Gene Therapy (2001) 8, 400–407 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Intramuscular electroporation delivery of IFN-␣ gene therapy for inhibition of tumor growth located at a distant site S Li, X Zhang, X Xia, L Zhou, R Breau, J Suen and E Hanna Department of Otolaryngology/Head and Neck Surgery, University of Arkansas School of Medicine, 4001 W Capital Avenue, Little Rock, AR 72205, USA Although electroporation has been shown in recent years to 2 or endostatin gene, also delivered by electro-injection. The be a powerful method for delivering genes to muscle, no increased therapeutic efficacy was associated with a high gene therapy via electro-injection has been studied for the level and extended duration of IFN-␣ expression in muscle treatment of tumors. In an immunocompetent tumor-bearing and serum. We also discovered that the high level of IFN-␣ murine model, we have found that delivery of a low dose of expression correlated with increased expression levels of reporter gene DNA (10 ␮g) to muscle via electroporation the antiangiogenic genes IP-10 and Mig in local tumor under specific pulse conditions (two 25-ms pulses of 375 tissue, which may have led to the reduction of blood vessels V/cm) increased the level of gene expression by two logs of observed at the local tumor site. Delivery of increasing doses magnitude. Moreover, administration of 10 ␮g of interferon (10–100 ␮g) of IFN-␣ plasmid DNA by injection alone did (IFN)-␣ DNA plasmid using these parameters once a week not increase antitumor activity, whereas electroporation for 3 weeks increased the survival time and reduced squam- delivery of increasing doses (10–40 ␮g) of IFN-␣ plasmid ous cell carcinoma (SCC) growth at a distant site in the DNA did increase the survival time. Our data clearly demon- C3H/HeJ-immunocompetent mouse. IFN-␣ gene therapy strate the potential utility of electroporation for delivery of delivered to muscle using electroporation demonstrated stat- gene therapy to muscle for the treatment of residual or dis- istically significant (P Ͻ 0.05) therapeutic efficacy for treating seminated tumors. Gene Therapy (2001) 8, 400–407. SCC located at a distant site, compared with interleukin (IL)- Keywords: gene therapy; electroporation; muscle; IFN-␣; antiangiogenesis; IL-12 Introduction Skeletal muscle is an attractive site for somatic gene delivery because of its large size, good capacity for pro- ␣ Interferon (IFN)- recombinant protein therapy is the tein synthesis, easy accessibility for intramuscular injec- first cytokine to be used clinically for cancer patients and tion, and ability to take up plasmid after intramuscular has been shown to be effective in the treatment of several administration.13 Intramuscular injection of high-dose, cancers including renal cell carcinomaa, hairy cell leuke- but not low-dose, IFN-␣ gene using a syringe needle sig- mia, malignant melanoma, basal cell carcinoma, and mul- nificantly inhibited B16F10 melanoma and glioma 261 1–3 tiple myeloma. However, long-term daily admini- growth in mice and reduced Cloudman melanoma tumor ␣ stration of IFN- protein is required to maintain growth in vivo,14 demonstrating the high level of IFN-␣ ␣ therapeutic efficacy, and some IFN- protein clinical that is required to achieve therapeutic effect. 4 ␣ trials have been disappointing. Ex vivo IFN- gene ther- Electroporation is a highly effective method for apy studies have demonstrated tumor inhibition and an increasing gene expression by creating transient pores in 5–9 elicited tumor-specific immune memory. However, cell membranes through which plasmids can gain entry this approach is patient-specific and the application is into the cell. Although electroporation delivery of ␣ time-consuming. Direct intratumoral injection of IFN- to reporter and therapeutic genes to muscle has been 10 11 treat solid Renca, basal cell carcinoma, and hetero- described by several groups over the past few years,15–17 xenograft prostatic (PC-3) or hepatocellular carcinoma there are no data available to determine the therapeutic 12 (Hep3B) with the use of viral or nonviral delivery sys- efficacy of such an approach in a tumor model. tems has demonstrated effective tumor suppression in IFN-␣ has several subtypes and multiple biological mouse models in vivo, but the intratumoral approach is functions that control cell growth, modulate host immun- not practical for metastatic lesions and microscopic ity, and inhibit angiogenesis.18–21 The mechanisms of anti- residual lesions after surgery. tumoral cell proliferation and immunity modulation by IFN-␣ have been extensively examined, but the mech- anism for antiangiogenesis is less well studied and only Correspondence: S Li a few antiangiogenic genes regulated by IFN-␣ have been Received 7 September 2000; accepted 20 December 2000 identified.18–22 The expression of interferon inducible pro- Electroporation delivery of IFN-␣ gene into muscle for inhibition of tumor growth SLiet al 401 tein-10 (IP-10) and monokine induced by IFN-␥ (Mig) started when the tumor volume was 30 mm3, or twice have been known to be enhanced by IFN-␥ and inhibit the size as in the above experiment. Although the angiogenesis in tumors in vivo,23–28 but it is not clear reduction was not as dramatic as in the previous experi- whether IFN-␣ will have a similar effect in vivo. ment, a 50% reduction in tumor growth was obtained We used squamous cell carcinoma (SCCVII) in an with electroporation delivery (Figure 2c). These data immunocompetent mouse (C3H/HeJ) as a model for this clearly demonstrate that although electro-injection of study. We present data demonstrating that electro-injec- IFN-␣ was not able to eradicate the established SCCVII tion of the IFN-␣ gene is a very powerful method for tumor in vivo, it was able to reduce significantly the rate generating a high and durable level of gene expression. of tumor growth. We also show that this method of delivering the IFN-␣ gene into muscle induces significant anti-SCCVII tumor High-level and extended gene expression in serum after growth by increasing the level of IFN-␣ expression, electro-injection of IFN-␣ gene in muscle which may in turn inhibit tumor vascularization by To determine whether the enhanced therapeutic efficacy inducing IP-10 and Mig expression. of IFN-␣ by electroporation delivery directly correlates with IFN-␣ production, Western blot analysis was perfor- med to analyze the level of IFN-␣ gene expression in both Results injected muscle tissue and serum. As expected, high lev- els of IFN-␣ gene expression were detected in both mus- Increased gene expression in C3H/HeJ mice using cle and serum on day 3 after electro-injection. There was electro-injection in muscle no detectable level of IFN-␣ expression in either muscle In an earlier study, we observed a two- to three-fold tissue or serum following injection alone (Figure 3a). increase in the level of gene expression by electro-injec- The duration of IFN-␣ gene expression after a single tion of plasmid DNA in the muscle of the CD1 mouse, administration of IFN-␣ gene therapy was also deter- using electroporation conditions of two 25-ms pulses at mined. Serum was collected on days 1, 3 and 7. IFN-␣ 375 V/cm field strength.29 To determine whether the protein was detectable in the serum of electro-injected same results could be obtained in a murine tumor model, mice at all three sampling times by Western blot analysis. we determined enzyme activity after delivery of lucifer- However, it was not detected in mice receiving the injec- ase or secreted alkaline phosphatase (SEAP) gene to the tion without electroporation (Figure 3b). Clearly, the muscle of the C3H/HeJ mouse, an immunocompetent therapeutic efficacy of electroporation therapy could be mouse model, using 10 ␮g of DNA for each muscle, with attributed, at least in part, to the resulting high-level and or without electroporation. As shown in Figure 1, the extended expression of IFN-␣. level of luciferase expression in muscle reached 135 ng/mg total protein (Figure 1a), which was more than Inhibition of vascularization in tumor tissue after 100-fold higher than that in the injection-only group. The electroporation delivery of IFN-␣ gene in muscle maximum level of SEAP in serum was 285 ng/ml blood, To determine the mechanism by which electroporation which was reached on day 14 after delivery and was 100- gene therapy with IFN-␣ inhibited tumor growth, a cyto- fold higher than that in the injection-only group (Figure toxic T lymphocyte (CTL) assay was performed. This 1b). As expected, the magnitude of increase in gene showed no increase in tumor cell-lysing activity in either expression in C3H/HeJ mice was similar to that which the electro-injected animals or controls (data not shown). we observed in CD1 mice. The increased level of gene However, immunostaining with anti-CD8+ T cell anti- expression by electro-injection was correlated to the body and anti-endothelial cell marker CD31 antibody increased number of fiber cells transfected with exogen- demonstrated a slightly increased number of infiltrating ous DNA as determined by green fluorescent protein CD8+ T cells (P Ͼ 0.05) and significantly decreased vessel (GFP) expression (Figure 1c). Thus, our delivery tech- density (P Ͻ 0.05) after electroporation delivery of IFN- nique was applicable to this murine tumor model. ␣ gene in muscle (Figure 4A–C). The tumor samples for immunostaining were obtained from mice killed 3 days Inhibition of SCCVII tumor growth via electro-injection of after the second administration, with a week interval IFN-␣ gene in muscle between administrations. Tumor growth in the animal group receiving 10 ␮gof INF-␣ plasmid DNA injected by electroporation was sig- Enhanced expression of IP-10 and Mig after nificantly reduced, compared with that in either the syr- electroporation delivery of IFN-␣ gene in muscle inge needle injection group or the control groups (empty The slight increase in CD8+ T cell infiltration and decrease plasmid with or without electroporation delivery), and in vessel density in tumor observed in our study impli- the survival time was increased (P Ͻ 0.05; Figure 2a, b).