Increased functional expression of transgene in primary human lymphocytes using retroviral vectors modified with IRES and splicing motifs

• Published in: Gene therapy Vol. 9; no. 16; pp. 1085 - 1092
• Main Authors: ROYAL, R. E, KERSHAW, M. H, REEVES, M. E, WANG, G, DALY, T, TREISMAN, J, LAM, J, HWU, P
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01.08.2002
• Subjects: Adult; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Antigens; Antigens, Neoplasm - genetics; Antigens, Neoplasm - metabolism; Applied cell therapy and gene therapy; Biological and medical sciences; Biotechnology; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Colonic Neoplasms - immunology; Colonic Neoplasms - pathology; Colorectal cancer; Colorectal carcinoma; Cytokines; Cytotoxicity, Immunologic; Epithelial Cell Adhesion Molecule; Expression vectors; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Gene therapy; Genetic Therapy - methods; Genetic Vectors - genetics; Granulocyte-Macrophage Colony-Stimulating Factor - biosynthesis; Health. Pharmaceutical industry; Humans; Immunophenotyping; Immunotherapy - methods; Industrial applications and implications. Economical aspects; Interferon-gamma - biosynthesis; Lymphocytes; Lymphocytes T; Medical sciences; Mice; Moloney murine leukemia virus - genetics; Neomycin; Neomycin phosphotransferase; Other treatments; Phosphotransferase; RNA Splice Sites; T cell receptors; Transduction, Genetic; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Transgenes; Transgenes - genetics; Treatment. General aspects; Tumor Cells, Cultured; Tumors; Carcinoma; Immune response; Cell therapy; Enzyme; Antibody; Retroviridae; Genetic transfer; Virus; Isomerases; Neomycin; Intramolecular transferases; Primary culture; Colon; LTR sequence; Lymphocyte; Phosphotransferases; Vector
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3301734

Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.