Hong Sung Kim

Host Lymphodepletion Enhances the Therapeutic Activity of an Oncolytic Vaccinia Virus Expressing 4-1BB Ligand

Oncolytic viral vectors have shown promise as antitumor therapeutic agents but their effectiveness is complicated by induction of antiviral antibody responses and rapid host clearance of recombinant vectors. We developed a recombinant oncolytic vaccinia virus expressing the 4-1BBL T-cell costimulatory molecule (rV-4-1BBL) and showed modest tumor regression in the poorly immunogenic B16 murine melanoma model. To improve the therapeutic potential of this vector, we tested the antitumor activity of local intratumoral injection in the setting of host lymphodepletion, which has been shown to augment vaccination and adoptive T-cell therapy. In this model, rV-4-1BBL injection in the setting of lymphodepletion promoted MHC class I expression, reduced antiviral antibody titers, promoted viral persistence, and rescued effector-memory CD8(+) T cells, significantly improving the therapeutic effectiveness of the oncolytic vector. These data suggest that vaccination with rV-4-1BBL in the setting of host nonmyeloablative lymphodepletion represents a logical strategy for improving oncolytic vaccination in melanoma, and perhaps other cancers as well.

Inhibition of B16BL6 tumor progression by coadministration of recombinant angiostatin K1-3 and endostatin genes with cationic liposomes.

Transfection of the antiangiogenic angiostatin and endostatin genes was shown to be an alternative to high-dose administration of angiostatin or endostatin proteins for cancer therapy. We have systematically investigated whether coadministration of the mouse angiostatin kringle 1–3 gene (pFLAG-AngioK1/3) and the endostatin gene (pFLAG-Endo) complexed with cationic liposomes exhibits enhanced therapeutic efficacy. In vitro, the coexpressed mixture of angiostatin K1-3 and endostatin more effectively reduced angiogenesis in chorioallantoic membranes than either angiostatin K1-3 or endostatin alone. In vivo, subcutaneous co-administration of pFLAG-AngioK1/3 and pFLAG-Endo lipoplexes more effectively inhibited vascularization in Matrigel plugs implanted in mice than either one alone. Additionally, subcutaneous administration of these genes inhibited the growth and formation of pulmonary metastases of B16BL6 melanoma cells in mice. Compared to treatment with an empty vector, treatment with pFLAG-AngioK1/3 plus pFLAG-Endo inhibited 81% of tumor growth, while treatment with pFLAG-AngioK1/3 or pFLAG-Endo inhibited tumor growth 70 and 69%, respectively. Cotreatment with the two plasmids after primary tumor excision induced a 90% inhibition of pulmonary metastases versus 79% for pFLAG-AngioK1/3 or 80% for pFLAG-Endo individually. These results suggest that combined administration of angiostatin K1–3 and endostatin genes complexed with cationic liposomes may be an innovated antiangiogenic strategy for cancer therapy.

Inhibition of angiogenesis by salmosin expressed in vitro.

Recently, salmosin, a novel snake venom-derived disintegrin containing the Arg-Gly-Asp (RGD) sequence, was reported to be both antiangiogenic and antitumorigenic. The antitumor activity was substantiated by in vivo administration of recombinant salmosin into mice bearing tumors. However, it was difficult to prepare functionally active recombinant salmosin and to maintain a therapeutically effective concentration of the protein in the circulatory system by daily injections. Hence, we have suggested that salmosin gene transfer mediated by cationic liposomes may be a practical alternative for cancer treatment. Plasmids encoding the salmosin gene were constructed and then transferred by means of cationic liposomes into transformed human embryonic kidney (HEK) 293 cells. The transfected genes were able to produce functionally active salmosin proteins in vitro. In fact, the expressed salmosin remarkably inhibited proliferation of bovine capillary endothelial (BCE) cells and effectively inhibited the migration of highly metastatic B16BL6 mouse melanoma cells. Neovascularization in chick chorio-allantoic membranes (CAM) and in Matrigel implanted subcutaneously into mice was greatly inhibited in the presence of the expressed salmosin. Based on these experimental results, we suggest that the antitumor effect induced by salmosin gene transfection may be due to the antiangiogenic activity of the expressed salmosin proteins.

An efficient liposomal gene delivery vehicle using Sendai F/HN proteins and protamine.

By means of a simple mixing procedure, we have constructed cationic Sendai virosomes consisting of fusogenic viral F/HN proteins and cationic lipids. Sendai virus F/HN proteins were purified by Triton X-100 treatment and sequential centrifugation, and then quantitatively added to cationic liposomes. The presence of HN proteins is essential for hemolytic activity of Sendai virus as well as efficient gene transfection. The amount of detergent added for purification of F/HN proteins was also crucial for hemolytic activity. The relevance of F/HN proteins in the gene-transfer capability of the cationic Sendai F/HN virosomes (CSVs) was verified by heat inactivation of the F/HN proteins, and cell-binding competition. DNA condensation by protamine sulfate was able to further enhance the transfection efficiency and serum resistance of CSV. The enhanced transfection efficiency of protamine-condensed DNA-encapsulating cationic Sendai F/HN virosomes (PCSVs) may result from specific and efficient cell binding mediated by F/HN proteins and efficient DNA encapsulation by protamine. The DNA condensation by protamine was crucial for systemic in vivo gene transfer by CSVs. The PCSVs exhibited a higher gene expression in various organs, especially the liver, compared to DOTAP/Chol lipoplexes. These results demonstrate the potential for the use of PCSV as gene delivery vehicles for systemic gene transfer.

Hepatic control elements promote long‐term expression of human coagulation factor IX gene in hydrodynamically transfected mice

Long‐term expression of the delivered target gene is critical for successful gene therapy. Recently, hepatic control region I (HCR I) originating from the apolipoprotein (apo)C‐I pseudogene was shown to be a critical element for long‐term gene expression in the liver of mice. HCR II is another hepatic control region of apoC‐I.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor‐directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off‐target effects of gene transfection.

Sendai Viroplexes for EGF Receptor‐directed Delivery of IL12 and Salmosin Genes to Cancer Cells

The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor‐directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off‐target effects of gene transfection.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles

Background Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. Purpose In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. Materials and methods The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. Results Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. Conclusion This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

556. Another Hepatic Control Region for Sustained Expression of Human Factor IX in the Mouse Liver

For long-term gene expression mediated by plasmid vectors, the plasmids should remain stable in the episomal state with transcriptional and post-transcriptional activities inside nucleus. There was an important report that a hepatic control region (referred to HCR I) maintained plasmids in the stable episomal state in nucleus, preventing loss of plasmids. Furthermore, the HCR has an enhancer activity, thus leading to high expression of transgenes in hepatic cells. There is another hepatic control region, called the second hepatic control region (referred to HCR II). In this research, we have tried to compare the functional activities of HCR I and HCR II in hepatic transgene expression. Their sequences in the functional region have 85% homology. We constructed the plasmid vector containing HCR II and encoding human factor IX (pBS-HCRII-IX), that was compared with the plasmid containing HCR I (pBSHCRHPI-IXA, provided by Dr. Miao, U. Wa). Several other plasmids containing HCR I and HCR II (pBS-HCRI-HCRII-IX and pBS-HCRII-HCRI-IX) were also constructed. These four different plasmids were administered to mice by the hydrodynamic method. There was no significant differences among the expression levels of human factor IX by the four different vectors for 3 months; about 1.2 mg/ml (24% of the normal level) on day 1 and about 0.2 mg/ml (4% of the normal level) on day 85. Also, blood clotting activities of human factor IX produced by the four vectors were similar among each other for the same period of time. Meanwhile, the factor IX level induced by a control plasmid (pAAVCMV-hFIX) without HCR was drastically diminished in 72 hours. Interestingly, the significant amount of anti-human factor IX antibody was detected in the mice treated with the control plasmid, but not in the mice treated with the plasmids containing HCR. At this moment, we have not seen any functional differences among the four different plasmids containing HCR. Further analysis of human factor IX expression and blood coagulation in hemophilia B mice treated with the HCR-containing plasmids for a longer period of time will give us a better understanding about HCR functions in hepatic transgene expression.