Generation of Dual-RMCE Mediated TCR Gene Replacement System

Abstract: Adoptive immunotherapy with T cells modified by tumor antigen-specific T cell receptor (TCR) achieves a great progress in clinical trials， but it still existed some technical problem. For example， autoimmune side-effects induced by self-reactive TCRs from mismatched TCR chain dimers due to the transduced TCR paired with endogenous TCR chains and the inactivation of tumor suppressor genes causing by retroviral gene random integration. In order to overcome these drawbacks， we proposed to combine the two technologies of retroviral gene transfer and dual recombinase mediated cassette exchange (Dual-RMCE) to rapidly achieve site-specific TCR gene replacement in the mammalian cell lines. This technology was first successfully established in 16.113 and Jurkat76 cell lines. The single or low copy GFP cassette， which flanked by loxP and FRT sites， was introduced into cell genome through retroviral transduction， and then has been replaced by TCR cassette with the same direction of loxP and FRT sites. The efficiency of gene replacement was up to 5%， more effective than homologous recombination. The introduced TCR together with CD3 molecules were found to express on the surface of T cell line and could bound with the MAGE-A1 HLA-A2 multimer. We are using this technology to pluripotent stem cell system for T cell differentiation. The system we established would provide a new strategy to generate tumor antigen specific T cells for a safe retroviral TCR gene transfer in clinical cancer treatment.

CSTR: 32200.14.cjcb.2015.11.0003