Cytokine Gene Therapy Research Articles

Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with "tracer" hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK(+), EGFP(+) stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with (18)F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([(18)F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK(+) tumor stroma with an average of 0.36 +/- 0.24% ID/g [(18)F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF(+) and CD31(+) endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK(+), GFP(+) endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell-based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing "tracer" hMSCs in tumor stroma.

Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8(+) T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8(+) T cell-depleted mice, but not in CD4(+) T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8(+) T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4(+) and CD8(+) T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8(+) T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.

Liposome-Mediated Combinatorial Cytokine Gene Therapy Induces Localized Synergistic Immunosuppression and Promotes Long-Term Survival of Cardiac Allografts

Localized gene transfer has the potential to introduce immunosuppressive molecules only into the transplanted allograft, which would limit systemic side effects, and prolong allograft survival. However, an applicable gene transfer strategy is not available, and the feasible therapeutic gene(s) has not yet been determined. We developed an ex vivo liposome-mediated gene therapy strategy that is able to intracoronary deliver the combination of IL-4 and IL-10 cDNA expression vectors to the allograft simultaneously. We examined the efficiency, efficacy, and cardiac adverse effects of this combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate, the expression of both transgenes was long lasting and localized only in the target organ. The mean survival of cardiac allograft was prolonged from 7 to >100 days. Synergism of overexpressed IL-4 and IL-10 in the inhibition of T lymphocyte infiltration and cytoxicity, and modulation of Th1/Th2 cytokine production promote long-term survival of cardiac allografts.

269. Stimulation of the Immune System Using Gene Therapy Is an Effective Approach for Treating Brain Tumors in Mouse and Rat Models

Malignant glioma is a highly invasive and neurologically destructive tumor frequently resulting in death within nine months after diagnosis. Suicide gene therapy has been explored as an alternative to chemotherapy for treating or curing the disease. While treatments utilizing gene therapeutic vectors expressing HSV1-thymidine kinase (HSV1-TK) in combination with the pro-drug ganciclovir (GCV) are 100% efficient in many preclinical glioma models, phase 1 and 2 clinical trials in human patients have not shown such dramatic results. We have developed a macroscopic preclinical model of glioma in rats and have shown that all single cytokine and cytotoxic gene therapies fail. Furthermore, combined treatments with HSV1-TK and various immuno-modulatory agents were also unsuccessful for improving survival. Only combined treatment with HSV1-TK and the potent cytokine for stimulating dendritic cells, human soluble Fms like tyrosine kinase 3 ligand (hsFlt3L), provided 80% survival in this syngeneic model of glioma. This model is a more faithful reflection of the clinical results obtained utilizing single gene therapies in human patients suffering from glioma. In the present study we have analysed the immune responses in both naive and glioma bearing animals when treated with RAdHSV1-TK and RAdhsFlt3L alone or in combination. We found that when adenoviral vectors expressing HSV1-TK and hsFlt3L are injected into naive brains, large increases in B, NK-T, NK and plasmacytoid dendritic cell (pDC) populations were observed. NK and NK-T cells are important mediators of antigen-independent anti-tumor cytotoxicity. pDC's may improve antigen presentation and prime an efficient adaptive immune response against the tumor. ELISA and ICC were used to assess long-term expression of HSV1-TK and hsFlt3L transgenes. In summary, our results indicate that the combined anti-tumor effect of HSV-1TK and hsFlt3L enhances both innate and adaptive branches of the immune system and improves survival in a syngeneic macroscopic glioma model.

In Situ Gene Therapy for Prostate Cancer

The incidence of prostate cancer has dramatically increased worldwide in the past decade, with mortality rates also increasing in many countries. Once prostate cancer is diagnosed, it is important to rapidly begin a treatment regimen that is either potentially curative or impedes disease progression. When the disease is confined to the prostate, it can be cured by radical prostatectomy or irradiation therapy. However, there are no curative therapies for locally advanced, recurrent, or metastatic diseases. Clearly, new therapies are needed for these patients. Gene therapy may provide additional therapeutic options with the potential to affect both localized and metastatic disease. Virus-mediated transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene transfer, followed by a course of the prodrug ganciclovir (GCV), so-called suicide gene therapy, has been demonstrated by several investigators. The present in situ gene therapy clinical trial for human prostate cancer demonstrated safety, clinical efficacy, and biological effects of antitumor activity. HSV-tk clinical trials for prostate cancer are also ongoing in Japan, the Netherlands, and Mexico. Currently, numerous preclinical studies have reported immunomodulatory cytokine gene therapy, such as interleukin-2, interleukin-12, B7-1 (CD80), B7-2 (CD86) and granulocyte-macrophage colony-stimulating factor. Several clinical studies have been approved that potentially will show that these immunomodulatory gene therapies may generate an effective local and systemic antitumor activity and that should provide options for patients with prostate cancer. We review the multiple issues involved in current in situ gene therapy (gene/immunotherapy), its outcome, and future directions for patients with prostate cancer.

Increased Anti-tumor Effect by a Combination of HSV Thymidine Kinase Suicide Gene Therapy and Interferon-γ/GM-CSF Cytokine Gene Therapy in CT26 Tumor Model

The potential therapeutic benefit of introducing IFN-γ and GM-CSF genes in combination with the HSVtk suicide gene into subcutaneously implanted CT26 tumor cells was compared with that from each treatment alone. Cells, unmodified or retrovirally transduced with HSVtk or IFN-γ/GM-CSF genes, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSVtk gene, with intraperitoneal ganciclovir treatment, reduced tumor volume by 81% at locally inoculated tumor sites (p<0.01) and by 25% at distantly inoculated tumor sites (p=0.052). IFN-γ/GM-CSF genes showed a 56% tumor volume reduction at local tumor sites (p<0.01) and 15% volume reduction at remote tumor sites, although this was not statistically significant. The combination of HSVtk (with GCV) and IFN-γ/GM-CSF genes showed an 81% volume reduction at local tumor sites (p<0.01) and a 43% volume reduction at remote tumor sites (p<0.01). Thus, the combination of HSVtk and IFN-γ/GM-CSF gene therapy produced greater therapeutic efficacy than either treatment alone.

Tumor cells secreting IL-13 but not IL-13Ralpha2 fusion protein have reduced tumorigenicity in vivo.

IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.

584. Intratumoral Electrotransfer of Interleukin-12 Gene and Bleomycin Synergistically Induced Therapeutic Immunity and Suppressed Subcutaneous and Metastatic Melanomas in Mice

In vivo electroporation-mediated transfer of chemotherapeutic agents provides an effective means for treatment of neoplasms, as demonstrated by clinical trials against various tumors including malignant melanoma. We postulated that the combination of the electro-chemotherapy and intratumoral cytokine gene therapy may further improve the therapeutic efficacy, thanks to the anti-tumor immune responses that may eradicate not only the manipulated tumors but also distant metastatic lesions.

348. IL-21 and IL-15 Gene Therapy Induces Powerful Antitumor Effect Against Head and Neck Squamous Cell Carcinoma

Top of pageAbstract Interleukin (IL)-21 is a member of the 4-helix-bundle cytokine family capable of supporting proliferation of mature T and B lymphocytes. The cytokine also facilitates expansion and maturation of natural killer (NK) cells in synergy with IL-15. We previously reported that intravascular transfection of IL-21 and IL-15 genes augmented antitumor immunity, resulting in a marked therapeutic outcome in a hepatic metastasis model of lymphoma in mice (Kishida et al. Molecular Therapy, 8:552, 2003). It remains to be elucidated, however, whether the combinatory cytokine gene therapy could also inhibit a pre-established solid tumor in vivo. In the present study, we investigated the efficacy of intravascular delivery of IL-21 and IL-15 genes in suppressing head and neck squamous cell carcinoma (HNSCC) implant in mice. SCCVII, a murine HNSCC cell line, was subcutaneously inoculated into the flank of syngenic C3H/He mice to develop palpable subcutaneous tumors. A plasmid vector encoding murine IL-21 was intravenously injected into the tumor-bearing mice via their tail vein, alone or in combination with an IL-15 expression vector construct. The genetic transfer of either IL-21 or IL-15 elicited a significant, but moderate, antitumor effect compared with the treatments with an empty plasmid. In contrast, a remarkable antitumor effect was obtained by repetitive transfection with both the IL-21 and IL-15 genes, leading to a significant prolongation of the survival periods of the animals (P<.05, IL-21+IL-15 vs IL-21 alone; P<0.05, IL-21+IL-15 vs IL-15 alone; P<0.005, IL-21+IL-15 vs control), although a single treatment with both of the cytokine genes failed to show such therapeutic results. Importantly, any detectable toxic effect such as body weight loss and capillary leaking was not revealed in the mice that had received these cytokine genes. IL-21 significantly elevated cytotoxic T lymphocyte (CTL) killing activity against the SCCVII in the spleen of the tumor-bearing mice (P<0.005, IL-21+IL-15 vs control; P<0.05, IL-21 alone vs control), while IL-21 and IL-15 augmented NK cytotoxicity (P<0.0005, IL-21+IL-15 vs control; P<0.001, IL-21+IL-15 vs IL-21 alone; P<0.005, IL-21+IL-15 vs IL-15 alone). The co-transfection with IL-21 and IL-15 genes slightly, but significantly, elevated the production of interferon (IFN)-γ in the liver compared with the delivery of either IL-21, IL-15, or control plasmids, whereas IFN-γ concentration in the sera was not significantly affected. Furthermore, IL-21 gene transfection induced production of immunoglobulin specific for SCCVII tumor antigen, as demonstrated by indirect cellular ELISA and flowcytometric analyses of the sera from the genetically modified tumor bearers (P<0.05, IL-21+IL-15 vs control; P<0.005, IL-21 alone vs control). Taken together, the present study strongly suggest that the IL-21 and IL-15 genes synergistically elicited powerful cellular and humoral antitumor immune responses, which effectively suppressed HNSCC-derived pre-established tumor without any detectable adverse effect in vivo.

Recent progress in immunotherapy for malignant glioma: treatment strategies and results from clinical trials.

Despite advances in surgical and adjuvant radiation therapy and chemotherapy strategies, malignant gliomas continue to be associated with poor prognoses. We review immune-mediated treatment approaches for malignant glioma and the relevance of recent clinical trials and their outcomes. We specifically address the increasing evidence implicating the role of cytotoxic T cells in ensuring adequate immune-mediated clearance of neoplastic cells and the need for the optimization of therapies that can elicit and support such antitumor T-cell activity. The poor outcome of this disease has spurred the search for novel experimental therapies that can address and overcome the root biological phenomena associated with the lethality of this disease. The use of immunotherapy to bolster the otherwise impaired antitumor immune responses in glioma patients has received increasing attention. An effective treatment paradigm for malignant gliomas may eventually require a multifaceted approach combining two or more different immunotherapeutic strategies. Such scenarios may involve the use of local cytokine gene therapy to enhance glioma-cell immunogenicity in conjunction with dendritic cell-based active vaccination to stimulate systemic tumoricidal T-cell immunity. Given the heterogeneity of this disease process and the potential risk of immunoediting out a selected, treatment-refractory tumor cell population, the concurrent use of multiple modalities that target disparate tumor characteristics may be of greatest therapeutic relevance.

Gene therapy eradicating distant disseminated micro-metastases by optimal cytokine expression in the primary lesion only: Novel concepts for successful cytokine gene therapy

The most serious problem in current gene therapy is that clinical applications have often led to unsatisfactory results. Here we show novel concepts and crucial factors that have been missing for successful cytokine gene therapy. A clinically-relevant mouse model of primary and micro-metastatic osteosarcoma was generated by subcutaneously and intravenously injecting murine osteosarcoma LM8 cells, in which adenoviral gene transduction efficiencies were extremely low; current therapies remain less effective for such disseminated micro-metastases. A single injection of adenoviral vector encoding interleukin-2 gene (Ad.IL-2) was given only into the established primary tumor. Notably, antitumoral immunity was successfully elicited by IL-2 secretion from connective tissues adjacent to the primary tumor, and this immunity not only suppressed primary tumor growth but also eradicated disseminated micro-metastases in distant organs. Most importantly, not only minimal side effects but also maximal therapeutic effects were exerted only in the case of injecting the optimal (i.e., not the highest) dose of Ad.IL-2, because spleen injuries caused by excessive levels of circulating IL-2 might diminish the therapeutic effect. Although the narrow range of the optimal therapeutic expression level of IL-2 may be crucial, it was feasibly determined by serum IL-2 levels. Thus, a crucial factor for successful cytokine gene therapy is not the high gene transduction efficiency in the tumor, which has been generally recommended, but the use of the optimal therapeutic expression level. In conclusion, just a single injection of Ad.IL-2 into a primary tumor lesion, which is feasible, not invasive and cost effective, is potently therapeutic for distant disseminated micro-metastases, as long as the optimal therapeutic level is monitored. These novel concepts, which contradict those of previous studies, warn researches about the possible problems with the ongoing clinical cytokine gene therapy.

Localized combinatorial IL-4 and IL-10 gene therapy readjusts the balance of endogenous TH1/TH2 cytokines and induces the tolerance of the cardiac allografts

The balance of Th1/Th2 cytokine plays an important role in the allograft tolerance, however, its operating mechanism remains unknown. Using a functional cervical heterotopic rabbit heart transplant model, we tested the hypothesis that liposome-mediated ex vivo intracoronary interleukin-4 (IL-4) and IL-10 combined gene therapy may induce localized IL-4 and IL-10 over expression that may synergistically regulates endogenous Th1/Th2 expression and induces allograft tolerance. The mean survival of cardiac allograft was prolonged from 7 ± 1 days in Control Group (CG) to 14 ± 3 days in IL-4 gene therapy group (IL4G), 28 ± 7 days in IL-10 gene therapy Group (IL10G) and 135 ± 20 days in IL-4 and IL-10 combined gene therapy Group (IL4&10G). The rejection score in IL4&10G was significantly lower (2.2 ± 0.2, p<0.05) than that of CG (3.6 ± 0.2) and IL-10G (2.7 ± 0.3) in POD3-6. In single cytokine gene therapy caused an excessive localized exogenous cytokine gene and protein expression which significantly downregulated the expression of the same endogenous cytokine, IL-4 or IL-10 gene (p<0.01), but in a certain extent upregulated the other Th2 cytokine genes expression (p<0.01). In IL4&10G, endogenous IL-4 and IL-10 gene expression was decreased in the early stage, then increased at POD 18-20 and remained in a high level for a long period of time. The expression of Th1 cytokine genes, IL-1b and TNF-a, were significantly decreased by the synergistic inhibition of IL-4 and IL-10, and maintained in the low level in the late stage. IFN-g gene expression level was increased in IL4G, decreased in IL10G. The slightly decreased IFN-g gene expression level observed in the combined gene therapy group that could be resulted from antagonistic immunoregulatory effects of two cytokines. We conclude that localized overexpression of exogenous IL-4 and IL-10 induced by ex vivo gene transfer could act synergistically for readjusting the balance of endogenous Th1/Th2 cytokines that may contribute to the allograft tolerance.

Interleukin-12 genetic administration suppressed metastatic liver tumor unsusceptible to CTL

A cytokine gene therapy approach was conducted against metastatic lesions of cytotoxic T lymphocyte (CTL)-unsusceptible tumor in mice. The EBV-based and conventional plasmid vectors that encode murine interleukin-12 (IL-12) gene (pGEG.mIL-12 and pG.mIL-12, respectively) were intravenously transfected into the mice that had received a subcutaneous inoculation of M5076 sarcoma cells. The pGEG.mIL-12 transfection drastically suppressed the subcutaneous as well as hepatic metastatic tumors, resulting in significant prolongation of survival period of the animals. Although single administration with pG.mIL-12 was not effective, repetitive transfection with the plasmid significantly prolonged the longevity of the mice-bearing the metastatic liver tumors. Multiple transfection with either pGEG.mIL-12 or pG.mIL-12 also suppressed peritoneal carcinomatosis in mice that had been injected with M5076 cells into the peritoneal cavity. It was suggested that a high level IL-12 production elicited by the intravenous delivery of the cytokine gene may be quite effective in inhibiting metastatic and CTL-unsusceptible neoplasms.

Tumor irradiation followed by intratumoral cytokine gene therapy for murine renal adenocarcinoma.

To circumvent the toxicity caused by systemic injection of cytokines, cytokine cDNA genes encoding the human interleukin IL-2 cDNA (Ad-IL-2) and murine interferon IFN-gamma gene (Ad- IFN-gamma) were inserted into adenoviral vectors. These constructs were used for intratumoral gene therapy of murine renal adenocarcinoma Renca tumors. Treatment with three doses of Ad-IL-2 or Ad- IFN-gamma, given a day apart, was more effective than single-dose gene therapy. We found that tumor irradiation enhanced the therapeutic efficacy of Ad-IL-2 and Ad-IFN-gamma intratumoral gene therapy. Tumor irradiation, administered 1 day prior to three doses of Ad-IL-2 treatment, was more effective than radiation or Ad-IL-2 alone, resulting in tumor growth arrest in all mice, increased survival and a consistent increase in complete tumor regression response rate. Complete responders rejected Renca tumor challenge and demonstrated specific cytotoxic T-cell activity, indicative of specific tumor immunity. The effect of radiation combined with three doses of Ad-IFN-gamma was less pronounced and did not lead to tumor immunity. Histological observations showed that irradiation of the tumor prior to gene therapy increased tumor destruction and inflammatory infiltrates in the tumor nodules. These findings demonstrate that tumor irradiation improves the efficacy of Ad-IL-2 gene therapy for induction of antitumor immune response.

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues.

The augmentation and optimization of specific targeted transgene expression systems are important strategies for clinical research into gene therapy and DNA vaccination, due to safety considerations. In this study, we introduced 3' untranslated regions and transcriptional control modifications and direct tandem or combinational vector design strategies into a number of specific cytokine cDNA expression plasmids. The experiments were performed in parallel using both in vivo and in vitro transgene expression systems. In vivo studies were carried out using gene gun delivery of test vectors into mouse skin tissues. A combination of specific cell lines and fresh cell explants were used for in vitro and ex vivo transgene expression assay systems. The results from these comparative experiments demonstrated that a number of molecular biology manipulations can be readily adapted to define and significantly enhance the level or/and duration of transgene expression for a group of clinically relevant cytokine genes, with very similar effects for both in vivo and in vitro test systems. This cytokine transgene expression system may offer a favorable means for improving the efficiency of cytokine gene therapy and DNA vaccines in future clinical studies.

Effect of interleukin-7 gene transfection into ovarian carcinoma cell line SKOV3 in vitro and in vivo

Objective. To investigate the effect of interleukin-7 (IL-7) gene transfection into an established ovarian carcinoma cell line (SKOV3) in vitro and evaluate the tumorigenicity of SKOV3-IL-7 in severe combined immunodeficient (SCID) mice. Methods. IL-7 gene was transfected into SKOV3 cells by liposome. IL-7 mRNA and protein of SKOV3-IL-7 and their parental control cells were detected by reverse transcriptive-polymerase chain reaction (RT-PCR) and Western blot, respectively. The levels of IL-7, IL-2, TNFα and TGFβ1 in the supernatant were detected by ELISA. The cell cycle, HLA-ABC, HLA-DR and ICAM-1 expressions were assayed by flow cytometry. The sensitivities of tumor cells to lymphokine-activated killer (LAK) cells were measured by lactate dehydrogenase (LDH) release assay. Tumorigenicities of SKOV3-IL-7 and their parental cells in SCID mice were evaluated by macro- and histological examination, while IL-7 expression and secretion were detected by immunohistochemistry and ELISA. Results. IL-7 mRNA and protein were detectable in SKOV3-IL-7 only. ICAM-1 expression was significantly higher and TGFβ1 level in culture supernatants was significantly decreased in SKOV-IL-7, all other variables remained unchanged. The proliferative activity and cell cycle of SKOV3-IL-7 were unchanged. The cytotoxic sensitivity of SKOV3-IL-7 to LAK cells was significantly higher. Both gene-transfected and nontransfected SKOV3 cells were successfully inoculated into the peritoneal cavities of SCID mice. IL-7 proteins in plasmas and tumor tissues were detectable only in SCID mice inoculated with SKOV3-IL-7. The IL-7 engineered murine tumor models revealed better general aspects, reduced tumor development and dissemination. Conclusions. IL-7 gene transfection into SKOV3 cells downregulates TGFβ1 secretion, upregulates ICAM-1 expression and enhances sensitivity to LAK cells in vitro. The tumorigenicity of IL-7 engineered cells in SCID mice is reduced. These findings may offer support to the development of cytokine gene therapy for ovarian carcinoma.

Cytokine Immuno-Gene Therapy for Treatment of Brain Tumors

The prognosis for patients with an intracerebral (i.c.) neoplasm is poor. Conventional treatments such as surgery, radiation therapy and chemotherapy have done little to affect long-term survival, and new methods of treatment are urgently needed. In this report approaches involving cytokine gene therapy in treatment of malignant brain tumors are reviewed and contrasted to a strategy developed in this laboratory involving the use of allogeneic cells genetically modified to secrete cytokines. In our studies, mice with an i.c. glioma, melanoma or breast carcinoma treated solely by intratumoral injections with allogeneic cells genetically modified to secrete interleukin-2 (IL-2) were found to survive significantly longer than mice in various control groups. The anti-tumor response was mediated predominantly by T-cell subsets (CD8+ and NK/LAK cells). The injections resulted in the killing of only the neoplastic cells; non-neoplastic cells were unaffected. Experiments involving treatment of animals with i.c. tumor using subcutaneous injections of cytokine-secreting allogeneic cells in the presence of tumor antigens demonstrated no effect in prolonging survival in spite of the development of a vigorous systemic anti-tumor immune response. Of special interest, mice injected intracerebrally with the cytokine-secreting allogeneic cells alone exhibited no neurologic defect and there were no adverse effects on survival. The injection of cytokine-secreting allogeneic cells into the microenvironment of an i.c. tumor is hypothesized to induce an anti-tumor immune response capable of prolonging survival. This pre-clinical animal data directly translates into clinical treatments for patients with a malignant i.c. tumor.

Interleukin (IL)-21 and IL-15 genetic transfer synergistically augments therapeutic antitumor immunity and promotes regression of metastatic lymphoma

IL-21 supports proliferation of mature T and B cells and facilitates expansion and maturation of natural killer (NK) cells in synergy with IL-15. However, the biological implications of IL-21 in vivo have not been fully elucidated. IL-21 and IL-15 expression plasmids were intravenously injected under high pressure into the tail veins of mice, which were subsequently challenged by an intravenous injection of RLmale1 lymphoma cells. The IL15 gene transfection significantly reduced the numbers of metastatic tumor foci in the liver. In contrast, when IL21 and IL15 genes were cotransfected, complete regression was achieved in 80% of the mice. The cytokine gene therapy was also performed in mice that had been intravenously inoculated with the tumor cells. Forty percent of mice that received a single injection of a mixture of cytokine genes successfully rejected the preestablished metastatic lymphoma and showed tumor-free survival for more than 300 days. IL-21 significantly elevated the cytotoxic T lymphocyte activity in the spleens of tumor-inoculated mice, while the two cytokines augmented NK killing activity in a synergistic manner. These results strongly suggest that the codelivery of IL-21 and IL-15 elicits powerful antitumor immune responses, resulting in marked therapeutic efficacy against metastatic tumors.

Keeping Obesity and Metabolic Syndrome at Bay with Central Leptin and Cytokine Gene Therapy

The incidence of obesity and metabolic syndrome along with the annual cost of treating obesity and related metabolic disorders are rising steeply world wide. In this article, the potential of gene therapy application directly into the hypothalamus, a CNS site involved in integration of energy homeostasis, as a therapeutic modality is reviewed. We engineered a non-immunogenic, non-pathogenic recombinant adeno-associated virus vector to encode leptin, leptin receptor, or the cytokines, leukemia inhibitory factor and ciliary neurotrophic factor. A single injection of virus vectors encoding any of these genes either intraventricularly or into selected hypothalamic sites of wild type or genetically obese rodents curtailed the gradual age-related and rapid high fat diet-induced obesity for long periods. Parameters of metabolic syndrome, hyperleptinemia, hyperinsulinemia and hyperlipidemia were normalized along with fat depletion and increased non-shivering thermogenic energy expenditure either alone or in association with voluntary reduction in food intake. Operation of hypothalamic appetite and energy regulating network was correspondingly modified to conform with the anorectic and obesity suppressing effects of central leptin and cytokine gene therapy. These results advocate central gene therapy as a viable alternative to curb the rising tide of obesity and associated metabolic diseases with or without a voluntary reduction in food intake. Keywords: gene therapy, cytokines, leptin, viral vectors, adeno-associated virus, hypothalamus, adiposity, metabolic syndrome, hyperinsulinemia, type 2 diabetes

Gamma interferon transduced 9L gliosarcoma. Cytokine gene therapy and its relevance to cellular therapy with alloreactive cytotoxic T lymphocytes.

In earlier studies, we demonstrated that intratumoral infusions of alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the major histocompatibility complex (MHC) antigens of the host, effectively retarded the intracranial growth of Fischer 9L gliosarcoma. We further demonstrated that continuous in vitro exposure to gamma-interferon (gammaIFN) upregulates MHC on 9L gliosarcoma cells and that they were better targets of anti-Fischer aCTL. We hypothesized that the efficacy of cellular therapy with aCTL could be further improved by in situ transduction of the tumor with retroviral vectors coding for gammaIFN, which would generate continuous secretion of the cytokine and maintain upregulated MHC expression by the tumor cells. 9L gliosarcoma and Herpes simplex virus thymidine kinase (tk) transductants of those cells were transduced with a retrovirus carrying the murine gammaIFN gene. By limiting dilution, clones of these cells, designated 9Lgamma 7, 9Lgamma tk8, and 9Lgamma tk10, which produced similar levels of gammaIFN (383-411 ng gammaIFN/10(6) cells/24 h) were isolated. The production of gammaIFN by one clone, 9Lgamma 7, was stable when monitored over 6 weeks in vitro. The clones also demonstrated upregulated MHC class I expression, and the tk-transduced clones maintained their sensitivity to ganciclovir. Compared to the wildtype cells, 9Lgamma 7 had approximate 6- and 1.5-fold increases in the relative antigen densities of MHC I and II, respectively. Addition of exogenous gammaIFN to 9Lgamma 7 cultures did not significantly increase the MHC expression. In cytotoxicity assays, 9Lgamma 7 cells, or 9Lgamma 7 incubated with exogenous gammaIFN, were better targets of aCTL than the parental 9L cells. The growth rate of 9Lgamma-transduced cells was decreased compared to the wildtype cells both in vitro and in vivo. Proliferation studies with transwell plated 9L, 9Lgamma 7, and 9Lgamma tk10 cells in various combinations revealed that the secreted cytokine itself caused a decrease in proliferation. However, the transduced cells exhibited a much reduced growth rate, which likely was a consequence of redirected metabolic activity of the cells. In vivo growth of the 9L and 9Lgamma 7 tumors in rat brains given identical inoculums similarly demonstrated significantly reduced 9Lgamma 7 tumor volumes at various timepoints, indicative of slower growth of the gammaIFN-producing tumors.