Abstract
Background: Chimeric Antigen Receptor (CAR) T cells and other adoptive cell therapies have revolutionized cancer treatment, yet patient-specific (autologous) approaches are costly, slow, and inaccessible to many patients. A major translational barrier is the lack of an efficient method to produce “off-the-shelf” allogeneic T cells that can be administered to any patient. Two immune compatibility hurdles exist: donor T cells attacking the host (graft-versus-host disease) and host rejection of donor cells, necessitating knocking out the T cell receptor (TCR) and human leukocyte antigen (HLA) genes. Traditional manufacturing workflows rely on viral technologies for gene editing, which are costly and raise safety concerns from random genomic integration. CRISPR/Cas9 technologies offer a more cost-effective alternative; however, they also present risks of off-target mutations and chromosomal rearrangements arising from the DNA double-strand breaks required for gene disruption.
Objective: To develop a non-viral GMP-ready base editing platform to generate universal donor T cells. Base editors enable precise single-nucleotide edits to knock out genes without introducing double-strand breaks, improving safety and consistency. Our platform will leverage base editors to simultaneously disrupt TCR, HLA-I, and HLA-II in healthy donor T cells, eliminating the risks associated with immune compatibility.
Specific Aims: (1) Optimize multiplex base editing protocols to produce triple-knockout universal T cells; (2) Characterize the functional safety and killing capacity of the edited cells in vitro; (3) Adapt the process to a scalable, clinical-grade (GMP) manufacturing workflow.
Impact: The pilot funding will support Aim 1, establishing core editing methods and proof-of-concept product. Successful completion will yield a generalizable platform technology that accelerates immunotherapy development by reducing manufacturing time, cost, and risk. This platform will serve as a shared infrastructure for the USC/CHLA translational research community, enabling investigators (including the SC CTSI community) to swiftly translate new cell-based therapies for cancers, autoimmune conditions, and rare diseases.