Universal CAR-T Generated Using the One-shot CRISPR/Cas9 System as a Replacement for Conventional Patient-specific CAR-T Therapy for B-Cell Acute Lymphoblastic Leukemia

Abstract

Since the traditional patient-specific CAR-T has an obvious disadvantage of long wait time while universal CAR-T has a risk of graft rejection. This experiment will investigate the safety and efficacy of the enhanced universal CAR-T generated using the one-hit CRISPR/Cas9 system and the traditional CAR-T in humanized-leukemic mice. Mice are first humanized by transplantation of human fetal thymus CD34+ liver cells of two origins. B-ALL cells are extracted from mice transduced with MLL-AF9 fusion gene/GFP-carrying retroviruses. T cells are extracted from mice and anti-CD19 CAR-T are generated by lentiviral transduction. They are transplanted into autologous mice. Other T cells extracted undergo TCR/HLA-I/Fas-triple gene ablation using one-shot CRISPR protocols and transplanted into allogenic mice. Three measurements are taken at day 1, 3, 7, 14, 21, and 28 post-transplantation: anti-CD19 CAR-T serum level by flow cytometry; CD19+GFP+ PBMC serum level by FACS, and acute GVHD-related biomarker expression (IL-2Rα, TNFR-1, IL- 8, HGF) by ELISA assay. There are 8 possible results but only when the universal CAR-grafted mice express a significantly higher serum level of CAR-T and a significant increase in the killing of leukemic cells while showing no indication of graft rejection risk, then would the result support the hypothesis. The experimental design of this study can be used as a reference for future research on universal CAR-T in mice. The results of this experiment provide important insight into the safety and efficacy of universal CAR-T; and aids in the clinical translation of this technology.