An overview on in-vivo generation of CAR-T cells using CRISPR-loaded functionalized nanocarriers for treating B-cell lineage acute lymphoblastic leukemia.

Chimeric antigen receptor T (CAR-T) cell therapy has become a milestone in the management of B cell lineage acute lymphoblastic leukemia. Yet, the traditional method-dependent on ex vivo manipulation, amplification, and reinfusion of autologous T cells-is high-cost, low-scalability, and severely immune-related toxicity. Here, we report a new nano-immunoengineering platform that allows in vivo production of chimeric antigen receptor T cells through the use of functionalized nanoparticles carrying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing elements. These nanoparticles are engineered to specifically target blood circulating T lymphocytes and deliver CRISPR/Cas9 complexes that have the ability to integrate chimeric antigen receptor constructs into the TRAC locus and knock out immune checkpoint genes like programmed cell death protein 1 (PD-1) simultaneously. Targeted delivery, endosomal escape, and efficient genome editing with minimal off-target effects are ensured through gold-based and DNA nanostructure-based carriers. Preclinical models show effective in vivo programming of functional chimeric antigen receptor T cells with vigorous antitumor efficacy, improved persistence, and decreased cytokine release syndrome. This method is a revolutionary breakthrough in cancer immunotherapy that provides a scalable, economical, and clinically flexible replacement for conventional chimeric antigen receptor T cell production.