Developing Off-the-Shelf T-cell Receptor Therapy Targeting Resistant Pathogens Using CRISPR Genome-Editing

Immunocompromised patients, such as those undergoing chemotherapy, allogeneic hematopoietic stem cell transplantation (alloHSCT), or solid organ transplantation (SOT), are highly susceptible to severe opportunistic infections and viral reactivations. These infections often progress rapidly and exhibit resistance to standard treatments, leading to significant morbidity and mortality. To address this urgent need, we propose an innovative approach utilizing allogeneic, "off-the-shelf" T-cell therapy targeting pathogen-specific infections. Our strategy involves identifying and isolating pathogen-specific T-cell receptors (TCRs), which are then transduced into allogeneic T-cells via gammaretroviral vectors. To mitigate the risk of alloreactivity, we employ CRISPR-Cas9 technology to knock out the endogenous TCRα (TRAC) gene. Preliminary data demonstrates robust TCR insertion and potent antigen-specific responses against viral peptides in TRAC-KO T-cells. These cells exhibit upregulation of activation markers, such as OX40 and 4-1BB, and reduced alloreactivity in mixed lymphocyte reaction (MLR) assays. To further enhance safety and precision, we are exploring viral-free genome editing techniques to directly insert the TCR-V gene into the TRAC locus under its endogenous promoter. This approach eliminates the risks associated with random integration and provides greater control over editing outcomes. Our platform offers a scalable and GMP-compliant pathway for developing TCR-based immunotherapies, providing a promising strategy to combat opportunistic viral infections in immunocompromised patients.