Targeting malignant B cells of lymphoma and leukemia with genetically engineered T-cell clones.
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Animals↗
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Results for “Cell engineering”
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[Transduction of the IL-2 gene into human gastric cancer cell: an experimental study].
We evaluated the possibility of inducing a productive transfer of the IL-2 gene into human gastic cancer cells (GCC) and assessed the phenotypic and proliferative changes generated in the nude mice. The plasmid vector (BMGNeo-IL-2) carrying the human IL-2 gene was used to transduce the SGC-7901 GCC line by the lipofectin reagent. The IL-2 gene was analyzed by Southern blot and productive IL-2 release using IL-2-dependent CTLL. Cytotoxicity of LAK cells was tested by MTT colorimetry. The kinetics of in vitro growth and proliferation of parental and engineered cells were also measured. Parental and IL-2 gene transduced GCC were injected into nude mice. The tumorigenic potential of IL-2 gene-transfected GCC was evaluated by examining their in vivo growth in nude mice. The productive insertion of the IL-2 gene was achieved in SGC-7901. The amounts of IL-2 constitutively released by the engineered neoplastic cells ranged from 20 to 131 U/ml of IL-2 produced from 10(6) cells in 24 hours. Transduction of GCC with IL-2 gene did not modify the morphology and growth rate. IL-2 gene transfected cells demonstrated increased susceptibility to cell killing by LAK cells. IL-2 producing cells lost their tumorigenicity as evidenced by failure to grow in nude mice. The results demonstrate that IL-2 gene can be productively transduced into human gastric cancer cells without modifying their morphology and growth rate and this transduction leads to reduced or abrogated in vivo tumorigenic potential.
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[Biomaterials].
Biomaterials are essential components in the development of artificial organs. Synthetic polymers are widely used for a number of biomedical applications: a) as medical supplies, b) devices to support or replace malfunctioning body parts or c) systems with a local therapeutic function. The design of biomaterials has developped over the past three decades from devices with a mechanical function, over systems with a dedicated function (degradable, drug releasing, surface modified) to devices which are tailor made to interact in a smart way with the biological environment. At present polymers are attractive materials to construct scaffolds for tissue regeneration and regenerative medicine. The material design is adapted to the requirements for cell ingrowth, cell migration, angiogenesis etc. This is mainly controlled by the surface topography and chemistry that play a key role in the interfacial material-cell communication. This remarkable development is the direct result of a multi-disciplinary approach, combining material science with engineering, cell biology, molecular biology, medicine and regulatory guidelines.
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Microfabricated airflow nozzle for microencapsulation of living cells into 150 micrometer microcapsules.
Microencapsulation of genetically engineered cells has attracted much attention as an alternative nonviral strategy to gene therapy. Though smaller microcapsules (i.e. less than 300 microm) theoretically have various advantages, technical limitations made it difficult to prove this notion. We have developed a novel microfabricated device, namely a micro-airflow-nozzle (MAN), to produce 100 to 300 microm alginate microcapsules with a narrow size distribution. The MAN is composed of a nozzle with a 60 microm internal diameter for an alginate solution channel and airflow channels next to the nozzle. An alginate solution extruded through the nozzle was sheared by the airflow. The resulting alginate droplets fell directly into a CaCl2 solution, and calcium alginate beads were formed. The device enabled us to successfully encapsulate living cells into 150 microm microcapsules, as well as control microcapsule size by simply changing the airflow rate. The encapsulated cells had a higher growth rate and greater secretion activity of marker protein in 150 microm microcapsules compared to larger microcapsules prepared by conventional methods because of their high diffusion efficiency and effective scaffold surface area. The advantages of smaller microcapsules offer new prospects for the advancement of microencapsulation technology.
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The genetic engineering of hematopoietic stem cells: the rise of lentiviral vectors, the conundrum of the ltr, and the promise of lineage-restricted vectors.
Recent studies on the integration patterns of different categories of retroviral vectors, the genotoxicity of long-terminal repeats (LTRs) and other genetic elements, the rise of lentiviral technology and the emergence of regulated vector systems providing tissue-restricted transgene expression and RNA interference, are profoundly changing the landscape of stem cell-based therapies. New developments in vector design and an increasing understanding of the mechanisms underlying insertional oncogenesis are ushering in a new phase in hematopoietic stem cell (HSC) engineering, thus bringing the hitherto exclusive reliance on LTR-driven, gamma-retroviral vectors to an end. Based on their ability to transduce non-dividing cells and their genomic stability, lentiviral vectors offer new prospects for the manipulation of HSCs. Tissue-specific vectors, as exemplified by globin vectors, not only provide therapeutic efficacy, but may also enhance safety, insofar that they restrict transgene expression in stem cells, progenitor cells and blood cells in all but the transcriptionally targeted lineage. This review provides a survey of these advances as well as several remaining challenges, focusing in particular on the importance of achieving adequate levels of protein expression from a limited number of vector copies per cell-ideally one to two.
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Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.
Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.
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A continuous delivery system of IL-1 receptor antagonist reduces angiogenesis and inhibits tumor development.
The involvement of interleukin-1 (IL-1) in inflammation, tumor growth, and metastasis makes it an attractive target for therapeutic intervention. Here, we show that a continuous delivery of a low, but steady-state level of the naturally occurring IL-1 receptor antagonist (IL-1Ra) reduced inflammatory responses and inhibited tumor development in mice, phenomena that are induced by IL-1, mainly secretable IL-1beta. The IL-1Ra was delivered from microencapsulated genetically engineered cells, which overexpress and secrete this mediator. For a tumor model, we used fibrosarcoma cell line, which secretes high levels of IL-1beta; when injected s.c. into mice, the cells developed into large tumors characterized by very active angiogenic patterns. The proangiogenic features of IL-1beta were manifested at low levels of the cytokine, and release of 25 ng per day of the IL-1Ra was needed to oppose its effects and inhibit tumor development. The continuous delivery of the IL-1Ra contributed to improved biocompatibility of the microencapsulated cell systems; the fibrotic sac surrounding the systems was much thinner with significantly less blood capillaries and inflammatory cells. Not only do our findings point to the antiangiogenic properties of IL-1Ra in inflammation and tumor growth, but they also provide a more efficient and convenient way for treating diseases involving IL-1.
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Integration of therapeutic cargo into the human genome with programmable type V-K CAST.
CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.
Humans↗
Surface-epitope masking: a strategy for the development of monoclonal antibodies specific for molecules expressed on the cell surface.
BACKGROUND: Producing monoclonal antibodies against specific targets, including tumor-specific antigens, is a tedious and extremely inefficient process. PURPOSE: Our purpose was to determine whether DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies. The quest was for monoclonal antibodies that would react with molecules existing on the surface of genetically altered cells. METHODS: We developed a masking technique called surface-epitope masking (SEM). The SEM procedure involves the selective blocking of surface antigens present in a genetically engineered cell (referred to as a "tester") with high-titer polyclonal antibodies that have been produced against the untransfected parental cell (referred to as a "driver"). Surface-epitope-masked tester cells were injected into BALB/c mice; immune spleen cells then taken from these mice were fused with myeloma cells. RESULTS: This process resulted in the efficient generation of hybridomas that secreted monoclonal antibodies that reacted with cell-surface antigens on transfected tester cells and with additional cell types that expressed the same surface molecules. In one case, CREF-Trans 6 cells were engineered to express a typical multidrug-resistant (MDR) phenotype. Using CREF-Trans 6:MDR cells as a tester cell line, we utilized the SEM procedure to produce monoclonal antibodies that displayed surface reactivity to both CREF-Trans 6:MDR cells and MDR human breast carcinoma (MCF7) cells. In a second case, human prostatic carcinoma CREF-Trans 6 cells, which were DNA transfected and derived from nude mouse tumors, were used as the tester cell line. The SEM procedure was again used to produce monoclonal antibodies. These antibodies were designed to and did react with: (a) tumor-associated antigens on the surface of the original LNCaP cell line used to obtain human prostatic carcinoma DNA, (b) primary and secondary nude mouse transfectants derived from tumors, and (c) two additional human prostatic carcinoma cell lines, DU-145 and PC-3. CONCLUSIONS: The SEM approach was used for the efficient and selective development of monoclonal antibodies that react with cell-surface molecules with both known and unknown functions. IMPLICATIONS: The SEM procedure should be useful in producing monoclonal antibodies and identifying genes associated with important cellular processes, including immunologic recognition, tumorigenesis, metastasis, atypical multidrug resistance, and autoimmune diseases.
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Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis.
In this study, Lactococcus lactis was engineered to express Giardia lamblia cyst wall protein 2 (CWP2) at three different subcellular locations, intracellular, secreted or cell-surface-anchored, using nisin as an inducing agent. CWP2 expression did not appear to be detrimental to L. lactis viability. No particular subcellular location of CWP2 expression offered any advantages over the others with respect to decreased toxicity towards the bacteria. All recombinant lactococci experienced a similar reduction in growth rate when induced. It was determined whether recombinant lactococcal cells engineered for cell surface expression of CWP2 were capable of inducing a CWP2-specific mucosal IgA antibody response. Recombinant lactococci were successful at inducing CWP2-specific IgA antibodies. Moreover, in a pilot challenge experiment, mice immunized with these recombinant lactococci demonstrated a significant (63 %) reduction in cyst output. Thus, it has been demonstrated that G. lamblia CWP2 may be expressed in L. lactis and that recombinant lactococcal cells elicit Giardia-specific antibodies which reduce cyst shedding in a murine model.
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[The application of basic fibroblast growth factor in tendon tissue engineering].
OBJECTIVE: To review the recent researches of basic fibroblast growth factor (bFGF) in tendon tissue engineering. METHODS: Recent original related literature was extensively reviewed and analyzed. RESULTS: bFGF played an important role in establishing standard tendon tissue engineering cell lines, inducing the compound and analysis of extracellular matrix, enhancing interactions between cells and extracellular matrix and accelerating tissue engineering materials' neovascularization. CONCLUSION: The progresses in increasing endogenetic bFGF expression, controlling the release of exogenous bFGF and improving the bio-utilization of bFGF has laid foundation for wider use of bFGF in tendon tissue engineering.
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Optimizing HAPEX topography influences osteoblast response.
HAPEX (hydroxyapatite-reinforced polyethylene composite) is a second-generation orthopedic biomaterial designed as a bone analog material, which has found clinical success. The use of topography in cell engineering has been shown to affect cell attachment and subsequent response. Thus, by combining bioactivity and enhancing osteoblast response to the implant surface, improved tissue repair and implant life span may be achieved. In this study a primary human osteoblast-like cell model has been used to study the influence of surface topography and chemistry produced by three different production methods. Scanning electron microscopy, fluorescence microscopy, and confocal scanning laser microscopy have been used to study cell adhesion; tritiated thymidine uptake has been used to observe cell proliferation; and the reverse transcriptase-polymerase chain reaction and biochemical methods have been used to study phenotypic expression. Transmission electron microscopy has also been used to look at more long-term morphology. The results show that topography significantly influences cell response, and may be a means of enhancing bone apposition on HAPEX.
Alkaline Phosphatase↗
Enhanced expression of a furin-cleavable proinsulin.
Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.
Amino Acid Sequence↗
Fetal liver as a source of autologous progenitor cells for perinatal tissue engineering.
Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.
Adipocytes↗
Differential regulation of primitive human hematopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells.
Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems, but their role as microenvironmental regulators is poorly understood. To address this problem, we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early, intermediate, and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]), clonogenic cells, and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders, cells at all stages of hematopoiesis decreased to very low levels. In contrast, maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However, this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand, in the presence of GM-CSF-producing feeders, the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications.
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Adult mesenchymal stem cells and cell-based tissue engineering.
The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.
Adult↗
[Protection of spinal motorneurons of section sciatic nerve by transplantation of NT-4 genetically expression cells into the side of section].
The NT-4 genetically engineered cells were made by infecting L-6TG cell (a rat myoblast cell line) in vitro with a retroviral vector pN2A containing the rat NT-4 cDNA. The bioactivities were determined by bioassay of PC12 cell survival rate. The rat with left sciatic nerve transaction was used as a model for treatment by implanting NT-4 genetically modified cell. The condition of motorneurons was assessed by Nissl stain and ChE stain. The results showed that: (1) the percentage of surviving Nissl-stained neurons on the lessened side of NT-4 (+) grafts significantly increased as compared to that with NT-4 (-) grafts 2-3 weeks and 3 months after sciatic nerve transaction, and (2) the grafted cells produced significant increase in the positive ChE stained area after sciatic nerve transaction in 1-3 weeks. Our observations indicate that adult motor neurons are still able to respond to neurotrophic factors and they may require the factors for survival.
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Presentation of native TROP-2 tumor antigens to human cytotoxic T lymphocytes by engineered antigen-presenting cells.
Professional antigen-presenting cells (APC), e.g. dendritic cells, express immuno-proteasome components and process proteins for MHC presentation differently from non-immune cells. Thus, they induce reactivities against sets of peptides that do not overlap with those generated by non-professional APC, i.e., tumor cells, and stimulate cytotoxic T lymphocytes (CTL) that may not recognize them. The goal of this work was to establish a system for antigen presentation and in vitro stimulation of human CTL using "tumor-cell-like" engineered APC. Murine fibroblasts were transfected with human HLA Class I alleles, together with the B7.1, ICAM-1 and germ-line TROP2 genes. The last encodes a cell surface glycoprotein widely expressed by human cancers. Unseparated peripheral blood mononuclear cells from HLA Class I-matched individuals were stimulated in vitro by the engineered APC. These efficiently induced the activation and proliferation of antigen-specific HLA-restricted CTL lines and clones. The Trop-2-specific CTL demonstrated high specific cytotoxicity against the appropriate transfected target cells. They also efficiently lysed MCF-7 human tumor cells expressing endogenous HLA-A2.1, Trop-2 together with ICAM-1. These results demonstrate that Trop-2 is a target molecule recognized by human CTL. Moreover, they demonstrate that non-immune engineered APC efficiently process and present native tumor-specific proteins in the context of human MHC Class I, and stimulate the growth and cytotoxicity of specific anti-tumor CTL.
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