Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL

Abstract

Allogeneic chimeric antigen receptor T cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CART 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 90-99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared to CRISPR-Cas9. Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using four 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.

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

“…Increasing the efficacy and reducing the toxicity of gene editing allows optimized cell yields for therapeutic applications. In line with previous reports 22,28,38,46 , co-transfection of base-modified mRNA encoding for 8 th generation adenine BEs achieved highly efficient editing at two sites ( Figure 2, 5, 6 ). Most groups reported the combination of retroviral gene transfer and multiplex editing in separate steps 22,26,28 .…”

“…In line with previous reports 22,28,38,46 , co-transfection of base-modified mRNA encoding for 8 th generation adenine BEs achieved highly efficient editing at two sites ( Figure 2, 5, 6 ). Most groups reported the combination of retroviral gene transfer and multiplex editing in separate steps 22,26,28 . Diorio et al combined cytosine base editing for B2M silencing with a Cas12b nuclease for AAV6-assisted GFP KI but did not investigate translocations in this experiment 22 .…”

CRISPR nuclease off-target activity and mitigation strategies

“…These include gross chromosomal rearrangements such as translocations ( Bothmer et al, 2020 ; Stadtmauer et al, 2020 ; Samuelson et al, 2021 ), chromothripsis ( Leibowitz et al, 2021 ), and aneuploidy ( Amendola et al, 2022 ; Nahmad et al, 2022 ). Translocation events most often occur as a consequence of: 1) on-target cleavage and recombination with a homologous region of the genome ( Turchiano et al, 2021 ); 2) simultaneous cleavage at an on-target and off-target sequence ( Lattanzi et al, 2021 ); or 3) following multiple on-target cleavage events in multiplexed editing workflows ( Qasim et al, 2017 ; Bothmer et al, 2020 ; Stadtmauer et al, 2020 ; Samuelson et al, 2021 ; Diorio et al, 2022 ). In addition, large-scale deletions either surrounding the cut site or of the distal end of the chromosome can occur ( Cullot et al, 2019 ), as well as copy-neutral loss-of-heterozygosity ( Boutin et al, 2021 ).…”

Tumor buster - where will the CAR-T cell therapy ‘missile’ go?

“…Unlike the induced DNA double-strand breaks (DSBs) technique used in the manufacture of most allogeneic CAR-T cells, CBEs create point mutations in T cells that silence gene expression without DSBs with an efficiency of 90 to 99%, significantly reducing the incidence of unexpected target editing results [94][95][96]. Allogeneic CD7 CAR-T cells developed based on CBEs are highly effective against T-ALL cells in a CD7+ T-ALL cell line CCRF-CEM, a model constructed by transplanting CCRF-GFP-Luc cells in NSG mice, and a mouse model created from patient-derived xenografts [96]. In addition, the removal of CD7 expression on the surface of T cells by gene editing technology could significantly inhibit the fratricide of CAR-T cells and reduce the risk of side effects.…”