A MULTIPLEXED RNA REGULATION PLATFORM FOR PRIMARY IMMUNE CELL ENGINEERING

Smart overview of the Invention

The disclosed platform offers a versatile method for transcriptome regulation in primary human T cells using CRISPR/Cas13d technology. Unlike traditional Cas9 systems that introduce permanent DNA changes, Cas13d targets RNA, allowing for reversible and multiplexed gene knockdown. This approach is particularly effective in suppressing genes associated with T cell exhaustion, enhancing the anti-tumor activity of CAR T cells, and potentially overcoming resistance in cancer therapies.

Government Support and Background

This innovation was developed with support from the National Cancer Institute, highlighting its potential impact on cancer treatment. Adoptive T cell therapies, like CAR T cell therapy, have shown promise but face challenges such as resistance and limited efficacy against solid tumors. Traditional CRISPR/Cas9 methods, while powerful, have limitations in safety and scalability due to irreversible genome modifications, which can adversely affect T cell function and lead to genotoxicity.

Advantages of Cas13d over Cas9

The platform leverages Cas13d, a compact CRISPR effector that targets RNA instead of DNA, avoiding permanent genomic alterations. Cas13d's ability to process poly-crRNA guide arrays allows for simultaneous targeting of multiple RNA transcripts, facilitating large-scale multiplexing. This feature is crucial for safely perturbing complex gene programs necessary for optimizing T cell therapies without the risks associated with DNA-targeting CRISPR systems.

Applications and Methods

The platform is designed for various applications, including modulating CAR signal transduction and enhancing the anti-tumor activity of CAR T cells. It can disrupt metabolic pathways to alleviate immunosuppression and is capable of targeting gene sets involved in T cell exhaustion. The system includes compositions of genetically modified T cells expressing CRISPR effectors and guide arrays, along with methods for producing these cells and regulating gene expression to increase T cell proliferation and anti-tumor activity.

Embodiments and Specific Targets

Genetically modified T cells are engineered to express a CRISPR effector with RNA-guided endonuclease activity and a guide array of crRNA molecules. These crRNAs target mRNAs associated with T cell exhaustion, such as TOX, PDCD1 (PD-1), and CTLA4. The system can also include CARs targeting tumor antigens like GD2 and CD19. This approach allows for precise modulation of T cell activity, potentially improving the efficacy of CAR T cell therapies in cancer treatment.