Evaluation of the antitumoral effect mediated by IL-12 and HSV-tk genes when delivered by a novel lipid-based system

In the present work, we used a novel albumin-associated lipoplex formulation, containing the cationic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) and cholesterol (Chol), to evaluate the antitumoral efficacy of two gene therapy strategies: immuno-gene therapy, mediated by IL-12 gene expression, and "suicide" gene therapy, mediated by HSV-tk gene expression followed by ganciclovir (GCV) treatment. Our data show that, in an animal model bearing a subcutaneous TSA (mouse mammary adenocarcinoma) tumor, intratumoral administration of the albumin-associated complexes containing the plasmid encoding IL-12 results in a strong antitumoral effect, as demonstrated by the smaller tumor size, the higher T-lymphocyte tumor infiltration and the more extensive tumor necrotic and hemorrhagic areas, as compared to that observed in animals treated with control complexes. On the other hand, the application of the "suicide" gene therapy strategy results in a significant antitumoral activity, which is similar to that achieved with the immuno-gene therapy strategy, although involving different antineoplastic mechanisms. For the tested model, albumin-associated complexes were shown to efficiently mediate intratumoral delivery of therapeutic genes, thus leading to a significant antitumoral effect. This finding is particularly relevant since TSA tumors are characterized for being poorly immunogenic, aggressive and exhibiting high proliferation capacity.     

Therapy of cancer by cytokines mediated by gene therapy approach

Gene therapy offers a new approach for treatment of cancer. Transfer of genes encoding immunostimulatory cytokines has been used with remarkable success to eliminate cancer in animals. However, clinical trials in patients with this strategy had limited efficacy. Therefore, improvement ofgene transfer vector system is necessary. A hybrid viral vector, consisting of SFV replicon with either murine IL-12 or reporter LacZ gene, was constructed. This hybrid vector showed specificity and high level of expression in HCC both in vitro and in vivo. In a rat orthotropic liver tumor model, treatment of established tumors by the hybrid vector with raiL- 12 gene resulted in a strong anti-tumor activity without accompanying toxicity. Subsequently, a helper-dependent adenovirus vectors containing a mifepristone （RU486） inducible system was constructed for controlled and liver-specific expression of human interleukin 12 （hIL- 12） （HD-Ad/RUhIL- 12） and mouse IL-12 （mIL-12） （HD-Ad/RUmIL-12）. Data showed that high and sustained serum levels of hlL-12 could be attained by continuing administration of RU486 every 12 or 24 h. Repetitive induction ofhlL-12 could be obtained over, at least, a period of 48 weeks after a single injection of HD-Ad/RUhlL-12. Treatment of liver metastases with of HD-Ad/RUmIL- 12 plus RU846 resulted in complete tumor regression in all animals. Then, different cytokine genes were inserted into conditional replicative adenoviruses vectors （also called oncolytic adenovirus）. Replication ofadenovirus in tumor cells would kill tumor cells and release viruses, which infect surrounding tumor cells. The combination of cytopathic effect by oncolytic adenovirus and biological effect of transgene would exert strong antitumor activity. These new types of vectors may provide a potent and safe tool for cancer gene therapy.     

In vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas

BACKGROUND: Human breast cancer metastasizes mainly to lymph nodes, lungs, liver, and bone; in the majority of cases, it is the development of metastases which leads to death. In order to suppress mammary cancer metastasis, we applied in vivo electrogene transfer (non-viral method) as a means of interleukin-12 (IL-12) gene therapy on highly metastatic murine mammary cancer model. METHODS: Metastatic mammary tumors induced by inoculation in BALB/c female mice were treated by intratumoral injections of either a plasmid vector containing IL-12 or empty vector and then subjected to in vivo electrogene transfer once a week for 8 weeks. RESULTS: Treatment with IL-12 resulted in elevation of both IL-12 and IFNgamma levels in mammary tumors and in serum and intratumoral levels of CD4 and CD8 proteins were also increased. Tumor volumes and lymphatic and pulmonary metastases were significantly reduced. The histopathological changes induced by IL-12 characteristically included marked inflammation, increased apoptosis, decreased DNA synthesis, peripheral influx of significantly greater numbers of active macrophages, and reduced blood microvessel density, and apoptotic vascular endothelial cells were frequently seen. Western blotting showed decreases in VEGFR-3 of tumors exposed to IL-12 gene therapy. In adjuvant immunofluorescence studies, the CD31-positive endothelial cells of microvessels showed decreased VEGFR-3 expression in IL-12-treated tumors. However, apparent alterations in VEGFR-3 expression of podoplanin-positive lymphatic endothelial cells were not observed in IL-12-treated tumors. Although recombinant IL-12 did not inhibit tubular formation of human umbilical vein endothelial cells in a Matrigel assay, recombinant IFNgamma did completely suppress the tubular formation. CONCLUSIONS: In vivo electrogene transfer of IL-12 exerts strong anti-tumorigenic and anti-metastatic effects likely due to T-cell-mediated immune responses as well as anti-angiogenic action.     

Identification and characterization of a novel peptide ligand of Tie2 for targeting gene therapy

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has been considered as a rational target for gene therapy in solid tumors. In order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we screened a phage display peptide library and identified a candidate peptide ligand NSLSNASEFRAPY (designated GA5). Binding assays and Scatchard analysis revealed that GA5 could specifically bind to Tie2 with a dissociation constant of 2.1 × 10⁻⁸ M. In addition, we showed that GA5 was internalized into tumor cells highly expressing Tie2. In the biodistribution assay, ¹²⁵I-GA5 was mainly accumulated in SPC-A1 xenograft tumors that express Tie2. In gene delivery studies, GA5-conjugated polyethylenimine vector could achieve greater transgene transduction than non-targeted vectors both in vitro and in vivo . Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice that received eight intratumoral injections of GA5-polyethylenimine/ p53 complexes in 3 weeks. The difference in tumor volume between the experiment and control groups was significant ( P < 0.05). Our results showed that GA5 is a potentially efficient targeting element for cancer gene or moleculartherapy.     

Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

Background  

Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12.     

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following “suicide” gene therapy in a murine colon carcinoma model

In prodrug-activated (suicide) gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected bystander cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving suicide gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to bystander killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.Abdul-Razzak Alsheikhly and Jehad Zweiri contributed equally to this work and are listed in alphabetical order.     

选择性复制型腺病毒介导的自杀基因HSV -tk 在 肿瘤基因治疗中的应用

自杀基因治疗是肿瘤基因治疗的手段之一,治疗效果与自杀基因能否被高效、选择性的导入肿瘤细胞有关。肿瘤选择性复制型腺病毒（conditionally replication adenovirus,CRADs）可以特异性的在肿瘤细胞中复制,在复制的同时所携带的治疗基因也大量表达。由CRAds介导的自杀基因,实现了对肿瘤的病毒治疗和基因治疗的结合,提高了治疗效率和使用复制型腺病毒的安全性。     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Cationic lipid gene transfer of an IL-2 transgene leads to activation of natural killer cells in a SCID mouse human tumor xenograft

Natural killer (NK) cells play an important role in combating infectious and malignant diseases and interleukin-2 (IL-2) has been shown to promote proliferation and activation of NK cells in vitro and in vivo. Here we investigate the effects of local cationic lipid-mediated IL-2 gene transfer on intratumoral accumulation and activation of NK cells in a SCID mouse tumor model. UM449 human melanoma tumors in SCID mice received intratumoral injections of DMRIE/DOPE admixed with VR1103, a DNA plasmid encoding the gene for human IL-2. Dissagregated tumor cells were tested for IL-2 secretion and were characterized using antibodies to asGM1, MAC-1, and F4/80 antigens. Granzyme A, a proteolytic serine esterase, was also measured in tumor cell lysates. IL-2 secretion from tumors injected with VR1103:DMRIE/DOPE peaked at 48 h after injection and fell to baseline levels on day 8. Intratumoral granzyme A activity was significantly increased in tumors injected with IL-2 plasmid:DMRIE/DOPE complexes, but not by an irrelevant plasmid DNA:DMRIE/DOPE control. Importantly, the growth of UM449 tumors was slowed in VR1103:DMRIE/DOPE-injected tumors. These results indicate that local cationic lipid-mediated gene transfer of IL-2 induces activation of intratumoral NK cells and slows tumor growth.     

Tumor immunotherapy by converting tumor cells to MHC class II-positive,Ii protein-negative phenotype

A potent antitumor CD4⁺ T-helper cell immune response is created by inducing tumor cells in vivo to a MHC class II⁺/Ii^– phenotype. MHC class II and Ii molecules were induced in tumor cells in situ following tumor injection of a plasmid containing the gene for the MHC class II transactivator (CIITA). Ii protein was suppressed by the antisense effect of an Ii-reverse gene construct (Ii-RGC) in the same or another co-injected plasmid. The MHC class II⁺/Ii^– phenotype of the tumor cells was confirmed by FACS analysis of cells transfected in vitro and by immunostaining of tumor nodules transfected by injections in vivo. Subcutaneous Renca tumors in BALB/c mice were treated by intratumoral injection with CIITA and Ii-RGC, in combination with a subtherapeutic dose of IL-2, to up-regulate the activation of T cells. Significant tumor shrinkage and decrease in rates of progression of established Renca tumors were seen in the groups injected with Ii-RGC, compared with groups in which only IL-2 plus empty plasmid controls were injected. Our method provides an effective immunotherapy warranting further development for human cancers.Abbreviations CIITA MHC class II transactivator - DMRIE 1,2-dimeristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide/cholesterol - FCS fetal calf serum - RGC reverse gene constructThis research was funded in part by NCI grants R43 CA85100 and R43CA 89856.     

电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒,测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高,GFP可在肌细胞内持续高水平表达3周以上,而在肿瘤细胞内只能表达4～6 d,但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感,单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想,但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移,甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.     

腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究

目的:近年来通过应用白介素-12治疗肿瘤取得良好效果,因此对国内外应用腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究进展进行总结,以探索更为可行治疗方法。方法:运用Pubmed、Elsevier Sciencedirect、CNKI及万方全文数据库检索系统,以腺病毒,IL-12,肝癌,树突细胞为关键词,检索2008-01至2012-11月发表的文献。纳入标准:1)IL-12的生物学特性,在抗肿瘤过程中的免疫作用,2)应用腺病毒携带IL-12对抗肝癌治疗研究,3)肿瘤抗原致敏树突细胞对肿瘤的影响。根据纳入标准分析文献26篇。结果:通过腺病毒携带IL-12可以增强肿瘤抗原致敏树突细胞的免疫应答。并通过诱导肿瘤细胞的凋亡,减少新生血管的生成而对肿瘤产生直接抑制,有效抑制肝癌的生长和转移。结论:本文通过对IL-12的生物学特征、抗肿瘤通路、作用机制及在腺病毒介导下肿瘤抗原致敏树突细胞研究进展的概述,为腺病毒携带IL-12作为肝癌的基因治疗进一步提供理论依据和探索,期待在将来应用IL-12为基础的基因治疗一定会为包括肝癌在内的肿瘤治疗提供新的途径。     

Co-expression of IL-18 binding protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced arthritis

We constructed a recombinant adenoviral vector containing a murine interleukin （IL）-18 binding protein （mlL-18BP） and murine IL-4 （mIL-4） fusion gene （AdmIL-18BP/mIL.4） and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in modulating the T-helperl and T-helper2 （Th1/Th2） balance in mice with collagen-induced arthritis （CIA）. Mice with CIA were intra-articularly injected with 107 pfu/6 μl ofeitherAdmIL.18BP/mIL-4, or a controladenovirus, or with the control vehicle （phosphate-buffered saline）. After intra-articular gene therapy with AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-α （TNF-α）, T-interferon （IFN-γ）, IL-4, IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-T-expressing and IL-4-expressing CD4^＋ T cells from mice splenocytes were monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after intraarticular injection of AdmIL-18BP/mIL-4 showed significantly increased serum concentrations of IL-4 and IL-10 （P〈0.01 at all time points） but greatly decreased serum concentrations ofIFN-γ, TNF-α and IL-β （P〈0.01 at all time points ） compared to both the con trol adenovirus and phospha tebuffered saline control groups. The percentage of LFN-γ- producing CD4^＋ T cells was significantly decreased in response to local AdmIL-18BP/mIL-4 treatment. The percentage of IL-4-producing CD4^＋ T cells increased significantly at 1 week after local injection of AdmIL-18BP/ mIL-4 then returned to normal by week 4. These data indicated the significant modifying effects on the Th1/Th2 imbalance in murine CIA produced by local overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4 is a promising potential therapy for rheumatoid arthritis.     

IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice

Immunosuppressive therapy for organ transplantation is essential for controlling rejection. When liver transplantation is performed as a therapy for hepatocellular carcinoma (HCC), recurrent HCC is one of the most fatal complications. In this study, we show that intratumoral murine IL-12 (mIL-12) gene therapy has the potential to be an effective treatment for malignancies under immunosuppression. C3H mice (H-2(k)), injected with FK506 (3 mg/kg) i.p., were s.c. implanted with 2.5 x 10(6) MH134 cells (H-2(k)) and we treated the established HCC with electroporation-mediated gene therapy using mIL-12 plasmid DNA. Intratumoral gene transfer of mIL-12 elevated intratumoral mIL-12, IFN-gamma, and IFN-gamma-inducible protein-10, significantly reduced the number of microvessels and inhibited the growth of HCC, compared with HCC-transferred control pCAGGS plasmid. The inhibition of tumor growth in immunosuppressed mice was comparable with that of mIL-12 gene therapy in immunocompetent mice. Intratumoral mIL-12 gene therapy enhanced lymphocytic infiltration into the tumor and elicited the MH134-specific CTL response even under FK506. The dose of FK506 was sufficient to prevent the rejection of distant allogenic skin grafts (BALB/c mice, H-2(d)) and tumors, B7-p815 (H-2(d)) used as transplants, during mIL-12 gene therapy against MH134. Ab-mediated depletion studies suggested that the inhibition of tumor growth, neovascularization, and spontaneous lung metastasis by mIL-12 was dependent almost entirely on NK cells and partially on T cells. These results suggest that intratumoral mIL-12 gene therapy is a potent effective strategy not only to treat recurrences of HCC in liver transplantation, but also to treat solid malignant tumors in immunosuppressed patients with transplanted organ.     

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.