T Cell Homing Gene Identification with CRISPR/Cas9 Library

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Technology Description

T cells and the immune system usually have amazing potential to eliminate tumor cells in the body. The clinical success of CAR-T immunotherapy designed to enhance T cell function proves this therapeutic potential. Despite the remarkable progress made so far, only about 30% of patients can obtain treatment success, and extending success to more patients is still the top priority in this field. Studies have shown that T cell infiltration in tumors can improve the efficacy of checkpoint blockade therapy. Infiltration of T cells into tumors seems to be an important prerequisite for immune checkpoint blockade and successful CAR-T therapy. Preclinical models have proved that increasing the number of T cells in tumors can enhance the efficacy of immune checkpoint blocking. Therefore, increasing the infiltration of T cells into tumors may have beneficial therapeutic effects.

To this end, Creative Biolabs has developed an innovative method based on the CRISPR/Cas9 library system to screen novel therapeutic targets in T cells that affect the infiltration of T cells into tumors. We have used a variety of cancer models to identify hundreds of T cell genes. When they are mutated, they can enhance the transport of T cells to the tumor, which in turn will cause larger T cells to kill cancer cells. Such modifications can enhance the success of chimeric antigen receptor (CAR) T cell therapy, especially in solid tumors, where the infiltration of T cells into the tumor is critical to success.

Everything you need to know about CRISPR library screening

In recent years, CRISPR/Cas9-based tools have developed rapidly. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi). CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is very useful in screening function gains, and CRISPRi is more powerful than RNA interference (RNAi) libraries in screening function loss. Positive selection using the CRISPR library can detect surviving cells with specific conditions, such as drug therapy, and can easily elucidate the mechanism of resistance. Negative selection can effectively detect dead cells or slow-growing cells and can identify genes necessary for survival, which may be a promising candidate for molecularly targeted drugs. In addition, negative selection can be applied to synthetic lethal interactions, where the perturbation of two genes simultaneously leads to the loss of viability, but any single gene will not affect viability. This mechanism is very important for determining the best combination of molecularly targeted drugs. Survival coexisting genes in cancer cells can be identified using new methods, such as paired guide RNA systems and methods combined with single-cell RNA sequencing technology. These effective methods can elucidate interesting biological mechanisms and propose candidate targets for molecularly targeted drugs.

CRISPR/Cas9 screenings.

Fig.1 CRISPR/Cas9 screenings.

Advantages of CRISPR Library Screening

Loss and gain-of function mutations
Easier than Sleeping Beauty (SB) or shRNA screens
Wide range of applications including immunology and oncology

Stage of Development

The screen has been tested in lymphoma and melanoma xenograft models with and without PD-1 therapy.

Top 10 gene candidates are hypothesized to influence T-cell functions important for intratumoral infiltration.

Top 10 gene candidates hypothesized to influence T-cell infiltration.

Fig.2 Top 10 gene candidates hypothesized to influence T-cell infiltration.

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References

Kurata, M., et al. (2017). CRISPR/Cas9 library screening for drug target discovery. Journal of Human Genetics, 63(2), 179–186.
Rogers, L. M., et al. (2020). A genetic screen to identify gain- and loss-of-function modifications that enhance T-cell infiltration into tumors. Cancer Immunology Research, canimm.0056.2020.