Cyclin B1 knockdown mediated by clinically approved pulsed electric fields siRNA delivery induces tumor regression in murine melanoma.

RNA interference (RNAi) represents a promising therapy for the specific inhibition of gene expression in targeted tissues including tumors. To realize the therapeutic potential of RNAi drugs, non-immunogenic, efficient, and tissue-specific delivery technologies must be developed. We have previously shown that pulsed electric field (PEF) can deliver siRNAs into tumor cells thanks to long electrophoretic drift occurring during the use of millisecond duration pulses. Here, optical fluorescence imaging is used at first to evaluate the efficiency of microsecond-duration pulses for siRNA delivery. These pulsed electric fields (PEF) parameters, which are already used in clinics for electrochemotherapy (ECT) were compared to previous parameters optimized for electrogenotherapy (EGT) that use microsecond-duration pulses. Secondly, these PEF protocols were evaluated for the delivery of specific siRNAs targeting the cyclin B1 in subcutaneous tumors in mice. When a single treatment was performed, millisecond duration pulses led to a better efficiency. However, when multiple treatments were performed, both protocols were equally efficient and potentially silenced cyclin B1 endogenous gene, leading to a tumor growth reduction. Our findings provide insights into pulsed electric field-siRNA delivery that could benefit from existing clinical protocols for siRNA delivery to tumors.

Electrotransfer of RNAi-based oligonucleotides for oncology.

For more than a decade, there has been tremendous growth in our understanding of RNA interference (RNAi). The potent ability that small oligonucleotides have in gene silencing makes them desirable as novel cancer therapeutics, but many biological barriers exist for their efficient delivery into target cells or tissues. Electropulsation (EP) appears to be a promising method for cancer-associated gene therapy. EP is the direct application of 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 of their electrotransfer and the technical settings for pre-clinical purposes.

Fluorescence imaging agents in cancerology.

BACKGROUND: One of the major challenges in cancer therapy is to improve early detection and prevention using novel targeted cancer diagnostics. Detection requests specific recognition. Tumor markers have to be ideally present on the surface of cancer cells. Their targeting with ligands coupled to imaging agents make them visible/detectable. CONCLUSIONS: Fluorescence imaging is a newly emerging technology which is becoming a complementary medical method for cancer diagnosis. It allows detection with a high spatio-temporal resolution of tumor markers in small animals and in clinical studies. In this review, we focus on the recent outcome of basic studies in the design of new approaches (probes and devices) used to detect tumor cells by fluorescence imaging.

Targeted gene silencing into solid tumors with electrically mediated siRNA delivery.

Short interfering RNAs (siRNAs) represent new potential therapeutic tools, owing to their capacity to induce strong, sequence-specific gene silencing in cells. However, their clinical development requires new, safe, and efficient in vivo siRNA delivery methods. In this study, we report an efficient in vivo approach for targeting gene knockdown in solid tumors by the use of external electric field pulses. We show that gene silencing is efficiently obtained in vivo with chemically synthesized siRNA after targeted electrical delivery in the tumor-bearing mouse. The associated gene silencing was followed on the same animal by fluorescence imaging and confirmed by qPCR. Gene silencing obtained in tumors lasted from 2 to 4 days after a single treatment. Therefore, this method should allow gene function analysis or organ treatment by a localized delivery of siRNAs.

Gene electrotransfer: from biophysical mechanisms to in vivo applications : Part 2 - In vivo developments and present clinical applications.

Gene electrotransfer can be obtained not just on single cells in diluted suspension. For more than 10 years, this is a quasi routine strategy in tissue on the living animal and a few clinical trials have now been approved. New problems have been brought by the close contacts of cells in tissue both on the local field distribution and on the access of DNA to target cells. They need to be solved to provide a further improvement in the efficacy and safety of protein expression. There is a competition between gene transfer and cell destruction. Nevertheless, present results are indicative that electrotransfer is a promising approach for gene therapy. High level and long-lived expression of proteins can be obtained in muscles. This is used for a successful method of electrovaccination.

Tracking in vitro and in vivo siRNA electrotransfer in tumor cells.

RNA interference-mediated gene silencing offers the potential of targeted inhibition of disease-relevant genes. In vivo delivery of RNAi reagents can be obtained by a variety of approaches. Physical delivery methods appear safer and lack side effects. Electro-permeabilization is one of the non-viral methods successfully used to transfer small interfering RNAs (siRNAs) in vitro and in vivo. A promising approach may be, very little is known about the fundamental processes mediating siRNA transfer. In this study, we have investigated cellular delivery pathways involved in electro-delivery of siRNAs by a direct fluorescence imaging method. An Alexa-labeled siRNA was electro-transferred into murine melanoma cells stably-expressing the enhanced green fluorescent protein (eGFP) target reporter gene. The silencing of eGFP gene expression was quantified by time-lapsed fluorescence microscopy. Fluorescently-labeled siRNAs were found distributed homogeneously in cytoplasm 48 hours after electro-transfer, apparently by diffusion. Furthermore, siRNAs showed homogeneous distribution in vivo 48 hrs after intra-tumoral injection followed by electro- permeabilization. Histological fluorescence microscopy showed that siRNAs were mostly localized in the cytoplasm. Overall, this study shows that electro-permeabilization facilitates cytoplasmic distribution of siRNA, both in cultured cells and in vivo. This method offers a potential therapeutic tool to facilitate direct siRNA penetration into solid tumors.