US20260176622
2026-06-25
Chemistry; metallurgy
C12N15/113
Cancer therapy has seen significant advancements through the use of chimeric antigen receptor (CAR) technologies, which reprogram immune cells to target and kill cancer cells. CARs are specialized proteins with multiple domains, including an antigen binding domain that typically recognizes cancer-associated antigens. Upon binding, these CARs activate immune cells such as T cells to attack cancer cells. However, the effectiveness of CAR-based therapies can vary due to individual patient differences, necessitating personalized therapeutic approaches.
The application outlines methods and compositions for conducting CRISPR screens on CAR-immune cells like CAR-T and CAR-NK cells to identify genetic mutations that enhance their cancer-killing efficacy. The process involves using CRISPR guide RNA libraries to create diverse mutant CAR-T cell populations, which are then tested for their ability to kill cancer cells. By identifying specific mutations that improve CAR-T cell function, these methods aim to personalize CAR therapies for different patients and cancer types.
The methods and compositions described offer significant advantages over traditional approaches, especially those relying on model organisms. Previous experiments have shown that results in animals, such as mice, often do not translate effectively to humans. The described techniques are specifically designed for human application, allowing for direct therapeutic effects without the need for adaptation from non-human models. This ensures that the identified CAR-immune cell genotypes are directly relevant to human cancer treatment.
The disclosure provides strategies for designing effective CRISPR screens in humans, including determining the optimal size of guide RNA libraries. These libraries are tailored to represent mutant CAR-immune cells adequately, avoiding the pitfalls of genome-wide libraries that may be too large for conclusive results. By focusing on selected gene sets, the methods ensure sufficient representation and statistical resolution to identify impactful mutations on CAR-T cell efficacy.
The application details compositions of guide RNA (gRNA) libraries, specifying homology regions complementary to gene sequences identified as influential in CAR-T cell efficacy. These libraries can vary in size, with some embodiments comprising up to 135 different gRNA polynucleotides targeting distinct genes. This targeted approach enhances the ability to pinpoint mutations that contribute to improved cancer cell targeting and destruction by CAR-immune cells.