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.

BackgroundAutologous CAR-T therapies have demonstrated remarkable efficacy in treating some hematologic cancers. However, generating bespoke cell therapies creates manufacturing challenges, inconsistent products, high cost of goods, and delays in treatment that are often incompatible with effective clinical management of patients. Strategies to create universally-compatible allogeneic CAR-T therapies have been developed as a solution to these challenges. Allogeneic CAR-Ts require mitigation of graft-versus-host-disease (GvHD), host rejection of CAR-Ts, and elimination of fratricide in instances where the target (e.g. CD7) is expressed on both malignant cells and healthy T-cells. Many allogeneic CAR-T approaches utilize DNA double strand break (DSB)-inducing nucleases to overcome these barriers. However, simultaneous induction of multiple DSBs results in unpredictable outcomes such as large-scale genomic rearrangements, megabase-scale deletions, and reduced cell proliferation. Here we leverage base editors (BEs), which are a novel class of gene editing reagents that enable programmable single-base changes in genomic DNA without forming DSBs, to create multiplex edited, fratricide resistant, allogeneic CAR-T cells with no detectable genomic aberrations.MethodsT-cell acute lymphoblastic leukemia (T-ALL) is a disease with high and consistent expression of CD7 on malignant T cells, making CD7-targeting CAR-Ts (7CAR-Ts) an attractive therapeutic agent. We developed a GMP-compatible process to create 7CAR-Ts at clinical scale by isolating T cells from healthy human donors and electroporating the cells with base editor reagents, followed by transduction with a lentiviral vector encoding a second generation anti-CD7 CAR. 7CAR-Ts were characterized for their potency and specificity in vitro and in xenograft tumor models.ResultsSimultaneous base editing at four genomic loci resulted in 7CAR-Ts that are edited with 80–98% efficiency at each target gene, with greatly diminished risk of GvHD, CAR-T rejection, fratricide, and immunosuppression. In contrast to nuclease editing, concurrent modification of four genomic loci using BEs produced no detectable genomic rearrangements, no observable change in cell expansion, and no activation of the DNA damage-induced p53 pathway. Base edited 7CAR-Ts demonstrate robust antigen-dependent cytokine release, potent in vitro cytotoxicity, and dose-dependent in vivo tumor control.ConclusionsTaken together, our approach addresses limitations in CAR-T manufacturing and demonstrates that multiplexed base editing is a feasible strategy for generating universally-compatible, fratricide-resistant 7CAR-T cells, which we are advancing towards clinical development for the treatment of T-ALL. More generally, this program demonstrates the potential for base editing to create highly-engineered cell therapies featuring at least four simultaneous edits which can confer a wide range of desirable therapeutic attributes.

Ketosteroid Isomerase (KSI) performs an important biological function in the metabolism of many bacteria by degrading steroids. Pseudomonas putida KSI (PpKSI) is strikingly efficient and selective. Three putative KSIs identified from Structural Genomics were analyzed by THEMATICS (THEoretical Microscopic Anomalous TItration CurveS) and POOL (Partial Order Optimum Likelihood) and then characterized in vitro to determine their KSI activity. A putative KSI from Mycobacterium tuberculosis (MtKSI) was predicted to have ketosteroid isomerase‐like activity but putative KSIs from Mesorhizobium loti and Pectobacterium atrosepticum were predicted by these methods not to have such activity. Biochemical experiments reveal that the putative KSI MtKSI does indeed possess KSI activity, although with reduced efficiency compared to PpKSI. The two proteins predicted to be misannotated, MlKSI and PaKSI, exhibited no detectable KSI activity. To investigate this lower efficiency in the correctly annotated KSI, we engineered the MtKSI active site to resemble more closely that of PpKSI, and found that most of these mutations alone or in tandem significantly lowered rather than increased activity.