides of the permeabilized membrane facing the electrodes after the pulse. Plasmid DNA (pDNA), dragged by the electrophoretic forces, interacts with the permeabilized membrane only at the cathode side and remains for a few minutes on the membrane before its translocation into the cytoplasm. siRNA (as well as siLNA) migrates electrophoretically during the pulse through the membrane only at the cathode side, resulting in direct cytosolic localization. LNA-DNA oligomer (LNA) migrates electrophoretically during the pulse through the membrane only at the cathode side, resulting in direct cytosolic localization followed by a rapid nuclear relocalization. The pictures on the left represent stably transfected cells with GFP (adapted from references [50,57]).
Figure 1. Mechanisms of electrotransfer of molecules. Propidium iodide (PI) or small molecules mostly enter the cells by diffusion through both sides of the permeabilized membrane facing the electrodes after the pulse. Plasmid DNA (pDNA), dragged by the electrophoretic forces, interacts with the permeabilized membrane only at the cathode side and remains for a few minutes on the membrane before its translocation into the cytoplasm. siRNA (as well as siLNA) migrates electrophoretically during the pulse through the membrane only at the cathode side, resulting in direct cytosolic localization. LNA-DNA oligomer (LNA) migrates electrophoretically during the pulse through the membrane only at the cathode side, resulting in direct cytosolic localization followed by a rapid nuclear relocalization. The pictures on the left represent stably transfected cells with GFP (adapted from references [50,57]).
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Small interfering RNA (siRNA). siRNAs are double-stranded noncoding RNA that once introduced into the cells are loaded into the cytoplasmic RNA-induced silencing complex (RISC). The complex binds the targeted RNA messenger (mRNA) through a base-pairing interaction and leads to its cleavage [58]. Therefore, siRNA offers the possibility to silence the expression of any pathological protein in a specific way. RNAi-based experiments can suffer from a lack of specificity due to silencing of non-targeted genes unless a well-designed sequence is used [59]. Although siRNA in vitro efficiency is high, its in vivo delivery remains a critical issue for its therapeutic development. A safe approach requests a direct transfer to the cytoplasm to avoid unwanted effects such as interferon response [60].
Using a fluorescently labeled siRNA, we observed by fluorescence microscopy at the single cell level that electrotransferred siRNA was distributed homogeneously throughout the cytoplasm of cultured tumor cells [61]. Thus, upon EP, siRNA had immediate, free access to the cytoplasm allowing its direct interaction with the enzymatic machinery (RISC) and its mRNA target. In these experiments, no electrotransferred siRNA was seen in the nucleus of viable cells. The mechanism of siRNA electrotransfer differs from what is described for plasmid DNA or drugs. siRNA electrotransfer implies electrophoretic movements. However, contrary to plasmid DNA, no complex between the membrane and the siRNA was observed (Figure 1). siRNA electrotransfer led to its direct transfer to the cytoplasm. After pulse application no intracellular diffusion of siRNA occurred although the membrane is still permeabilized for cytotoxic drugs [61].
EP has been used successfully for in vivo siRNA electrotransfer in a wide variety of tissues such as muscle [50,62], joint tissue [63], eyes [19], brain [64], kidney [65] and skin [66]. EP has also proved its efficacy in siRNA delivery in tumors [11,67,12]. Similar intracellular localization was observed in vivo in mice after intratumor injection of siRNA followed by EP [20]. EP, as compared to other delivery methods such as hydrodynamic transfection, needs a much smaller amount of siRNA to be effective [18,68]. In addition, no immune response was observed with EP, contrary to other delivery technics [69].
MicroRNA (miRNA)-based oligonucleotides. Micro-RNAs (miRNAs) are small (~22 nt) non-coding RNAs that post-transcriptionally regulate gene expression by repressing translation or accelerating mRNA decay [5]. miRNAs play crucial roles in the control of critical biological processes, including immune response, cell-cycle control, metabolism, viral replication, stem cell differentiation and human development [70]. miRNA expression or function is significantly altered in many human diseases, including cancer [71,72], cardiovascular diseases [73] and diabetes [74]. Since microRNAs do not require perfect complementarity for target recognition, a single miRNA is able to regulate multiple mRNAs, in contrast to siRNA. Therefore miRNA-based therapy is anticipated to be highly efficacious. Depending on miRNA function and status in disease tissues, there are two approaches to develop miRNA-based therapy: use of antagonists or mimics. The binding of miRNA to its target mRNA by Watson-Crick base-pairing is needed for its biological function [75]. miRNA inhibitors are oligonucleotides that are complementary to their target miRNA and bind to it with high affinity and specificity [76]. Targeting pathways of human diseases with miRNA-based drugs represents a novel and potentially powerful therapeutic approach but again an efficient delivery method is needed [77].
Most of the published reports used systemic delivery [78,79], which implies repetitive high-dose injections, associated with non-specific targeting and toxic side-effects or direct intra-tumor injection alone without any delivery system [80] which is associated with poor tumor uptake and high degradation by tumor nucleases. Rescuing miR-143 expression with in vivo electrotransfer of mimic oligonucleotide abrogated prostate cancer growth showing that EP is effective in delivering therapeutic miRNA-based oligonucleotides to tumors in vivo [81].
Chemically modified oligonucleotides. RNAs are quickly degraded by extra- and intracellular ribonucleases [82,83]. To address this problem and improve RNAi potency and efficacy, approaches based on the introduction of chemical modifications in its sequence have been developed. Therefore, new generations of chemically modified oligonucleotides have been developed [84], including 2′-O-methyl, 2′-methoxyethyl, locked nucleic acids (LNA), and phosphorothioate linkages [85,86]. LNA are nucleotides with a modified backbone. Due to its methylene bridge, the sugar moiety is conformationally locked in an RNA/mimicking C3’-endo/N-type conformation that pre-organizes the base for hybridization [87]. LNA oligonucleotide incorporation into a DNA or RNA oligomer improves the mismatch discrimination compared to unmodified control oligonucleotides [88]. In addition, oligonucleotides containing LNA nucleotides are highly resistant to nuclease degradation and present low toxicity to biological systems [89,90]. The most advanced miRNA-based oligonucleotide is an antagonist LNA specific to the miR-122, which is currently in clinical phase II trial for patients infected with hepatitis C virus [91].
We investigated the electrodelivery of these chemically modified oligonucleotides by fluorescence microscopy. We observed that LNA-DNA oligomer can be efficiently electrotransferred [57]. The number or the position of LNA into the DNA sequence does not interfere with electrotransfer efficiency. The mechanism of LNA-DNA oligomer electrotransfer appears to be closely similar to that just described for siRNA, meaning that LNA-DNA oligomer entry is driven by the electrophoretic forces [57]. LNA-DNA oligomer had a direct access to the cytoplasm and the nucleus where its miRNA target and/or precursor miRNA target, such as pri-, pre-miRNA or miRNA gene, is located. Finally, we demonstrated that electrotransferred LNA/DNA oligomer is biologically functional. EP allowed the homogenous spreading of LNA-DNA oligomer throughout the cytoplasm contrary to lipid-mediated transfection in which LNA-DNA oligomer is shown to be localized in the nuclear periphery in a punctate way, suggesting an endosomal distribution [92,93].
Thus, if chemically modified oligonucleotides appear, in theory, to be promising for RNAi-based therapy, more work for their modifications needs to be performed. Indeed, we showed that modifications of the siRNA with LNA (siLNA) do not interfere with electrotransfer efficiency. However, despite its higher stability and its high electrotransfer efficacy, siLNA was less efficient for eGFP silencing as compared to the electrotransferred unmodified siRNA regardless of the electrical conditions used [94]. Our study highlighted the careful care that is needed when designing chemically-modified oligonucleotides.

4. Conclusions

Since the discovery of the RNAi pathway, there has been an explosion of interest in using this technology for clinical applications. Although highly attractive as a therapeutic approach, several hurdles must be overcome to successfully introduce RNAi-based therapies into the clinic. Progress is being made in developing new delivery approaches to provide efficient, safe and localized delivery to cells and tissues. In this context, EP is a promising physical method to target RNAi-based oligonucleotides to the tissue, to facilitate cellular uptake and to give direct access to the intracellular targets. The EP technique is already capable of overcoming many of the delivery problems. Elucidation of the mechanisms involved in RNAi-based oligonucleotides electro-delivery will lead to better planning of future treatments and allow the development of new approaches to EP-based treatments.


This work was supported by grants of the Ligue contre le Cancer and FP7 Oncomirs (Grant 201102). Research was conducted in the scope of the EBAM European Associated Laboratory (LEA).

Conflict of Interest

The authors declare no conflict of interest.


  1. Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D.W. RNAi therapeutics: A potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2006, 2, 711–719. [Google Scholar] [CrossRef]
  2. Fire, A.; Xu, S.; Montgomery, M.K.; Kostas, S.A.; Driver, S.E.; Mello, C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391, 806–811. [Google Scholar] [CrossRef]
  3. Lee, R.C.; Feinbaum, R.L.; Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993, 75, 843–854. [Google Scholar]
  4. Tong, A.W.; Zhang, Y.A.; Nemunaitis, J. Small interfering RNA for experimental cancer therapy. Curr. Opin. Mol. Ther. 2005, 7, 114–124. [Google Scholar]
  5. Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281–297. [Google Scholar] [CrossRef]
  6. Carthew, R.W.; Sontheimer, E.J. Origins and mechanisms of miRNAs and siRNAs. Cell 2009, 136, 642–655. [Google Scholar] [CrossRef]
  7. Whitehead, K.A.; Langer, R.; Anderson, D.G. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discov. 2009, 8, 129–138. [Google Scholar] [CrossRef]
  8. Garzon, R.; Marcucci, G.; Croce, C.M. Targeting microRNAs in cancer: Rationale, strategies and challenges. Nat. Rev. Drug Discov. 2010, 9, 775–789. [Google Scholar] [CrossRef]
  9. Cemazar, M.; Golzio, M.; Escoffre, J.M.; Couderc, B.; Sersa, G.; Teissie, J. In vivo imaging of tumor growth after electrochemotherapy with cisplatin. Biochem. Biophys. Res. Commun. 2006, 348, 997–1002. [Google Scholar] [CrossRef]
  10. Cemazar, M.; Golzio, M.; Sersa, G.; Hojman, P.; Kranjc, S.; Mesojednik, S.; Rols, M.P.; Teissie, J. Control by pulse parameters of DNA electrotransfer into solid tumors in mice. Gene Ther. 2009, 16, 635–644. [Google Scholar] [CrossRef]
  11. Golzio, M.; Mazzolini, L.; Paganin-Gioanni, A.; Teissie, J. Targeted gene silencing into solid tumors with electrically mediated siRNA delivery. Methods Mol. Biol. 2009, 555, 15–27. [Google Scholar]
  12. Coster, H.G. A quantitative analysis of the voltage-current relationships of fixed charge membranes and the associated property of “punch-through”. Biophys. J. 1965, 5, 669–686. [Google Scholar] [CrossRef]
  13. Teissie, J.; Golzio, M.; Rols, M.P. Mechanisms of cell membrane electropermeabilization: A minireview of our present (lack of ?) knowledge. Biochim. Biophys. Acta 2005, 1724, 270–280. [Google Scholar] [CrossRef]
  14. Hu, W.Y.; Myers, C.P.; Kilzer, J.M.; Pfaff, S.L.; Bushman, F.D. Inhibition of retroviral pathogenesis by RNA interference. Curr. Biol. 2002, 15, 1301–1311. [Google Scholar]
  15. Calegari, F.; Haubensak, W.; Yang, D.; Huttner, W.B.; Buchholz, F. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. USA 2002, 22, 14236–14240. [Google Scholar]
  16. Pekarik, V.; Bourikas, D.; Miglino, N.; Joset, P.; Preiswerk, S.; Stoeckli, E.T. Screening for gene function in chicken embryo using RNAi and electroporation. Nat. Biotechnol. 2003, 21, 93–96. [Google Scholar]
  17. Golzio, M.; Mazzolini, L.; Ledoux, A.; Paganin, A.; Izard, M.; Hellaudais, L.; Bieth, A.; Pillaire, M.J.; Cazaux, C.; Hoffmann, J.S.; et al. In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery. Gene Ther. 2007, 14, 752–759. [Google Scholar] [CrossRef]
  18. Lewis, D.L.; Hagstrom, J.E.; Loomis, A.G.; Wolff, J.A.; Herweijer, H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 2002, 32, 107–108. [Google Scholar]
  19. Matsuda, T.; Cepko, C.L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. USA 2004, 101, 16–22. [Google Scholar] [CrossRef]
  20. Paganin-Gioanni, A.; Bellard, E.; Couderc, B.; Teissie, J.; Golzio, M. Tracking in vitro and in vivo siRNA electrotransfer in tumor cells. J. RNAi Gene Silencing 2008, 4, 281–288. [Google Scholar]
  21. Wells, D.J. Gene therapy progress and prospects: Electroporation and other physical methods. Gene Ther. 2004, 11, 1363–1369. [Google Scholar] [CrossRef]
  22. Heller, L.C.; Heller, R. In vivo electroporation for gene therapy. Hum. Gene Ther. 2006, 17, 890–897. [Google Scholar] [CrossRef]
  23. Mir, L.M.; Glass, L.F.; Sersa, G.; Teissie, J.; Domenge, C.; Miklavcic, D.; Jaroszeski, M.J.; Orlowski, S.; Reintgen, D.S.; Rudolf, Z.; et al. Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br. J. Cancer 1998, 77, 2336–2342. [Google Scholar] [CrossRef]
  24. Sersa, G.; Stabuc, B.; Cemazar, M.; Miklavcic, D.; Rudolf, Z. Electrochemotherapy with cisplatin: the systemic antitumour effectiveness of cisplatin can be potentiated locally by the application of electric pulses in the treatment of malignant melanoma skin metastases. Melanoma Res. 2000, 10, 381–385. [Google Scholar] [CrossRef]
  25. Gothelf, A.; Mir, L.M.; Gehl, J. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev. 2003, 29, 371–387. [Google Scholar] [CrossRef]
  26. Sersa, G.; Miklavcic, D.; Cemazar, M.; Rudolf, Z.; Pucihar, G.; Snoj, M. Electrochemotherapy in treatment of tumours. Eur. J. Surg. Oncol. 2008, 34, 232–240. [Google Scholar] [CrossRef]
  27. Daud, A.I.; DeConti, R.C.; Andrews, S.; Urbas, P.; Riker, A.I.; Sondak, V.K.; Munster, P.N.; Sullivan, D.M.; Ugen, K.E.; Messina, J.L. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J. Clin. Oncol. 2008, 26, 5896–5903. [Google Scholar]
  28. Bodles-Brakhop, A.M.; Heller, R.; Draghia-Akli, R. Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments. Mol. Ther. 2009, 17, 585–592. [Google Scholar] [CrossRef]
  29. Mali, B.; Jarm, T.; Snoj, M.; Sersa, G.; Miklavcic, D. Antitumor effectiveness of electrochemotherapy: A systematic review and meta-analysis. Eur. J. Surg. Oncol. 2013, 39, 4–16. [Google Scholar] [CrossRef]
  30. Mir, L.M.; Devauchelle, P.; Quintin-Colonna, F.; Delisle, F.; Doliger, S.; Fradelizi, D.; Belehradek, J., Jr.; Orlowski, S. First clinical trial of cat soft-tissue sarcomas treatment by electrochemotherapy. Br. J. Cancer 1997, 76, 1617–1622. [Google Scholar] [CrossRef]
  31. Tozon, N.; Kodre, V.; Sersa, G.; Cemazar, M. Effective treatment of perianal tumors in dogs with electrochemotherapy. Anticancer Res. 2005, 25, 839–845. [Google Scholar]
  32. Kodre, V.; Cemazar, M.; Pecar, J.; Sersa, G.; Cor, A.; Tozon, N. Electrochemotherapy compared to surgery for treatment of canine mast cell tumours. In Vivo 2009, 23, 55–62. [Google Scholar]
  33. Rols, M.P.; Tamzali, Y.; Teissie, J. Electrochemotherapy of horses. A preliminary clinical report. Bioelectrochemistry 2002, 55, 101–105. [Google Scholar] [CrossRef]
  34. Aihara, H.; Miyazaki, J. Gene transfer into muscle by electroporation in vivo. Nat. Biotechnol. 1998, 16, 867–870. [Google Scholar]
  35. Mir, L.M.; Bureau, M.F.; Gehl, J.; Rangara, R.; Rouy, D.; Caillaud, J.M.; Delaere, P.; Branellec, D.; Schwartz, B.; Scherman, D. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. USA 1999, 96, 4262–4267. [Google Scholar] [CrossRef]
  36. Dean, D.A. Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals. Am. J. Physiol. Cell. Physiol. 2005, 289, C233–C245. [Google Scholar] [CrossRef]
  37. Heller, R.; Jaroszeski, M.; Atkin, A.; Moradpour, D.; Gilbert, R.; Wands, J.; Nicolau, C. In vivo gene electroinjection and expression in rat liver. FEBS Lett. 1996, 389, 225–228. [Google Scholar] [CrossRef]
  38. Liu, F.; Huang, L. Electric gene transfer to the liver following systemic administration of plasmid DNA. Gene Ther. 2002, 9, 1116–1119. [Google Scholar] [CrossRef]
  39. Titomirov, A.V.; Sukharev, S.; Kistanova, E. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta 1991, 1088, 131–134. [Google Scholar] [CrossRef]
  40. Vandermeulen, G.; Staes, E.; Vanderhaeghen, M.L.; Bureau, M.F.; Scherman, D.; Preat, V. Optimisation of intradermal DNA electrotransfer for immunisation. J. Control Release 2007, 124, 81–87. [Google Scholar] [CrossRef]
  41. Tupin, E.; Poirier, B.; Bureau, M.F.; Khallou-Laschet, J.; Vranckx, R.; Caligiuri, G.; Gaston, A.T.; Duong van Huyen, J.P.; Scherman, D.; Bariety, J.; et al. Non-viral gene transfer of murine spleen cells achieved by in vivo electroporation. Gene Ther. 2003, 10, 569–579. [Google Scholar] [CrossRef]
  42. Isaka, Y.; Yamada, K.; Takabatake, Y.; Mizui, M.; Miura-Tsujie, M.; Ichimaru, N.; Yazawa, K.; Utsugi, R.; Okuyama, A.; Hori, M.; et al. Electroporation-mediated HGF gene transfection protected the kidney against graft injury. Gene Ther. 2005, 12, 815–820. [Google Scholar] [CrossRef]
  43. Saito, T.; Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 2001, 240, 237–246. [Google Scholar] [CrossRef]
  44. Khoury, M.; Bigey, P.; Louis-Plence, P.; Noel, D.; Rhinn, H.; Scherman, D.; Jorgensen, C.; Apparailly, F. A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis. J. Gene Med. 2006, 8, 1027–1036. [Google Scholar] [CrossRef]
  45. Pavlin, D.; Cemazar, M.; Cör, A.; Sersa, G.; Pogacnik, A.; Tozon, N. Electrogene therapy with interleukin-12 in canine mast cell tumors. Radiol. Oncol. 2011, 45, 31–39. [Google Scholar]
  46. Bureau, M.F.; Gehl, J.; Deleuze, V.; Mir, L.M.; Scherman, D. Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim. Biophys. Acta 2000, 1474, 353–359. [Google Scholar] [CrossRef]
  47. Satkauskas, S.; Bureau, M.F.; Puc, M.; Mahfoudi, A.; Scherman, D.; Miklavcic, D.; Mir, L.M. Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. Mol. Ther. 2002, 5, 133–140. [Google Scholar] [CrossRef]
  48. Sel, D.; Golzio, M.; Pucihar, G.; Tamzali, Y.; Miklavcic, D.; Teissie, J. Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery. J. Control Release 2009, 134, 125–131. [Google Scholar] [CrossRef]
  49. Gehl, J.; Sorensen, T.H.; Nielsen, K.; Raskmark, P.; Nielsen, S.L.; Skovsgaard, T.; Mir, L.M. In vivo electroporation of skeletal muscle: Threshold, efficacy and relation to electric field distribution. Biochim. Biophys. Acta 1999, 1428, 233–240. [Google Scholar] [CrossRef]
  50. Golzio, M.; Mazzolini, L.; Moller, P.; Rols, M.P.; Teissie, J. Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery. Gene Ther. 2005, 12, 246–251. [Google Scholar] [CrossRef]
  51. Spugnini, E.P.; Citro, G.; Porrello, A. Rational design of new electrodes for electrochemotherapy. J. Exp. Clin. Cancer Res. 2005, 24, 245–254. [Google Scholar]
  52. Tjelle, T.E.; Salte, R.; Mathiesen, I.; Kjeken, R. A novel electroporation device for gene delivery in large animals and humans. Vaccine 2006, 24, 4667–4670. [Google Scholar] [CrossRef]
  53. Cemazar, M.; Golzio, M.; Sersa, G.; Rols, M.P.; Teissié, J. Electrically-assisted nucleic acids delivery to tissues in vivo: where do we stand? Curr. Pharm. Des. 2006, 12, 3817–3825. [Google Scholar] [CrossRef]
  54. Golzio, M.; Teissie, J.; Rols, M.P. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc. Natl. Acad. Sci. USA 2002, 99, 1292–1297. [Google Scholar] [CrossRef]
  55. Escoffre, J.M.; Portet, T.; Wasungu, L.; Teissie, J.; Dean, D.; Rols, M.P. What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol. Biotechnol. 2009, 41, 286–295. [Google Scholar] [CrossRef]
  56. Puc, M.; Kotnik, T.; Mir, L.M.; Miklavcic, D. Quantitative model of small molecules uptake after in vitro cell electropermeabilization. Bioelectrochemistry 2003, 60, 1–10. [Google Scholar] [CrossRef]
  57. Chabot, S.; Orio, J.; Castanier, R.; Bellard, E.; Nielsen, S.J.; Golzio, M.; Teissié, J. LNA-based oligonucleotide electrotransfer for miRNA inhibition. Mol. Ther. 2012, 20, 1590–1598. [Google Scholar] [CrossRef]
  58. Castanotto, D.; Rossi, J.J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 2009, 457, 426–433. [Google Scholar]
  59. Sigoillot, F.D.; King, R.W. Vigilance and validation: Keys to success in RNAi screening. ACS Chem. Biol. 2011, 6, 47–60. [Google Scholar] [CrossRef]
  60. Heidel, J.D.; Hu, S.; Liu, X.F.; Triche, T.J.; Davis, M.E. Lack of interferon response in animals to naked siRNAs. Nat. Biotechnol. 2004, 22, 1579–1582. [Google Scholar]
  61. Paganin-Gioanni, A.; Bellard, E.; Escoffre, J.M.; Rols, M.P.; Teissie, J.; Golzio, M. Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells. Proc. Natl. Acad. Sci. USA 2011, 108, 10443–10447. [Google Scholar]
  62. Kishida, T.; Asada, H.; Gojo, S.; Ohashi, S.; Shin-Ya, M.; Yasutomi, K.; Terauchi, R.; Takahashi, K.A.; Kubo, T.; Imanishi, J.; et al. Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA. J. Gene Med. 2004, 6, 105–110. [Google Scholar] [CrossRef]
  63. Inoue, A.; Takahashi, K.A.; Mazda, O.; Terauchi, R.; Arai, Y.; Kishida, T.; Shin-Ya, M.; Asada, H.; Morihara, T.; Tonomura, H.; et al. Electro-transfer of small interfering RNA ameliorated arthritis in rats. Biochem. Biophys. Res. Commun. 2005, 336, 903–908. [Google Scholar] [CrossRef]
  64. Akaneya, Y.; Jiang, B.; Tsumoto, T. RNAi-induced gene silencing by local electroporation in targeting brain region. J. Neurophysiol. 2005, 93, 594–602. [Google Scholar] [CrossRef]
  65. Takabatake, Y.; Isaka, Y.; Mizui, M.; Kawachi, H.; Shimizu, F.; Ito, T.; Hori, M.; Imai, E. Exploring RNA interference as a therapeutic strategy for renal disease. Gene Ther. 2005, 12, 965–973. [Google Scholar] [CrossRef]
  66. Broderick, K.E.; Chan, A.; Lin, F.; Shen, X.; Kichaev, G.; Khan, A.S.; Aubin, J.; Zimmermann, T.S.; Sardesai, N.Y. Optimized in vivo transfer of small interfering RNA targeting dermal tissue using in vivo surface electroporation. Mol. Ther. Nucleic Acids 2012, 14, e11. [Google Scholar]
  67. Nakai, N.; Kishida, T.; Shin-Ya, M.; Imanishi, J.; Ueda, Y.; Kishimoto, S.; Mazda, O. Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf. Gene Ther. 2007, 14, 357–365. [Google Scholar] [CrossRef]
  68. McCaffrey, A.P.; Meuse, L.; Pham, T.T.; Conklin, D.S.; Hannon, G.J.; Kay, M.A. RNA interference in adult mice. Nature 2002, 418, 38–39. [Google Scholar]
  69. Jackson, A.L.; Linsley, P.S. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat. Rev. Drug Discov. 2010, 9, 57–67. [Google Scholar] [CrossRef]
  70. Ambros, V. MicroRNAs: tiny regulators with great potential. Cell 2001, 107, 823–826. [Google Scholar] [CrossRef]
  71. Lu, J.; Getz, G.; Miska, E.A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B.L.; Mak, R.H.; Ferrando, A.A.; et al. MicroRNA expression profiles classify human cancers. Nature 2005, 435, 834–838. [Google Scholar]
  72. Hernando, E. MicroRNAs and cancer: Role in tumorigenesis, patient classification and therapy. Clin. Transl. Oncol. 2007, 9, 155–160. [Google Scholar] [CrossRef]
  73. Da Costa Martins, P.A.; Salic, K.; Gladka, M.M.; Armand, A.S.; Leptidis, S.; El Azzouzi, H.; Hansen, A.; Coenen-de Roo, C.J.; Bierhuizen, M.F.; van der Nagel, R.; et al. MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling. Nat. Cell. Biol. 2010, 12, 1220–1227. [Google Scholar] [CrossRef]
  74. Long, J.; Wang, Y.; Wang, W.; Chang, B.H.; Danesh, F.R. Identification of microRNA-93 as a novel regulator of vascular endothelial growth factor in hyperglycemic conditions. J. Biol. Chem. 2010, 285, 23457–23465. [Google Scholar]
  75. Brodersen, P.; Voinnet, O. Revisiting the principles of microRNA target recognition and mode of action. Nat. Rev. Mol. Cell. Biol. 2009, 10, 141–148. [Google Scholar] [CrossRef]
  76. Stenvang, J.; Kauppinen, S. MicroRNAs as targets for antisense-based therapeutics. Expert Opin. Biol. Ther. 2008, 8, 59–81. [Google Scholar] [CrossRef]
  77. Stenvang, J.; Lindow, M.; Kauppinen, S. Targeting of microRNAs for therapeutics. Biochem. Soc. Trans. 2008, 36, 1197–1200. [Google Scholar] [CrossRef]
  78. Dalmay, T. MicroRNAs and cancer. J. Intern. Med. 2008, 263, 366–375. [Google Scholar] [CrossRef]
  79. Care, A.; Catalucci, D.; Felicetti, F.; Bonci, D.; Addario, A.; Gallo, P.; Bang, M.L.; Segnalini, P.; Gu, Y.; Dalton, N.D.; et al. MicroRNA-133 controls cardiac hypertrophy. Nat. Med. 2007, 13, 613–618. [Google Scholar]
  80. Mercatelli, N.; Coppola, V.; Bonci, D.; Miele, F.; Costantini, A.; Guadagnoli, M.; Bonanno, E.; Muto, G.; Frajese, G.V.; de Maria, R.; et al. The inhibition of the highly expressed miR-221 and miR-222 impairs the growth of prostate carcinoma xenografts in mice. PLoS One 2008, 3, e4029. [Google Scholar]
  81. Clape, C.; Fritz, V.; Henriquet, C.; Apparailly, F.; Fernandez, P.L.; Iborra, F.; Avances, C.; Villalba, M.; Culine, S.; Fajas, L. miR-143 interferes with ERK5 signaling, and abrogates prostate cancer progression in mice. PLoS One 2009, 4, e7542. [Google Scholar]
  82. Layzer, J.M.; McCaffrey, A.P.; Tanner, A.K.; Huang, Z.; Kay, M.A.; Sullenger, B.A. In vivo activity of nuclease-resistant siRNAs. RNA 2004, 10, 766–771. [Google Scholar] [CrossRef]
  83. Raemdonck, K.; Remaut, K.; Lucas, B.; Sanders, N.N.; Demeester, J.; De Smedt, S.C. In situ analysis of single-stranded and duplex siRNA integrity in living cells. Biochemistry 2006, 45, 10614–10623. [Google Scholar] [CrossRef]
  84. Corey, D.R. Chemical modification: The key to clinical application of RNA interference? J. Clin. Invest. 2007, 117, 3615–3622. [Google Scholar] [CrossRef]
  85. Braasch, D.A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.; White, M.A.; Corey, D.R. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 2003, 42, 7967–7975. [Google Scholar]
  86. Kumar, R.; Singh, S.K.; Koshkin, A.A.; Rajwanshi, V.K.; Meldgaard, M.; Wengel, J. The first analogues of LNA (locked nucleic acids): Phosphorothioate-LNA and 2'-thio-LNA. Bioorg. Med. Chem. Lett. 1998, 8, 2219–2222. [Google Scholar] [CrossRef]
  87. Petersen, M.; Nielsen, C.B.; Nielsen, K.E.; Jensen, G.A.; Bondensgaard, K.; Singh, S.K.; Rajwanshi, V.K.; Koshkin, A.A.; Dahl, B.M.; Wengel, J.; et al. The conformations of locked nucleic acids (LNA). J. Mol. Recognit. 2000, 13, 44–53. [Google Scholar] [CrossRef]
  88. Kaur, H.; Babu, B.R.; Mait, S. Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem. Rev. 2007, 107, 4672–4697. [Google Scholar] [CrossRef]
  89. Crinelli, R.; Bianchi, M.; Gentilini, L.; Magnani, M. Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acids Res. 2002, 30, 2435–2443. [Google Scholar] [CrossRef]
  90. Wahlestedt, C.; Salmi, P.; Good, L.; Kela, J.; Johnsson, T.; Hokfelt, T.; Broberger, C.; Porreca, F.; Lai, J.; Ren, K.; et al. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. USA 2000, 97, 5633–5638. [Google Scholar] [CrossRef]
  91. Lanford, R.E.; Hildebrandt-Eriksen, E.S.; Petri, A.; Persson, R.; Lindow, M.; Munk, M.E.; Kauppinen, S.; Orum, H. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010, 327, 198–201. [Google Scholar] [CrossRef]
  92. Braasch, D.A.; Liu, Y.; Corey, D.R. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: Effect of mRNA target sequence and chimera design. Nucleic Acids Res. 2002, 30, 5160–5167. [Google Scholar] [CrossRef]
  93. Corsten, M.F.; Miranda, R.; Kasmieh, R.; Krichevsky, A.M.; Weissleder, R.; Shah, K. MicroRNA-21 knockdown disrupts glioma growth in vivo and displays synergistic cytotoxicity with neural precursor cell delivered S-TRAIL in human gliomas. Cancer Res. 2007, 67, 8994–9000. [Google Scholar] [CrossRef]
  94. Pelofy, S.; Teissié, J.; Golzio, M.; Chabot, S. Chemically modified oligonucleotide-increased stability negatively correlates with its efficacy despite efficient electrotransfer. J. Membr. Biol. 2012, 245, 565–571. [Google Scholar] [CrossRef]

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Chabot, S.; Pelofy, S.; Teissié, J.; Golzio, M. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization. Pharmaceuticals 2013, 6, 510-521.

AMA Style

Chabot S, Pelofy S, Teissié J, Golzio M. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization. Pharmaceuticals. 2013; 6(4):510-521.

Chicago/Turabian Style

Chabot, Sophie, Sandrine Pelofy, Justin Teissié, and Muriel Golzio. 2013. "Delivery of RNAi-Based Oligonucleotides by Electropermeabilization" Pharmaceuticals 6, no. 4: 510-521.

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