Science and Technology Platform Program for Advanced Biological Medicine


Next generation CAR & T cell therapies with a novel genome editing technology

<Project Leader> Tomoji Mashimo

Division of Animal Genetics, Division of Genome Engineering Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo

Tomoji Mashimo

CRISPR-Cas9 system has been utilized as a tool for genome editing around the world. It can efficiently induce modification of genes in cultured cells, microorganisms, plants and model animals. However, CRISPR-Cas9 often cause 1) off-target and 2) mosaic mutations, 3) and It is difficult for it to cleave in repeat or heterochromatin regions. 4) The development of model animals and gene therapy in Japan by CRISPR-Cas9 technology are limited because the intellectual property is held down by the United States.
We found that genome editing using Class 1 CRISPR-Cas3 is possible in human cells as a novel genome editing technology, which is different from Class 2 CRISPR-Cas9 system. With this patent application, they have started up a company, named C4U.This genome technology has following advantages: 1) CRISPR-Cas3 can carry a deletion in the genome of heterochromatin and repeat sequences. 2) It is expected that the system can delete the target sequence with high specificity due to Cas3 recognizing 27nucleotide spacer. 3) The system is possible of knocking out and knocking in a gene by causing a large deletion of hundreds to thousands of bases in the target sequence. 4) We have already granted the basic patent of the system in Japan. The primary purpose of this research is to establish a proof of concept to use CRISPR- Cas3, a genome editing technology from Japan, and to provide this genome editing system for gene therapy so that many pharmaceutical companies can apply it in japan.
Malignant tumors are the leading cause of death in Japan. The development of effective treatments for malignant tumors is an urgent issue. Chimeric antigen receptor T (CAR-T) cell therapy is an effective cancer immunotherapy and based on using genetically modified T cells to precisely target and kill cancer cells. Currently, CAR-T therapy is effective for B-cell tumors, but is poorly effective for solid tumors. Tamada et al developed Prime CAR-T which exerts a therapeutic effect on solid tumors by secreting the cytokine IL-7 and the chemokine CCL19 to increase the ability of proliferation immune cells and accumulation on cancer (Fig. 2). With the patent, they also built a start-up company, named Noile-Immune Biotech.
CAR-T cell therapy is an autologous T cell therapy in which a CAR gene is introduced into T cells collected from a patient using a viral vector, cultured, and returned to the patient. There are problems that 1) the quality of T cells is affected by the patient’s immune status, 2) culturing and quality check cost time, and 3) the production costs are very high. The second purpose of this research is to combine the two technologies of a novel genome editing technology, CRISPR-Cas3, and Prime CAR-T cell technology to create a next generation CAR- T cell therapy (Fig. 3). In this study, we aim to establish an allogeneic CAR-T cell therapy by deletion of endogenous TCR genes and to enhance its antitumor effect by disruption of the PD-1 gene with CRISPR-Cas3system.

Figure 1 Figure 1: Comparison of CRISPR-Cas3 and Cas9 system
The features of the CRISPR-Cas3 (left) and the conventional CRISPR-Cas9 system (right) are shown.
Figure 2 Figure 2: Conventional CAR-T cell(left)and Prime CAR-T cell(right)
Mechanism of Prime CAR-T cells to exert a therapeutic effect on solid tumors
Figure 3 Figure 3: Project organization diagram
This project will be carried out by The University of Tokyo, Yamaguchi University, C4U and Noile-Immue Biotech.


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