Intramuscular and intratumoral gene electrotransfer for gene therapy of cancer

D. Scherman

UMR 7001 CNRS / ENSCP Pierre et Marie Curie University / Aventis Company, Vitry-sur-Seine, France

Gene delivery to tumors and to skeletal muscle is a promising strategy for the treatment of cancer. Anti-oncogenic genes such as tumor suppressor p53 can be administered directly by intratumoral injection. Alternatively, genes coding for immunostimulatory cytokines, for antigenic epitopes, or for antiangiogenic factors might be administered either within the tumor, or at distal site of the tumor. Of particular interest for tumor treatment is the systemic secretion of antiangiogenic factors by in vivo transfected skeletal muscle. Non-viral gene transfer for gene therapy means using a plasmid DNA as gene expression vector, in association or not with a chemical delivery vector or with physical administration technique, is a rapidly expanding field. Cationic lipids are the most commonly used chemical DNA delivery vectors. They self-associate with plasmids and form nanostructures called lipoplexes. Successful non viral gene transfer requires mastering several steps: preparation, purification and formulation of the therapeutic DNA and synthetic vector, plasmid administration, plasmid access to target cells, then intracellular penetration and nuclear localization. We will present our contribution in different aspects: 1) optimized plasmid for biosafety and bioavailability; 2) effect of plasmid size on lipoplexe structure and on gene delivery efficiency; and 3) plasmid electrotransfer to skeletal muscle and tumor tissue. Increased gene transfer by minimal size « minicircle » plasmids: in vivo results and structural physico-chemical studies Minicircles are a new form of supercoiled DNA molecule for non-viral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. Two minicircles containing the luciferase or b-galactosidase gene under the control of hCMV-IE enhancer/promoter gave 2-5 times more reporter gene activity than the unrecombined plasmid in NIH3T3 cells in rabbit smooth muscle cells. Moreover, injection into mouse cranial tibial muscle, or into human head and neck carcinoma grafted in nude mice resulted in 15-50 fold more reporter gene expression with minicircles than with the unrecombined plasmid or with larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid. Similar results were observed using a pCOR plasmid devoid of prokaryotic replication origin. Effect of plasmid size on lipoplexe structure and on gene delivery efficiency. In order to further investigate the mechanism of the increase in gene transfer efficiency observed with minicircles, we have explored the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52,500 base pairs. The morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was strongly affected by the size and number of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the largest amount of the shortest plasmid DNA. Plasmid electrotransfer to skeletal muscle and tumors. We have obtained very efficient plasmid DNA transfer in muscle fibers and tumors using square-wave electric pulses of low field strength (less than 500 V/cm) and of long duration (more than 1 ms). This « electrotransfer » method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles and tumors. We will present recent results concerning the three following aspects. Mechanism of electrotransfer. A new combination of pulses of various strength and duration leads to similar gene transfer efficiency while delivering less energy to the tissue and leading to lower electro-permabilization. Moreover, these results give insights on the mechanism of DNA entry into electrotransfered cells. Sustained plasmatic protein secretion. Sustained blood secretion of transgenic secreted alcaline phophatase (SeAP) for more than 12 months has been observed, and of Factor IX were observed after a single i.m. electrotransfer. I.M. electrotransfer of a pCMV EPO pCOR plasmid led to an increase of approximately 10- to 100- fold in circulating murine erythropoietin level, as compared to naked DNA alone, and induced a stable and reproducible increase in mouse hematocrit, from 47% up to 80%. Plasmid electrotransfer into tumors. We have confirmed the potential of long duration/low voltage pulses to transfer DNA to several murine and human tumor models (10-1200 fold increase over values obtained with naked DNA injection). An example of tumor growth retardation after electrotransfer of a recently discovered gene will be presented.

KEY WORDS: gene therapy, electric pulses, electrotransfer, tumor, gene transfer.

For more information, contact daniel

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Gene Therapy, Part 2.