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We developed a new potent nonviral gene transfer method into mouse muscles in vivo named "electrosonoporation." We tried in this report to treat murine orthotopic hepatocellular carcinoma (HCC) by muscle-targeted mouse interleukin-12 (mIL-12) gene transfer using in vivo electrosonoporation. I.m. administration of the mIL-12 gene with electrosonoporation elevated serum IL-12 and IFN-gamma and significantly prolonged the survival periods with both growth inhibition of orthotopic HCC and inhibition of spontaneous lung metastasis. The IL-12 gene therapy reduced the number of microvessels and induced more Mac-1-positive cells into HCC. These results show that muscle-targeted mIL-12 gene therapy for orthotopic HCC using in vivo electrosonoporation is very efficient and is thus promising for further clinical trial.


Introduction
Serious concerns have been voiced regarding the use of viral vectors, especially when clinical trials are involved.Recently, we shown that gene transfer by in vivo electrosonoporation was found to be effective at introducing DNA into mouse muscles (1).Like other nonviral methods, electrosonoporation has a variety of advantages over viral vectors, in that all tissues and cells can in theory become targets, it is easy to handle and quickly completed, and no immunogenicity is expected.
Interleukin-12 (IL-12) was originally identified as a factor-stimulating natural killer cells (2, 3), promoting maturation of CTLs (4) and inducing antiangiogenic effects (5).It has recently been shown that local or systemic treatment with recombinant IL-12 protein mediates profound antitumor effects in vivo, causing regression of established tumors and their distant metastases (6).However, daily local or systemic administration of IL-12 requires patient self-administration and compliance with a regimen of daily injections.In addition, systemic administration of IL-12 protein has caused dose-dependent toxicity in mice (7) and in human trial (8).Alternative approaches, including gene therapy, for the delivery of IL-12 have been pursued (9,10), and we have shown previously the efficacy of direct transfection of the pCAGGS-mouse IL-12 (mIL-12) gene into s.c.hepatocellular carcinoma (HCC) using in vivo electroporation in a mouse model (11).
In the present study, we have shown some novel effects of muscle-targeted IL-12 gene therapy using electrosonoporation on the growth of orthotopic HCC and on spontaneous lung metastasis.

Establishment of Orthotopic HCC in C3H Mice
Six-week-old female C3H/HEN Crj mice purchased from Charles River, Inc. (Tokyo, Japan) were used throughout this study.The mice were maintained under specific pathogen-free conditions.Under satisfactory anesthesia using pentobarbital sodium, mice were challenged into both left lateral and right lobes of the liver with 100 AL of a single-cell suspension containing 5 Â 10 5 MH134 cells of the C3H mouse HCC cell line using an insulin syringe with a 30 gauge needle (12 -14).The Kyushu University Institutional Animal Care and Use Committee approved all animal protocols, which were designed according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy for Sciences and published by the NIH.

I.m. Plasmid DNA Injection and Electrosonoporation
Mice were anesthetized with pentobarbital sodium.Seven days after inoculations of MH134 cells into both left lateral and right lobes of the liver, they were injected with 100 Ag closed circular DNA (pCAGGS or pCAGGS-mIL-12) into the quadriceps muscle at 1.0 Ag/AL in 0.85% NaCl using an insulin syringe with a 27 gauge needle.Gene transfers via electrosonoporation into mouse quadriceps muscle were done.The quadriceps muscle was covered with ultrasound conducting lotion (Aloe-Sound Lotion, Rich-Mar, Inola, OK) and sonoporated for 5 minutes at 1 MHz input frequency with a 50% duty cycle and 2.0 W/cm 2 output intensity using a sonoporation device (Sonitron 1000, Rich-Mar; ref. 16).At the middle of the duration of sonoporation, steel electrodes in the form of parallel plates (0.5 Â 2.5 cm) were brought into contact with the muscle and 25 V of electric pulses of the opposite polarity (pulse length of 100 ms/pulse and 6 pulses) using an electric pulse generator (CUY-21, BEX Co., Ltd., Tokyo, Japan) were given to the muscle (1).
ELISA of mIL-12 and mIFN-; Serum samples obtained from the tail vein of mice were assayed for total mIL-12 (Genzyme, Cambridge, MA) and mIFN-g (Biosource International, Camarillo, CA) using an ELISA kit according to the manufacturer's instructions.

Survival, Spontaneous Lung Metastasis, and Tumor Morphology
Mice were divided into three groups: (a) i.m. transfer of pCAGGS-mIL-12 by electrosonoporation (n = 10), (b) i.m. injection of pCAGGS-mIL-12 without electrosonoporation (n = 10), and (c) i.m. transfer of control pCAGGS by electrosonoporation (n = 10).Survival was observed to assess long-term outcomes among the above three groups.To analyze the spontaneous lung metastasis macroscopically, bilateral lungs were removed 28 days after gene therapy.In addition, five livers from each of the three groups at 14 days after gene therapy were resected and the antitumor effects were evaluated by H&E staining and CD31 immunohistochemistry (PharMingen, San Diego, CA).The ratio of the viable region of MH134 cells at five random areas was calculated using an image analyzer (MAC SCOP, Mitani, Nagano, Japan).CD31positive microvessels within or surrounding the HCC were counted by hotspot methods (17,18).The averages within the hotspot areas (mean microvessel density) were recorded and expressed as counts per mm 2 .
Assay System for CTL Responses In vitro sensitization and the cytotoxicity assay of splenocytes in C3H mice against MH134 cells were essentially the same as described previously (21,22).
Statistical Analysis Statistical evaluations of numerical variables in both groups were done using Mann-Whitney's U test and those of the qualitative variables were done using Fisher's extract probability test.Survival was calculated by means of the product limit method of Kaplan-Meier and the differences in survival between the groups were compared using the log-rank test.

Results
Time Course of mIL-12 and mIFN-; Serum mIL-12 levels increased gradually after pCAGGS-mIL-12 transfer into the quadriceps muscle by electrosonoporation and peaked at 7 days after gene therapy (mean F SEM 25.5 F 2.8 ng/mL).Thereafter, serum mIL-12 levels were maintained at f 54.5% of the maximum value after 1 month (13.9 F 1.0 ng/mL; Fig. 1A).Serum mIFN-g levels also increased gradually and peaked 7 days after electrosonoporation (285.7 F 53.4 pg/mL; Fig. 1B).In contrast, both in mice with control pCAGGS transfer into quadriceps muscle with electrosonoporation and in mice with i.m. injection of pCAGGS-mIL-12 without electrosonoporation, no elevation of mIL-12 was found and mIFN-g was not detectable.
Survival Rate and Spontaneous Lung Metastasis Figure 2 presents cumulative Kaplan-Meier survival plots for each group.Both mice with i.m. transfer of control pCAGGS by electrosonoporation and mice with i.m. injection of pCAGGS-mIL-12 without electrosonoporation developed HCCs markedly and died within 45 days of gene therapy.Five of 10 mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation also died f 45 to 120 days after gene therapy, but 5 of 10 (50%) mice survived >120 days after gene therapy.The survival advantage for mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation was statistically significant (P < 0.01).
In 8 of 10 (80%) mice, multiple spontaneous lung metastases became evident macroscopically during the HCC growth period in mice both with i.m. transfer of control pCAGGS by electrosonoporation and with i.m. injection of pCAGGS-mIL-12 without electrosonoporation.However, 9 of 10 (90%) mice with HCCs with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation did not develop lung metastases.The inhibition of spontaneous lung metastasis from HCCs in mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation was statistically significant (P = 0.03).
Morphologic and Histopathologic Analysis Orthotopic HCCs at both left lateral and right lobes of the liver developed markedly and occupied a large region of the liver 14 days after gene therapy in mice both with i.m. transfer of control pCAGGS by electrosonoporation and with i.m. injection of pCAGGS-mIL-12 without electrosonoporation.In contrast, HCC development in mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation was markedly inhibited (Fig. 3A -C).In addition, the weight of the resected livers in mice with i.m. transfer   The histopathologic analysis of HCC stained with H&E revealed the presence of massive necrosis and that the distribution of viable MH134 cells in mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation was remarkably reduced (mean F SEM 15.9 F 5.3%) in comparison with that in mice with i.m. transfer of control pCAGGS by electrosonoporation (91.3 F 4.6%) and in mice with i.m. injection of pCAGGS-mIL-12 without electrosonoporation (89.8 F 3.8%; P < 0.01; Fig. 4A -C).
Significantly fewer microvessels stained by anti-mouse CD31 monoclonal antibody, as evaluated by mean microvessel density, within and surrounding the HCC were The inhibition of viable regions was statistically significant (P < 0.01).

Flow Cytometric Analysis of Tumor-Infiltrating Lymphocytes
Table 1 shows more infiltrating Mac-1-positive cells in the HCC in mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation (35.6/cm 3 tumor) than in mice with i.m. transfer of control pCAGGS by electrosonoporation (3.1/cm 3 tumor) and mice with i.m. injection of pCAGGS-mIL-12 without electrosonoporation (3.4/cm 3 tumor).However, the increases in infiltrating natural killer cells and CD3+ T cells were not dominant in mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation.

CTL Responses
Effector cell activity generated by spleen cells from mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation was marginal and not particularly potent.There was no remarkable change in effector cell activity between mice with i.m. transfer of pCAGGS-mIL-12 by electrosonoporation and mice with i.m. transfer of control pCAGGS by electrosonoporation or i.m. injection of pCAGGS-mIL-12 without electrosonoporation (data not shown).

Discussion
We have reported that muscle-targeted gene transfer using in vivo electrosonoporation is an effective therapeutic method for continuous delivery of IL-12 (1).Muscletargeted gene transfer using in vivo electrosonoporation may enable the continuous and gradual elevation of therapeutic proteins without a rapid dosage increase in patients, which may induce toxicities such as those related to IL-12, and without patient self-administration and compliance with a regimen of daily local or systemic injection.Additionally, the large-scale synthesis required for continuous delivery of therapeutic proteins such as IL-12 may be too expensive, making an alternative method like muscletargeted gene therapy quite attractive.
In the present study, we showed that administration of mIL-12 cDNA into mice quadriceps muscles by in vivo electrosonoporation significantly elevates serum levels of mIL-12 (peaking at 25.5 F 2.8 ng/mL) and IFN-g (peaking at 285.7 F 53.4 pg/mL) and inhibited the growth of orthotopic HCCs and spontaneous lung metastasis.Due to systemic elevation of IL-12 and IFN-g, which had antiangiogenetic effects, fewer microvessels were found in HCC.To avoid IL-12 toxicity, ''predosing'' is believed to be important (23).Thus, the gradual elevation of systemic IL-12 levels by i.m. gene transfer by electrosonoporation would be an ideal model for IL-12 cancer therapy.No mice died unexpectedly in the present study.
We have shown previously the efficacy of direct transfection of the pCAGGS-mIL-12 gene into s.c.HCC in the same mouse model with systemic tumor immunity, lymphocytic infiltration (natural killer, CD3+, and Mac-1positive cells) into tumors, and antiangiogenetic effects (11).Although inhibitory effects with regard to tumor growth of orthotopic HCCs and spontaneous lung metastasis were observed in mice with the muscle-targeted mIL-12 gene therapy using electrosonoporation, but systemic tumor immunity as evaluated by the CTL responses of splenocytes was not acquired, infiltration of only the Mac-1-positive lymphocytes was detected.These differences in the effects of tumor immunity may be due to differences in the tumor model (s.c.HCC or orthotopic HCC) or in the target of mIL-12 gene transfer (direct HCC or distant muscle).Advanced study is necessary regarding the effects of direct mIL-12 transfer into orthotopic HCC by electroporation or electrosonoporation in this mouse model; many technical difficulties stands in way of this research, however, and we have not yet achieved constant gene transfer by electroporation or electrosonoporation into orthotopic HCC with this model.
In sum, our data suggest that muscle-targeted IL-12 gene therapy for orthotopic HCC using electrosonoporation may be a new strategy of gene therapy and a promising treatment modality for advanced HCC with intrahepatic metastases in humans with severe liver cirrhosis.

Table 1 .
Fluorescence-activated cell sorting analysis of tumor infiltrating lymphocytes : Data have been confirmed in three independent experiments.killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes.J Exp Med 1989;170:827 -45. 3. Chan SH, Perussia B, Gupta JW, et al.Induction of IFNg production by NKSF: characterization of the responder cells and synergy with other inducers.J Exp Med 1991;173:869 -79. 4. Wolf SF, Temple PA, Kobayashi M, et al. Cloning of cDNA for NKSF, a heterodimeric cytokine with multiple biologic effects on T and NK cells.J Immunol 1991;146:3074 -81.