2A and Gene Therapies.
The repair of many genetic lesions requires the co-expression of multiple proteins, or, may require the therapeutic protein(s) along with (selectable) marker proteins. The classical problem with transforming cells with multiple transgenes is that of co-expression within the the same cell.
Linking transgenes via multiple promoters into a single construct may improve success, but very often this is not the case. An alternative strategy is to create an artificial polyprotein system using 2A. In this manner multiple proteins can be concatenated into a single, long, open reading frame - whose expression is driven by a single promoter: a single transgene.
The individual proteins are separated during translation - although the 2A oligopeptide remains as a C-terminal extension of the upstream sequences and the protein(s) downstream of 2A have an N-terminal proline. Since the 'cleavage' of the polyprotein is co-translational, this strategy is compatible with the insertion of signal (leader) sequences. Such signal sequences can be either co- or post-translational - or both.
Adoptive Immunotherapy, Adoptive Cell Therapy (ACT) for Cancer and Cancer Vaccines.
Many of these treatments are a form of ex vivo gene therapy - they use a cancer patient’s own T lymphocytes which are (i) purified, (ii) genetically engineered in vitro to express proteins with anti-tumour activity, (iii) the transformed cell population expanded in vitro and (iv) reinfused into the patient.
In essence, the strategy is to charaterise the immune response to a tumor antigen from one patient, then genetically engineer the gene sequences responsible into the T-cells of other (non-responsive) patients.
Overall, results from studies empoying these strategies have been disappointing, although in some patients impressive clinical responses were reported. Since the majority of cancer cells only express MHC class I-restricted epitopes, many such strategies to improve such therapies are aimed at producing a strong, long-lived, cytotoxic T-lymphocyte (CTL) response. Cell-based antitumor immunity is driven by CD8+ CTLs bearing T-cell receptors (TCRs) that recognize specific tumor-associated peptides bound to class I MHC molecules. A method of redirecting CD4+ T-cells to MHC class I-restricted epitopes through engineered expression of class I-restricted, epitope-specific, T-cell receptors in CD4+ T-cells. Futhermore, MHC class I-restricted, epitope-specific, TCR-engineered CD4+ T-cells provide help toward the generation of memory CTL response in animal models.
The ability to efficiently and reliably co-express the multiple proteins that comprise, for example, the T-cell receptor is a major step forward. Here, 2A has been used to co-express the A and B chains of the TCR, plus the four diferent CD3 chains that also form part of the TCR complex. This is currently being applied to the treatment of metastatic melanoma, but other treatments will undoubtedly follow.
References citing the use of 2A;
Szymczak, A.L., Workman, C.J., Wang, Y., Vignali, K.M., Dilioglou, S., Vanin, E.F. & Vignali, D.A. (2004). Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nature Biotech. 22, 589-94.
Szymczak, A.L. & Vignali, D.A.A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinion 5, 627-638.
Holst, J., Szymczak-Workman, A.L., , K.M., Burton, A.R., Workman, C.J. & Vignali, D.A.A. (2006). Generation of T-cell receptor retrogenic mice. Nature Protocols 1, 406-417.
Holst, J., Vignali, K.M., Burton, A.R. & Vignali, D.A.A. (2006). Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nature Methods 3, 191-197.
Huang, X., Wilber, A.C., Bao, L., Tuong, D., Tolar, J., Orchard, P.J., Levine, B.L., June, C.H., McIvor, R.S., Blazar, B.R., & Zhou, X.Z. (2006). Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system. Blood 107, 483-491.
Scholten, K.B., Kramer, D., Kueter, E.W., Graf, M., Schoedl, T., Meijer, C.J., Schreurs, M.W. & Hooijberg, E. (2006). Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells. Clin. Immunol. 119, 135-145.
Kuhns, M.S. & Davis, M.M. (2007). Disruption of extracellular interactions impairs T cell receptor-CD3 complex stability and signaling. Immunity 26, 357-369.
Quintarelli, C., Vera, J.F., Savoldo, B., Attianese, G.M.P.G., Pule, M., Foster, A.E., Heslop, H.E., Rooney, C.M., Brenner, M.K. & Dotti, G. (2007). Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes. Blood 110, 2793-2802.
Burton, A.R., Vincent, E., Arnold, P.Y., Lennon, G.P., Smeltzer, M., Li, C.S., Haskins, K., Hutton, J., Tisch, R.M., Sercarz, E.E., Santamaria, P., Workman, C.J. & Vignali, D.A. (2008). On the pathogenicity of autoantigen-specific T-cell receptors. Diabetes 57, 1321-1330.
Chaparro, R.J., Burton, A.R., Serreze, D.V., Vignali, D.A. & DiLorenzo, T.P. (2008). Rapid identification of MHC class I-restricted antigens relevant to autoimmune diabetes using retrogenic T cells. J. Immunol. Methods 335, 106-115.
Chhabra, A., Yang, L., Wang, P., Comin-Anduix, B., Das, R., Chakraborty, N.G., Ray, S., Mehrotra, S., Yang, H., Hardee, C.L., Hollis, R., Dorsky, D.I., Koya, R., Kohn, D.B., Ribas, A., Economou, J.S., Baltimore, D. & Mukherji, B. (2008). CD4+ CD25- T Cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model. J. Immunol. 181, 1063-1070.
Griffioen, M., van Egmond, H.M., Barnby-Porritt, H., van der Hoorn, M.A., Hagedoorn, R.S., Kester, M.G., Schwabe, N., Willemze, R., Falkenburg, J.H. & Heemskerk, M.H. (2008). Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application. Haematologica 93, 1535-1543.
Hart, D.P., Xue, S-A., Thomas, S., Cesco-Gaspere, M., Tranter, M., Willcox, B., Lee, S.P., Steven, N., Morris, E.C. & Stauss, H.J. (2008). Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells. Gene Therapy 15, 625-631.
Holst, J., Wang, H., Eder, K.D, Workman, C.J., Boyd, K.L., Baquet, Z., Singh, H., Forbes, K., Chruscinski, A., Smeyne, R., van Oers, N.S.C., Utz, P.J. & Vignali, D.A.A. (2008). Scalable signaling mediated by T cell antigen receptor– CD3 ITAMs ensures effective negative selection and prevents autoimmunity. Nature Immunol. 9, 658-666.
Leisegang, M., Engels, B., Meyerhuber, P., Kieback, E., Sommermeyer, D., Xue, S-A., Reuß, S., Stauss, H. & Uckert, W. (2008). Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J. Mol. Med. 86, 573-583.
Moisini, I., Nguyen, P., Fugger, L. & Geiger. T.L. (2008). Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor. J. Immunol. 180, 3601-3611.
Robbins, P.F., Li, Y.F., El-Gamil, M., Zhao, Y., Wargo, J.A., Zheng, Z., Xu, H., Morgan, R.A., Feldman, S.A., Johnson, L.A., Bennett, A.D., Dunn, S.M., Mahon, T.M., Jakobsen, B.K. & Rosenberg, S.A. (2008). Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J. Immunol. 180, 6116-6131.
Simmons, A.D., Moskalenko, M., Creson, J,. Fang, J.M., Yi, S.L., VanRoey, M.J., Allison, J.P. & Jooss, K. (2008). Local secretion of anti-CTLA-4 enhances the therapeutic efficacy of a cancer immunotherapy with reduced evidence of systemic autoimmunity. Cancer Immunol. Immunother. 57, 1263-1270.
Varela-Rohena, A., Carpenito, C., Perez, E. E., Richardson, M., Parry, R.V., Milone, M., Scholler, J., Hao, X., Mexas, A., Carroll, R.G.. June, C.H. & Riley, J.L. (2008). Genetic engineering of T cells for adoptive immunotherapy. Immunologic Res. 42, 166-181.
Yang, S., Cohen, C.J., Peng, P.D., Zhao, Y., Cassard, L., Yu, Z., Zheng, Z., Jones, S., Restifo, N.P., Rosenberg, S.A. & Morgan, R.A. (2008). Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Therapy 15, 1411-1423.
Wargo, J.A, Robbins, P.F, Li, Y., Zhao, Y.B, El-Gamil, M., Caragacianu, D., Zheng, Z.L., Hong, J., Downey, S., Schrump, D., Rosenberg, S. & Morgan, R. (2009). Recognition of NY-ESO-1+tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression. Cancer Immunol. Immunother. 58, 383-394.
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