T. Yamauchi1, H. Saito2,3, T. Hoki1, F. Ito1,2,4,5 2University Of Michigan,Surgery,Ann Arbor, MI, USA 3Kanazawa Medical University,Biochemistry,Kanazawa, ISHIKAWA, Japan 4State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 5Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 1Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA
Introduction: Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. This limitation can be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. We and others have shown that iPSC-derived regenerated T cells have potent antitumor efficacy in vitro and in vivo. Recently, the type II bacterial CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system was developed as an efficient and versatile technology for genome editing in eukaryotic cells and whole organisms. The potential of iPSCs can be further enhanced by genome engineering and then used to study individual gene function, track cells or endogenous proteins with a knock-in reporter, and correct genetic defects for gene therapy.
Methods: We reprogrammed T-cell receptor (TCR) transgenic CD8+ T cells into pluripotency, and established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Plasmids encoding rtTA and tet-O-Cas9-2A-mCherry were obtained and co-transduced using the lentiviral system. The Cas9 expression was analyzed after the doxycycline treatment, and clonally-derived Cas9-iPSCs were obtained.
Results: Pluripotency of TCR transgenic T cell-derived iPSCs (TiPSCs) were confirmed with immunostaining of embryonic stem cell (ESC) markers, RT-PCR (reverse transcription-polymerase chain reaction) analysis of pluripotency-associated transcription factors, and whole genome expression profiling by microarray analysis that demonstrated a high degree of similarity in their gene expression patterns and close correlation values with ESCs, but distinct from parental CD8+ T cells. Cytogenetic analysis revealed derived TiPSCs maintained normal karyotype. The TiPSCs differentiated to embryoid bodies in vitro, and upregulation of marker genes for all three germ layers was detected by immunostaining. Their differentiation capacity was further confirmed by teratoma formation in immune-deficient mice in vivo. Moreover, we confirmed that the TiPSCs retained the same rearranged configuration of TCR chain genes as the original TCR transgenic T cells. TiPSCs were, then, subjected to the lentivirus-mediated transduction of tetracycline-inducible Cas9 vectors. The transduction efficacy was confirmed by the mCherry fluorescence and the RT-PCR against the Cas9 sequence.
Conclusion: Our results indicate successful reprogramming of antigen-specific T cells and lentivirus-mediated transduction of tetracycline-inducible Cas9 vectors into TiPSCs, which will allow us to generate an unlimited number of phenotypically defined, functional and expandable genome-edited autologous antigen-specific T cells.