An Engineered Adeno-Associated Virus Variant Enables Efficient Gene Editing in Human T Cells.

Abstract

Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective donor template for homology-directed repair because they can avoid integrating into the host genome. However, only a few AAV serotypes can efficiently transduce human primary T cells at low multiplicities of infection (MOIs) with high packaging efficiency. To address this problem, variants derived from an AAV2 peptide library were screened in Jurkat cells and later validated in primary T cells. A high-ranking sequence identified outside the VR-VIII region, NNSKLTV, was discovered after three rounds of selection and was named Tot3. Tot3 demonstrated transduction efficiency similar to AAV2, but at a 27-fold lower MOI. In addition, Tot3 exhibited greater packaging efficiency and reduced thermal stability. Simultaneously, programmed cell death protein 1 (PD-1) knockout and CAR overexpression were achieved in human primary T cells using Tot3, with knockout and knock-in efficiencies reaching up to 70% and 55%, respectively. These CAR-T cells demonstrated significantly enhanced antitumor activity and increased survival times in a mouse model of diffuse B cell lymphoma.