Creating Retroviral Vector Encoding Two Homologous Chimeric Immune Receptors with Degenerated Codons (47.28)

Abstract

Abstract T cells genetically modified to express chimeric immune receptors (CIR) have been shown to be promising for immunotherapy. T cell receptor (TCR)-based CIR can be created in that the extracellular domains (Ec) of alpha (a) and beta (b) chains are fused to signaling cytoplasmic domain (Cy) of CD3 zeta (z) (TCRa-z and TCRb-z). To reduce homologous recombination associated with high homology between two nucleotide sequences in the same vector, we applied a novel approach by substituting the nucleotide sequence of one of the two homologous genes with degenerated genetic codes without change in amino acid sequence. In this study, we created a mutated DNA sequence encoding the Cy of z with degenerated codons (muz). A retroviral vector encoding a pair of TCR based CIRs (TCRa-z and TCRb-muz; tcsv-p36z) was created, with specificity via the pair of Ec of TCR a/b to a JC virus peptide p36/HLA-A2. 293T cells were transduced with retrovirus from PG13 cells with the tcsv-p36z. The transduced cells that expressed substantial TCR Vb of the CIR were able to bind p36/A2 tetramer, indicating the expression of both a and b chains in the single cell level. Western blot confirmed that both a and b chains were cellularly expressed intact. This approach, which we employed in creating retroviral vector encoding tc-CIRs with high homology, can be universally applied to creating viral vectors encoding two or multiple homologous molecules for gene therapy.