Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors.
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We recently showed that retroviral vectors can be targeted through protease substrate interactions. Infectivity is blocked by a polypeptide fused to the viral envelope glycoprotein (SU) and is restored when a protease cleaves the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity is achieved by engineering the sequence of the linker. Here, using two different matrix-metalloproteinase (MMP)-activatable vectors, we demonstrated highly efficient and selective transduction of MMP-rich target cells in a heterogeneous cell population. In vivo, the MMP-targeted vectors showed strong selectivity for MMP-rich tumor xenografts. Protease-activatable vectors offer new possibilities for in vivo targeting of gene delivery. (+info)
Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection.
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Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-gamma, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1-3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-gamma response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies. (+info)
Interleukin 2 gene therapy of colorectal carcinoma with autologous irradiated tumor cells and genetically engineered fibroblasts: a Phase I study.
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The purpose of this study was to determine the safety, toxicity, and antitumor immune response following S.C. immunizations with a mixture of irradiated, autologous tumor cells and autologous fibroblasts that were genetically modified to express the gene for interleukin 2 (IL-2) in patients with colorectal carcinoma. Ten patients were treated with a fixed dose of tumor cells (10(7)) and escalating doses of fibroblasts secreting IL-2 (per 24 h): 100 units (three patients), 200 units (three patients), 400 units (three patients), and 800 units (one patient). Pre- and posttreatment peripheral blood mononuclear cells were evaluated for evidence of antitumor immune responses. Fatigue and/or flu-like symptoms were experienced by seven patients and delayed-type hypersensitivity-like skin reactions were observed at the sites of the second or subsequent vaccinations in five patients. Low frequencies of tumor cytotoxic T-cell precursors (range, 1/190,000-1/1,320,000 peripheral blood mononuclear cells) were detected prior to therapy in four of seven patients. There was a 5-fold increase following treatment in the frequency of tumor cytotoxic T-cell precursors in two of six evaluable patients. Some patients with colorectal cancer have low frequencies of tumor cytotoxic T-cell precursors that may be increased by this well-tolerated form of IL-2 gene therapy, which warrants continued clinical evaluation. (+info)
Lessons from genetically engineered animal models. IV. Nitric oxide synthase gene knockout mice.
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Nitric oxide is a ubiquitous molecule implicated in a variety of biological processes. The specific action of nitric oxide depends on its enzymatic sources, namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), each having distinct tissue localization. Conventional pharmacological antagonists could not distinguish these enzymes or provide models of chronic nitric oxide depletion in whole animals. Several lines of knockout mice have been generated to distinguish the roles of nitric oxide from each enzyme: nitric oxide from nNOS is a major inhibitory neurotransmitter, nitric oxide from eNOS regulates blood flow under physiological conditions, and nitric oxide from iNOS causes hypotension during severe inflammatory conditions. Moreover, the nitric oxides from each isoform have different roles in tissue injury and inflammation. Studies of NOS-deficient animals have also identified redundant and compensatory pathways and revealed the consequences of life-long deficiency of these enzymes. The nNOS-deficient mice develop gastric dilation and stasis, the eNOS-deficient mice develop hypotension and lack vasodilatory responses to injury, and iNOS-deficient mice are more susceptible to inflammatory damage but more resistant to septic shock. (+info)
A host-vector system for analysis and manipulation of rifamycin polyketide biosynthesis in Amycolatopsis mediterranei.
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Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5' flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation. (+info)
Expression of a soluble form of CTLA4 on macrophage and its biological activity.
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Interaction between cytotoxic T lymphocyte-associated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte, including antigen-specific T-cell activation and induction of autoreactive T-cell. We describe here the first introduction of a murine soluble CTLA4 gene, CTLA4Ig, to Mm1 cells, a macrophagic cell line. CTLA4Ig was successfully expressed on Mml cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies. The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture. A high CTLA4Ig producing macrophagic cell line was obtained. As Mm1 cells were regarded as difficult for gene transfection and there had so far been no report on expression of CTLA4Ig gene on Mm1 cells, these results suggested that the CTLA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B7 molecule interaction in both macrophage and T-cell. (+info)
Genetically modified organisms: an analysis of the regulatory framework currently employed within the European Union.
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BACKGROUND: Genetic engineering technology is starting to bring many commercial products to the market. These genetically modified organisms (GMOs) and their derived products are subject to topical debate as to their benefits and risks. The strengths and weaknesses of the regulatory framework that controls their development and application is central to the question of whether this technology poses significant risk to the public health during this critical phase of its evolution. METHODS: A critical review was carried out of the legal framework regulating the contained use, deliberate release and some aspects of consumer protection relevant to the control of GMOs in Europe and the United Kingdom. RESULTS: The current legal framework is failing to provide a speed of adaptation commensurate with the development of the science of genetic engineering; failing to properly respond to democratic control; failing to resolve significant conflict between the protection of free markets and protection of public health and the environment; and failing to implement obligations on biodiversity. CONCLUSION: The present legal framework must be replaced. Current European Union proposals for new standards of regulation are welcome, but provide only for further incremental change, and do not address some significant fundamental flaws in our current laws. (+info)
A genetically engineered cell line resistant to subgroup J avian leukosis virus infection (C/J).
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A cell line (DF-1 inverted question markJ) expressing the envelope protein isolated from the ADOL-Hc1 strain of the avian leukosis virus subgroup J (ALV-J) was used to analyze receptor interference to six different isolates of ALV-J as well as ALV subgroups A-D. The traditional gag-specific enzyme-linked immunosorbent assay (ELISA) as well as flow cytometry was used to evaluate viral infection. The parental cell line (DF-1) was susceptible to all ALV subgroups tested while the DF-1 inverted question markJ cell line was selectively resistant to the subgroup J isolates. The DF-1 inverted question markJ cell line was resistant to infection by all six ALV-J isolates as determined using the gag-specific ELISA. There was no interference with the other ALV subgroups (A-D) induced by the expression of the ADOL-Hcl envelope. The ALV-J isolates used in this analysis are serologically distinct when analyzed by flow cytometry. Convalescent sera to ADOL-Hcl cross-reacts with all of the ALV-J isolates tested; however, sera to HPRS-103 did not bind to four of the six isolates. Based on the intensity and differential binding of these antisera using flow cytometry, the six ALV-J isolates used can be grouped into four categories. Thus the DF-1 inverted question markJ cell line is resistant to infection by a serologically and genetically diverse group of ALV-J isolates and should be useful as a diagnostic tool. (+info)