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1.
Gene Ther ; 22(2): 209-15, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25427612

ABSTRACT

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.


Subject(s)
Antigens, CD19/immunology , DNA Transposable Elements , Lymphoma, B-Cell/therapy , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Coculture Techniques , Genetic Engineering , Genetic Therapy , Humans , Immunotherapy, Adoptive , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , NIH 3T3 Cells , Neoplasm Transplantation , Receptors, Antigen, T-Cell/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics
2.
Oncogene ; 22(46): 7155-69, 2003 Oct 16.
Article in English | MEDLINE | ID: mdl-14562044

ABSTRACT

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.


Subject(s)
Cell Transformation, Neoplastic/genetics , Epithelial Cells/physiology , Gene Expression Regulation, Neoplastic/genetics , Mesoderm/physiology , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Differentiation , Cell Line , Cluster Analysis , Epithelial Cells/pathology , Female , Gene Expression Profiling , Genes, ras , Humans , Mammary Glands, Animal/cytology , Mammary Glands, Animal/pathology , Mesoderm/pathology , Mice , Neoplasm Invasiveness , Polyribosomes/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Transcription, Genetic
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