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Electroporation-Enhanced Gene Delivery in Mammary Tumors

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Electroporation-Enhanced Gene Delivery in Mammary Tumors Gene Therapy (2000) 7, 541–547 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Electroporation-enhanced gene delivery in mammary tumors JM Wells, LH Li, A Sen, GP Jahreis and SW Hui Membrane Biophysics Laboratory, Molecular and Cellular Biophysics Department, Roswell Park Cancer Institute, Buffalo, NY 14263-0001, USA Electroporation was applied to enhance gene transfer into pulses 1 ms long were applied across tumors, using caliper subcutaneous MC2 murine breast tumors. Cultured MC2 electrodes on the skin surface. Electric field strengths cells were also transfected by electroporation or by cationic ranged from 400–2300 V/cm. Luciferase expression was liposomes in the presence of serum using pSV-luc plasmids. approximately two orders of magnitude higher than controls Electroporation parameters and liposome formulation were in tumors treated with pulses у800 V/cm. Differences optimized to achieve the highest relative levels of transfec- between enhanced relative levels of transfection using tion. An electric field threshold for successful electrotransfec- uncomplexed plasmid and lipoplexes were not statistically tion in cultured cells appeared around 800–900 V/cm. The significant. Distribution of DNA into tumor tissues was moni- liposomes used contained the cationic lipid dioleoyl-3-trime- tored by fluorescence in situ PCR. The highest numbers of thylammonium propane (DOTAP). Multilamellar vesicles fluorescent cells were found in tumors electroporated follow- (MLV) had a 10-fold advantage over small unilamellar ves- ing the injection of plasmid. The significant transfection icles (SUV) in cell culture transfection. For in vivo gene deliv- improvement shows that in vivo electroporation is a powerful ery, the plasmids were injected either alone, or in complex tool for local gene delivery to tumors. Gene Therapy (2000) with MLV or SUV DOTAP liposomes. A series of six electric 7, 541–547. Keywords: electroporation; breast tumor; gene transfer; liposomes; transfection; mice Introduction electrodes and create electric field gradients through soft tissues makes electroporation an attractive method for Gene therapy is fast becoming a therapeutic mode for gene delivery to tumors of the head, neck, breasts and cancer therapy. Many new vectors have been designed skin. In vivo electroporation has been applied to introduce for effective gene transfer. Among these newly developed bleomycin to solid tumor cells with significant tumor vectors, nonviral vectors have several advantages over suppressing effects.4,5 This therapeutic mode is at phase retroviral and adenoviral vectors in gene delivery for I/II clinical trial.6 However, the application so far is cancer therapy. For instance, some retroviruses are cap- restricted to membrane transport-limited drugs, (ie able of activating oncogenes or deactivating tumor sup- bleomycin). For cancer gene therapy, a promising pressor genes. In addition, nonviral methods such as cat- approach is the partial transfection of tumors and sur- ionic liposomes, cationic polymers and electroporation rounding cells with genes coding for biological modu- do not usually elicit immune responses. Therefore, they lators (eg cytokines), or the limited transfection of anti- can be repeatedly administered. However, the gene trans- gen-presenting cells for enhancing immunotherapy. fer efficiency of nonviral vectors is still below that of viral Current attempts to inject cytokines have limited success, vectors. Long-term expression is still a challenge for non- because these agents produce strong inflammatory viral methods. With advanced liposome designs and responses and edema. Localized gene therapy allows for improved electroporation technology, the combination of long-term production of these agents in the immediate cationic liposomes and electroporation holds promise for vicinity of tumors. Transfecting a fraction of tumor cells enhanced local gene delivery. with apoptotic (eg Tk), tumor necrosis factor, or tumor Electroporation is a highly efficient method for suppressor genes may also be beneficial, taking advan- delivering exogenous molecules, including DNA, into tage of bystander effects upon neighboring cells. cells. This method has been used widely as means to An earlier electrotransfection experiment, using surface 1 deliver molecules into cells in vitro. Recently, this electrodes on skin, was reported by Titomirov et al,7 method has been applied to permeate the upper layer delivering plasmids coding for a reporter gene into new- (stratum corneum) of the skin for the purpose of drug born mouse skin. They reported transient and long-term 2,3 and gene delivery. The possibility of applying surface transfection of cells isolated and cultured from electropo- rated skin. Zhang et al8 also reported transfection of skin cells with the LacZ reporter gene, using a combination of Correspondence: SW Hui electric pulse and pressure. Recent studies have shown Received 6 May 1999; accepted 26 November 1999 that electroporation applied in vivo can introduce reporter Gene delivery by electroporation of mammary tumors JM Wells et al 542 genes to murine liver, and indirectly to rat brain Transfection of cultured MC2 tumor cells tumor.9,10 Rols et al11 have demonstrated the possibility In order to establish the transfectability of MC2 cells by of electrotransfection of melanoma in vivo. liposome and electroporation methods, using the same To show that electroporation can significantly enhance plasmid that would be used in in vivo experiments, we gene delivery to tumors, we applied electroporation to a first tested the cells and methods in vitro. The in vitro large number of implanted murine breast tumors. experiments, using cultured MC2 cells, provided us with Because of tumor heterogeneity, studies involving many a starting point in terms of lipoplex types and concen- tumor samples are needed to establish a statistical basis trations, as well as electrical parameters for subsequent for the usefulness of electroporation in gene delivery to in vivo experiments. tumors. Altogether, 105 tumors were used in this experi- Table 1 shows the transfection efficiency using lipo- ment. Variability in tumor size and appearance was plexes made from DOTAP MLV or SUV, as well as MLV examined both visually and by palpation for its effect on or SUV with DOTAP/DOPE at a 1:1 molar ratio. The transfection rates. Furthermore, the effects of different charge ratio for lipid:DNA was 1:2, giving the complex types of liposomes as adjuvant vectors were examined. a net negative charge. As with most other cells we tested, The purpose is to establish the usefulness of electropor- lipoplexes made of MLV were more efficient than those ation with or without liposomes for gene delivery to solid made of SUV.12 With this particular cell line, DOTAP tumors. The efficiencies and conditions for in vitro and in alone was as effective as DOTAP/DOPE mixture. There- vivo transfections, using the same tumor cells and plas- fore, lipoplex of DOTAP alone was used in subsequent mids, are compared. This study illustrates the potential experiments. of local gene delivery to solid tumors by applying electric Electrotransfection results are shown in Figure 2. A pulses with surface electrodes in a way comparable to a pulse field strength threshold is found at 800–900 V/cm, clinical setting. for both plasmid concentrations (162.5 and 650 ␮g/ml) we used. At high pulse field strength of 1600 V/cm, the transfection efficiency drops due to increased cell death. Results Lower transfection efficiency for samples with higher plasmid concentration at the optimal field strength may Tumor classification indicate membrane damage by DNA transport during In order to improve the reproducibility of gene electroporation.13 expression in a heterogeneous population of tumors, we classified tumors into medium–large (8–15 mm wide) and Transfection of tumors without electroporation small (Ͻ8 mm) groupings. Smaller tumors tended to be The relative levels of transfection by injecting naked firm, well vascularized, and fast growing, without areas DNA or lipoplexes made from SUV or MLV are shown of apparent necrosis. Medium-sized tumors were almost in Figure 3. All tumors were injected in the same day, as firm, with slightly uneven regions of growth and little without electroporation, then analyzed 2 days later. A P or no apparent necrosis. Large tumors (Ͼ11 mm) tend to value Ͻ0.05 is considered to be significantly independent be the least firm with variable regions of growth and may of chance for two results to be distinct. The differences contain non-growing regions. Most data presented in our between SUV and naked DNA and between SUV and studies of electroporation are from the medium–large MLV are significant. SUV lipoplexes transfected more group. Differences in luciferase expression between than twice as well as naked DNA or MLV lipoplexes on medium and large tumors were minor. Data from the average. (Note that the Y-axis of Figure 3 is in log10 scale.) small tumor group are presented separately. The consistency of the results gave a small standard error Gross anatomical features of the tumors were recorded for each. The background level of relative light units at the time of injection. The apparent viability of several (RLU) was subtracted from all data values for this and tumors changed during the period between all other experiments. injection/electroporation and time of death. In few cases, we noted the unambiguous appearance of scabs,
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