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Laboratory of T Cell Engineering at SIAIS and collaborators report new technology for genome editing in CAR T cell

Recently, Research Associate Professor Xuekai Zhu from T Cell Engineering Laboratory at SIAIS, and Professor Xingxu Huang from SLST have developed a new technology for the preparation of genome-edited chimeric antigen receptor (CAR) T cells based on non-viral vectors. The researchers used the CRISPR/Cas9 system to achieve efficient knockout of the PD-1 gene in human T cells, while stably integrating and expressing CD133-specific CAR via the transposon system. The study was published in the journal Human Gene Therapy titled with Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells.

Using viral vector-transduced T cells is the mainstream direction of CAR-T cell therapy. However, the production and quality control of clinical grade viral vectors is very expensive. There are currently only a few units in the world that are qualified and capable of providing clinical grade CAR virus vectors. In addition, CRISPR/Cas9-mediated genome editing in CAR T cells requires the presence of Cas9. In order to avoid the immunogenicity of non-human Cas9 protein in the edited T cells, it is common to use electroporation to ensure that Cas9 is transiently present. T Cell Engineering Laboratory has long been working on methods for delivering genes into T cells using non-viral vectors. In this study, CRISPR/Cas9 was used in combination with the transposon system, and a one-step plasmid-only method was used to achieve gene editing and CAR expression simultaneously in T cells. The researchers further confirmed that the technology can prepare genome-edited CAR T cells, in which CAR expression and gene editing efficiency can meet clinical needs (Fig.1). Advantages of the non-viral plasmid vector for delivering genes relative to viral vector transduction include cost reductions to 1/10 of the latter and larger capacity for delivered genes.

In recent years, the success of immunotherapy represented by CAR T cells and PD-1/PD-L1 blocking antibodies has made cancer treatment a new era. The 2018 Nobel Prize in Physiology or Medicine was awarded to scientists who made important contributions to the development of the immune checkpoint blockade therapy, including CTLA-4 and PD-1 antibodies. The combination of CAR T cells and immune checkpoint inhibition is expected to counter the immunosuppressive environment of solid tumors and reduce the systemic immune-related side effects caused by checkpoint blocking antibodies. It has been a research hotspot in the field of tumor immunotherapy. In this study, the researchers successfully knocked out the PD-1 gene in CD133-CAR T cells using genome-edited CAR T cell technology. They found that CD133-CAR T cells with PD-1 gene silence were more potent in killing tumor cells in vitro than traditional CAR T cells. And significantly prolonged survival of tumor-bearing mice and inhibition of tumor growth were shown for PD-1KO CAR T cells in animal experiments (Fig.2).

In future research, T Cell Engineering lab will use this technology to study unknown regulatory factors that affect T cell function, and develop powerful CAR T cells or universal CAR T cells for solving problems such as the treatment of refractory solid tumors and difficult large-scale preparation of autologous CAR T cells.

Graduate student Bian Hu from Xingxu Huangs group is the first author. Prof. Xuekai Zhu and Prof. Xingxu Huang are co-corresponding authors.

Link to full paper:

Fig.1: Achieve high expression of CD133-CAR and high efficiency of PD-1 gene knockout simultaneously in human T cells via non-viral vectors

Fig.2: PD-1 knockout CD133-CAR T cells show stronger anti-tumor function


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