In vivo site-specific engineering to reprogram T cells
Abstract
Abstract Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise 1 . However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR–Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.
Article Details
Authors (39)
William A. Nyberg
Pierre-Louis Bernard
Wayne Ngo
Charlotte H. Wang
Jonathan Ark
Allison Rothrock
Gina M. Borgo
Gabriella R. Kimmerly
Jae Hyung Jung
Vincent Allain
Jennifer R. Hamilton
Alisha Baldwin
Robert Stickels
Sarah Wyman
Safwaan H. Khan
Shanshan Lang
Donna Marsh
Niran Almudhfar
Catherine Novick
Yasaman Mortazavi
Shimin Zhang
Mahmoud M. AbdElwakil
Luis R. Sandoval
Sidney Hwang
Simon N. Chu
Hyuncheol Jung
Chang Liu
Devesh Sharma
Travis McCreary
Zhongmei Li
Ansuman T. Satpathy
Julia Carnevale
Rachel L. Rutishauser
M. Kyle Cromer
Kole T. Roybal
Stacie E. Dodgson
Jennifer A. Doudna
Aravind Asokan
Justin Eyquem