Gene manipulations for cancer gene therapy

Abstract

Tumor cells can be modified with cytokine genes such as the Interleukin-2 (IL-2) gene. The levels of IL-2 expressed are critical for successful treatment. We have tried to achieve higher levels of IL-2 than those currently available by conventional plasmids. Use of a transcriptional activator, e.g; the tat gene along with the HIV promoter driving the IL-2 gene, greatly increased IL-2 levels compared to widely used cytomegalovirus (CMV) driven plasmids. Control of the tat gene with an inducible promoter, i.e; the human HSP70B promoter, permitted control of gene expression. The inducibility of the HSP70B promoter by heat, γ-radiation and geldanamycin (a chemotherapeutic drug) allowed for a combinatorial approach to cancer treatment with hyperthermia, radiation therapy and chemotherapy. Also a brief heat treatment of 10 min at 42°C of target cells increased plasmid uptake, and higher levels of gene expression could be achieved. Another arm of immunotherapy is adoptive therapy with Tumor Infiltrating Lymphocytes (TILs). Insufficient numbers of tumor-specific T-cells limit the success of TIL therapy. An alternative approach to overcome this limitation is to transfer tumor-specific T cell receptor (TCR) into peripheral T-cells, redirecting their specificity to the tumor cell. To prove the feasibility of this technique, T-cell receptors were identified and cloned from hybridomas specific for the tumor cell line, MO5. A three domain single chain T-cell receptor was also constructed from the tumor-specific TCR genes to investigate the ability of a single chain T-cell receptor to activate T-cells. The CD3ζ chain was linked to the single chain to allow signal transduction upon antigen recognition by the TCR. The full length and the single chain TCR were cloned into a retroviral vector and transfected into mouse and human T cell lines. Cell surface expression of the chains were detected by flow cytometry. Functionality of the transfected TCR chains was assessed by IL-2 secretion on co-culture of the tumor cell line MO5 and the transfected T-cells. The two different approaches described here, i.e; higher levels of IL-2 for IL-2 gene therapy and specific redirection of T-cells can potentially greatly enhance the success rate of cancer treatment.