Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL
Caroline Diorio, Ryan Murray, Mark Naniong, Luis Barrera, Adam J. Camblin, John Chukinas, Lindsey Coholan, Aaron Edwards, Tori J. Fuller, Claudia Gonzales, Stephan A. Grupp, Alden Ladd, Melissa Le, Angelica Messana, Faith Musenge, Haley Newman, Yeh‐Chuin Poh, Henry Poulin, Theresa Ryan, Rawan Shraim, Sarah K. Tasian, Tiffaney L. Vincent, Lauren Young, Yingying Zhang, Giuseppe Ciaramella, Jason M. Gehrke, David T. Teachey
Abstract
Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.