Newly Developed Measles Viral Vector Can Efficiently Transduce Multiple Genes into naïve T Cells
Authors: Hiramoto Takafumi , Tahara Maino , Miura Yoshie , Nakatsu Yuichiro , Kubota Toru , Kurita Ryo , Hamana Hiroshi , Sakamoto Chika , Narusawa Megumi , Yamaguchi Saori , Ono Hiroaki , Liao Jiuan , Nii Takenobu , Takishima Yuto , Okada Michiyo , Matsumura Yumiko , Tanaka Yoshihiro , Yamada Kazunari , Hijikata Yasuki , Kohara Hiroshi , Ishihara Sonoko , Tsuruta Toshihisa , Okazaki Toshihiko , Kishi Hiroyuki , Muraguchi Atsushi , Ryo Akihide , Takeda Makoto , Tani Kenzaburo 
Published: October 2019

Abstract
Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%)　than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor.
These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells.

Disclosures
No relevant conflicts of interest to declare.