Murine Interferon-alpha Research Articles

A Semliki Forest virus vector engineered to express IFNα induces efficient elimination of established tumors

Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNα). IFNα displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNα, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNα both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNα by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.

Efficacy Assessment of Interferon-Alpha–Engineered Mesenchymal Stromal Cells in a Mouse Plasmacytoma Model

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules that can inhibit tumor cell proliferation and enhance the antitumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-α) gene (BM-MSCs/IFN-α) to assess in a mouse plasmacytoma model the efficacy of this approach toward neoplastic plasma cells. We found that IFN-α can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-α significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The antitumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-α did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-α are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.

Semi-mechanistic pharmacodynamic modelling of gene expression and silencing processes

Background Suppressed expression of transgene is a major obstacle in gene therapy. Understanding of the mechanisms involved in expression and silencing of exogenous genes is required to overcome gene therapy hurdles. Purpose To develop a semi-mechanistic model describing the effects of transgenes over the activity of an expression cassette. Methods Twelve Balb/c mice received 40 μg of plasmid DNA. Animals were assigned to one of the following treatments: (I) 20 μg of the plasmid expressing luciferase (pEF-Luc) and 20 μg of “empty” plasmid; (II) pEF-Luc (20 μg) and 20 μg of plasmid expressing murine interferon alpha (IFNα); and (III) pEF-Luc (20 μg), and 20 μg of plasmid expressing β-galactosidase (pCMVβ). The expression of luciferase over time, quantified by a noninvasive method, was used as a measured of pEF-Luc activity and modelled using NONMEM. Results The selected model suggests the co-existence of two forms of active DNA differing in their transcription efficiencies. The core model was expanded to describe reversible and irreversible silencing processes, induced by the coexpression of IFNα or β-galactosidase, respectively. Conclusion Coupling noninvasive in vivo imaging and mathematical modelling allows quantitative description of gene transfer, providing a tool to select the best regulatory elements to construct a therapeutic expression cassette.

Characterization of murine interferon-alpha 12 (MuIFN-α12): Biological activities and gene expression

Interferon alpha (IFN-α) belongs to the type I interferon family and consists of multiple subtypes in many species. In the mouse, there are at least 14 IFN-α genes and 3 IFN-α pseudogenes, the most recently identified of which are murine interferon-alpha 12 (MuIFN-α12), MuIFN-α13 and MuIFN-α14. To further study the biological activities of MuIFN-α12, we have produced a recombinant MuIFN-α12 (rMuIFN-α12) protein using COS-1 cells. rMuIFN-α12 was found to inhibit the growth of murine myeloid leukemia JCS cells. Flow cytofluorometric analysis with propidium iodide staining showed that the growth inhibitory activity of rMuIFN-α12 may be caused by the induction of apoptosis. Flow cytofluorometric analysis also revealed that rMuIFN-α12 was able to up-regulate the expression of MHC-I on both JCS cells and primary macrophages. Functional studies indicated that a MuIFN-α12 transgene could induce an anti-viral state in L929 cells against Influenza A virus. Moreover, expression of MuIFN-α12 was not detectable by RT-PCR in untreated, Influenza A virus infected, polyI:polyC induced L929 cells, or in a wide range of normal murine tissues. Taken together, this data shows that MuIFN-α12 is a protein with all the biological traits of a type I IFN.

Gene therapy of ovarian cancer with IFN-α-producing fibroblasts: comparison of constitutive and inducible vectors

Ovarian cancer represents a malignancy suitable for cell and gene therapy approaches owing to its containment within the peritoneal cavity, even at advanced tumor stages. As regulation of transgene expression would be preferable for conducting clinical trials for reasons of safety, we investigated whether intraperitoneal (i.p.) administration of retroviral vector-transduced fibroblasts encoding murine interferon-alpha (IFN-alpha) could have therapeutic activity, and compared its effect with the antitumor effects of fibroblasts producing IFN-alpha under a rapamycin analogue (AP21967)-inducible promoter. Human and murine fibroblasts were recruited into the solid component of transplantable ovarian cancer-grown i.p. in severe combined immunodeficiency mice. Multiple administrations of fibroblasts producing IFN-alpha in a constitutive manner showed therapeutic efficacy, leading to significant prolongation of survival in the majority of animals, associated with inhibition of tumor angiogenesis. Compared to cells transduced by the constitutive vector, fibroblasts transduced by the inducible vector released twofold higher IFN-alpha levels in vitro, following induction by AP21967, and production of the cytokine was under pharmacologic control both in vitro and in vivo. However, these cells elicited only modest therapeutic effects in vivo. Overall, these findings indicate that intracavitary IFN-alpha gene therapy using engineered fibroblasts requires sustained production of IFN-alpha to achieve durable antitumor effects.

Interferon-α Gene Therapy by Lentiviral Vectors Contrasts Ovarian Cancer Growth Through Angiogenesis Inhibition

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Combination nonviral cytokine gene therapy for head and neck cancer.

To establish the feasibility and efficacy of combination nonviral murine interferon-alpha (mIFN-alpha)and murine interleukin-2 (mIL-2) or murine interleukin-12 (mIL-12) gene therapy for head and neck squamous cell carcinoma in a murine model. Randomized controlled studies in a murine head and neck cancer model were performed to assess antitumor responses, secondary cytokine expression, and both natural killer (NK) cell and cytolytic T-cell (CTL) activity. Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with polymer-formulated murine interferon-alpha (mIFN-alpha), lipid-formulated mIL-2, and polymer-formulated mIL-12 alone or in combination. Primary and secondary cytokine expression,NK cell activity, and CTL activity were assayed. The use of mIFN-alpha gene therapy in combination with either mIL-2 or mIL-12 resulted in significant antitumor effects as compared with each of the single cytokine and control treatment groups (P = .002). Increased levels of NK cell activity and tumor specific CD8+ cytotoxic T-lymphocyte activity were found in the combination mIFN-alpha and mIL-2 or mIL-12 groups. Augmented immune responses correlated with clinical antitumor effects. The present study demonstrates that mIL-2 or mIL-12 augments tumor inhibition from mIFN-alpha and increases activation of NK and CD8+ T cells. These data support further investigation of polymer and lipid mediated delivery of cytokine genes for head and neck cancer.

Combination therapy with glatiramer acetate (copolymer-1) and a type I interferon (IFN-?) does not improve experimental autoimmune encephalomyelitis

We sought to determine whether combinations of glatiramer acetate and parenteral or ingested type I interferon were synergistic in experimental autoimmune encephalomyelitis. Glatiramer acetate, subcutaneous murine interferon-alpha, or ingested murine interferon-alpha individually improved clinical scores. In contrast, glatiramer acetate in conjunction with either subcutaneous or ingested interferon-alpha did not improve clinical scores compared with control. These data suggest that clinical trials designed to test a possible synergistic effect of glatiramer acetate and type I interferon in humans should be designed to detect possible adverse effects of this combination of immunomodulatory agents.

Effects of oral administration of interferon-alpha on antibody production in mice with induced tolerance.

In vivo systemic effects and the immunomodulating potential of the oral administration of murine interferon-alpha (IFN-alpha) were investigated through mRNA expression of both IFN-alpha-inducible factors, interferon regulatory factor-1 (IRF-1) and 2,5-adenylate synthetase [2-5(A) synthetase] and 2-5(A) synthetase enzymatic activity in spleen and antibody production. The daily administration of IFN-alpha (0.1, 1, 10, and 100 IU/body) for 1 week augmented IRF-1 and 2-5(A) synthetase mRNA expression levels, as well as 2-5(A) synthetase enzymatic activity in spleen cells but not in cervical lymph nodes. The in vivo immunomodulating potential of the oral administration of IFN-alpha was also evaluated through antibody production in mice with induced tolerance. Ovalbumin (OVA) was administered intraperitoneally (i.p.) to induce systemic antibody production on day 0 when OVA feeding was initiated. The OVA was fed every 2-3 days for a total of 14 doses to suppress serum antibody levels. Oral administration of murine IFN-alpha was initiated on day 0 and was continued for 5 consecutive days weekly for 5 weeks (24 doses). On every sampling date (days 10, 17, 24, and 32), specific antibody levels in the IFN-alpha-administered groups were significantly higher than those in the control (nonadministered) group. This was especially noted in early phases (days 10 and 17) of antibody production when the levels of antibody in serum from the IFN-alpha-administration groups were equivalent to those of the nontolerance group. Altogether, it is suggested that oral use of IFN-alpha can elicit immunomodulating actions (e.g., antibody levels) by affecting the systemic immune system(s).

INGESTED INTERFERON ALPHA INDUCES Mx mRNA

We have demonstrated that ingested murine interferon alpha (IFN-α) suppressed clinical relapse in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE), decreased inflammation and suppressed the adoptive transfer of EAE, and is a biological response modifier in patients with multiple sclerosis. We examined the relative levels of the Mx mRNA signal using semiquantitative reverse transcription-polymerase chain reaction analysis on splenocytes from mice and peripheral blood mononuclear cells from man after IFN-α ingestion. Both mice and man demonstrated inducible levels of Mx mRNA after ingesting IFN-α. Murine spleen T cells and CD8+T cells also demonstrated upregulation of Mx mRNA. Murine whole splenocytes demonstrated upregulation of Mx mRNA after IFN-α ingestion of 10 and 100U, but not after 0, 1000, 5000U. Ingested IFN-α acts via established pathways of type 1 IFN signalling.

A gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon alpha.

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon alpha (mIFN-alpha) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-alpha pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-alpha pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8(+) T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.

Ingested interferon alpha suppresses type I diabetes in non-obese diabetic mice.

Type I diabetes mellitus is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The non-obese diabetic mouse is a model of the human autoimmune disease Type I diabetes [1-3]. We have previously shown that ingested type 1 interferon inhibits chronic relapsing experimental autoimmune encephalomyelitis and the adoptive transfer of experimental autoimmune encephalomyelites by T cells, and decreases both antigen-specific and mitogen-induced pro-inflammatory cytokine secretion in this disorder. We therefore tried to determine whether ingested murine interferon alpha inhibits insulinitis and suppresses Type I diabetes mellitus in non-obese diabetic mice. Murine interferon alpha, given daily, decreased islet inflammation and suppressed diabetes. It increased the concanavalin A and ionomycin plus myristic acid palmitic ester-induced production of interleukin 4 and 10 and interferon gamma-secretion in spleen cells from treated mice. Adoptive transfer of unstimulated splenocytes secreting interleukin 4 and interleukin 10 from fed interferon alpha donors suppressed spontaneous diabetes mellitus in recipients. The protective effect of adoptively transferred unstimulated splenocytes shows the presence of ingested interferon alpha-activated regulatory splenic cell populations that may work via increased interleukin 4 or interleukin 10 production. Ingested interferon alpha administered during vulnerable periods in at-risk populations may potentially provide a continuous, convenient, non-toxic and effective treatment for Type I diabetes.

Oral-mucosal administration of IFN-alpha potentiates immune response in mice.

We studied the effects of oral-mucosal administration of murine interferon-alpha (Mu-IFN-alpha) on immune responses and infection with vaccinia virus (VV) in mice. When Mu-IFN-alpha was administered to sheep red blood cell (SRBC)-sensitized mice for 4 or 5 days, Mu-IFN-alpha significantly enhanced delayed-type hypersensitivity (DTH) and antibody production, with maximum enhancement of each at 1 IU/body. To investigate the antiviral effect of oral-mucosal Mu-IFN-alpha, mice were infected with VV, and Mu-IFN-alpha was administered for 15 days. Pocks were observed in the tail skin of infected mice, and Mu-IFN-alpha at doses of 1, 10, and 100 IU/body significantly suppressed pock formation. Also, VV-specific cytotoxic T cells (CTL) were observed in the spleen from the same mice at 7 days after infection, and Mu-IFN-alpha enhanced CTL activity at doses above 1 IU/body. These results suggest that the oral-mucosal Mu-IFN-alpha may have potentiating effects on cellular and humoral immune responses, which may contribute to its effects against VV.

Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-alpha 1 and herpes simplex thymidine kinase followed by ganciclovir.

Transduction of the murine interferon-alpha (IFN-alpha) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN-alpha-producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN-alpha-producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN-alpha-FLC did not result in any antitumor effect. The therapeutic potential of IFN-alpha-producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in > or = 70% of the animals after injection of high numbers (10(7)) of IFN-alpha-producing tk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of control tk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

Oral administration of human or murine interferon alpha suppresses relapses and modifies adoptive transfer in experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalitis (CR-EAE) is an inflammatory process of the central nervous system (CNS) that closely resembles the human disease multiple sclerosis (MS). EAE was induced in SJL/J mice and following recovery from the initial attack, animals were fed varying doses of human or murine interferon alpha (IFN-α), or mock IFN three times per week. After relapse, concanavalin A-activated spleen cells were transferred adoptively from orally fed animals into recipient animals. Oral administration of human or murine IFN-α suppressed relapse in actively immunized animals, modified adoptive transfer of EAE, and decreased mitogen/antigen proliferation and IFN-γ secretion in both donors and recipients. IFN-α acts orally by modifying the encephalitogenicity of donor spleen T cells.

Amino acid substitutions alter the tissue distribution of murine interferon-alpha 1.

Novel analogs created by site-directed mutagenesis of murine interferon-alpha 1 (IFN-alpha 1) were used to examine the effect of alterations in structure and biological activity of murine IFN-alpha 1 on tissue distribution in mice. The analogs were biosynthetically labeled with [35S]methionine using a cell-free transcription-translation system and injected intravenously into adult male BALB/c mice. Levels of murine IFN-alpha 1 (dpm/gram wet weight) were highest in the liver, spleen, kidney, and lung, lower in the heart, and quite low in testis, brain, skin, and muscle. The tissue distribution of the analogs differed from that of murine IFN-alpha 1. In general, analogs with reduced antiviral activity showed reduced uptake by the spleen and lung. The amount in the kidney of the analog R33E, which has no detectable antiviral activity in vitro, was substantially higher than that of native IFN, suggesting a greater rate of excretion of this analog. An analog of human IFN-alpha 4, which had increased antiviral activity on murine cells, showed increased uptake in the liver, spleen, and lung. These findings, together with the results of a previous study using autoradiography (Johns et al., 1990, Cancer Res. 50, 4718-4723) indicate that nonspecific uptake by parenchymal cells in the liver, spleen, and lung is unaffected by changes in antiviral activity, while specific, receptor-mediated localization of IFN in regions rich in macrophages is reduced in accordance with the reduction in antiviral activity.

Identification of a novel virus-responsive sequence in the promoter of murine interferon-alpha genes

We have previously shown that the infection of mouse L-cells with Newcastle disease virus activates transcription of the alpha 4 but not the alpha 6 interferon gene and that the induction is mediated by a 35-base pair inducible element (IE) found in the alpha 4 promoter (-109 to -75). In the present study, we show that the inactivity of the alpha 6 promoter can be mapped to 2 out of 6 nucleotides in which the alpha 6 differs from alpha 4 IE. The symmetrical sequence, GTAAAGAAAGT (-103 to -93), present in the alpha 4 IE is essential for its inducibility and binding of nuclear protein(s) to the alpha 4 IE.

Relative Antiviral Activity ofIn Vitro-Synthesized Murine Interferon-α4and -α1

Murine interferon-alpha 4 (MuIFN-alpha 4) is notable among the MuIFN-alpha subtypes because it lacks 5 amino acids corresponding to positions 103-107 of the other subtypes, yet is the most highly expressed subtype. Site-directed in vitro mutagenesis has been used to modify the genes coding for MuIFN-alpha 4 and MuIFN-alpha 1. The modifications have allowed (i) the in vitro expression of the mature form of each MuIFN-alpha subtype and (ii) the insertion of five amino acids, corresponding to amino acid positions 103-107 of MuIFN-alpha 1, into the MuIFN-alpha 4 sequence. In contrast to previously published data MuIFN-alpha 4 and MuIFN-alpha 1 show only a twofold difference in antiviral activity, with MuIFN-alpha 4 being the more active subtype. In keeping with this observation, it was also found that insertion of the five "missing" amino acids into MuIFN-alpha 4 resulted in an analogue MuIFN-alpha with antiviral activity equivalent to MuIFN-alpha 1. It may be inferred from this work that the deletion of amino acids 103-107 (QVGVQ) is solely responsible for the difference in antiviral activity between MuIFN-alpha 4 and MuIFN-alpha 1.

Positive modulation of hemoglobin, heme, and transferrin receptor synthesis by murine interferon-alpha and -beta in differentiating Friend cells. Pivotal role of heme synthesis.

Administration of highly purified preparations of murine interferon (IFN)-alpha 1, -alpha 4, -alpha 6, or -beta to Friend leukemia cells induced to differentiate by dimethyl sulfoxide leads to a 100% increase of benzidine-positive (B+) cells. Different efficiencies for the two IFN species have been observed; a 10-fold higher dose of IFN-alpha is needed for stimulation of hemoglobin production and inhibition of cell growth as compared with IFN-beta. Both species of IFN induce a substantial increase in heme, hemoglobin, and transferrin receptor levels. In vitro run-on transcription assays indicate that IFN-beta moderately stimulates transcription of the alpha-globin gene but not the transferrin receptor gene. It is postulated that IFN induces the enhancing effect on differentiation via a marked increase of heme synthesis and number of transferrin receptors, which in turn leads to an enhancement of globin chain synthesis. In this regard, the negative feedback reported in a variety of other cell types for the regulation of transferrin receptor expression by heme does not seem to be operative in maturing Friend erythroleukemia cells, which present evidence for a positive mechanism.

Chemiluminescence in a macrophage cell line modulated by biological response modifiers.

We have studied a murine macrophage cell line, J774, and found these cells capable of a zymosan-triggered chemiluminescent oxidative burst. Such activity was enhanced by preincubation with Corynebacterium parvum (CP), bacillus Calmette-Guerin, and lipopolysaccharide (LPS). Under similar conditions, CP and LPS were shown to enhance J774-mediated tumor cell lysis. We have also demonstrated that murine interferon alpha + beta rendered J774 cells more sensitive to the actions of CP and LPS. These results indicate that J774 cells may be useful for the in vitro evaluation of biological response modifiers as well as the study of oxygen radical production by macrophages.