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The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies. Here, the authors show that base editing of CD33 and gamma globin genes in HSPCs results in the long-term engraftment of dual edited cells along with HbF reactivation and GO resistance in mice and nonhuman primates.

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance,without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2alpha) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2alpha. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-alpha. This was concomitant with increased IL-8 synthesis and cPLA2alpha activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-beta-cyclodextrin induced further cPLA2alpha activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-alpha-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2alpha and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-alpha-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.

Many therapeutic bispecific T-cell engagers (BiTEs) are in clinical trials. A modular and efficient process to create BiTEs would accelerate their development and clinical applicability. In this study, we present the design, production, and functional activity of a novel bispecific format utilizing synthetic orthogonal heterodimers to form a multichain modular design. Further addition of an immunoglobulin hinge region allowed a stable covalent linkage between the heterodimers. As proof-of-concept, we utilized CD33 and CD3 binding scFvs to engage leukemia cells and T-cells respectively. We provide evidence that this novel bispecific T-cell engager (termed IgGlue-BiTE) could bind both CD3+ and CD33+ cells and facilitates robust T-cell mediated cytotoxicity on AML cells in vitro. In a mouse model of minimal residual disease, we showed that the novel IgGlue-BiTE greatly extended survival, and mice of this treatment group were free of leukemia in the bone marrow. These findings suggest that the IgGlue-BiTE allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. These results suggest a compelling modular system for bispecific antibodies, as the CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis , thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. , dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.

Closed-loop insulin delivery is the new standard of care for patients with type 1 diabetes (T1D). However, in France, its implementation remains predominantly hospital based. Expanding access to this treatment through alternative care models looks essential. This study (cost-effectiveness analysis) compares 2 care models for people with T1D implementing a closed-loop system in France: outpatient care in the Inter-Regional Center for Automated Insulin in Diabetes (CIRDIA) and inpatient care. We conducted a cost-effectiveness analysis using retrospective observational data from individuals with T1D aged 16 years and older from the implementation of the closed loop to a 12-month follow-up either in the CIRDIA (CIRDIA group) or in a hospital center setting (hospital center [HC] group). The cost analyses were based on patient records and public databases: the French Medical Information Systems Program and the French General Nomenclature of Professional Acts. Closed-loop efficacy was assessed using a time in range (TIR) of 70 to 180 mg/dL, and closed-loop safety was assessed using the glycemia risk index (GRI), a single indicator that represents the risk of hypoglycemia or hyperglycemia and ranges from 0 (minimal risk) to 100 (maximal risk). A total of 201 patients were included: 128 in the CIRDIA group and 73 in the HC group. The mean (SD) age was 43 (14) years and 46 (15) years, respectively. Mean (SD) baseline TIR was 52.9% (16%) in the CIRDIA group versus 65.9% (15.1%) in the HC group (P<.001), whereas mean (SD) baseline GRI was 56.4 (21) in the CIRDIA group versus 37.8 (19.8) in the HC group (P<.001). After 12 months, both groups achieved similar efficacy and safety outcomes with a mean (SD) TIR at 72.7% (11.6%) in the CIRDIA group versus 71.9% (10.5%) in the HC group, and a mean GRI at 30.1 (14.1) versus 30.3 (13), respectively. There were no significant between-group differences (P=.60 for TIR; P=.91 for GRI). However, the CIRDIA was associated with significantly lower management costs with a mean cost of €8373.12 (SD €427.30; €1=US $1.10 at the time of the study) per patient in the CIRDIA group versus €8814.32 (SD €192) per patient in the HC group (P<.001). The estimated saving was €626 per percentage point of increase in TIR and €2011 per point of reduction in GRI, indicating that the HC closed-loop initiation was dominated by the CIRDIA. The CIRDIA was less costly than HC in 8600 (86%) out of 10,000 simulations in a probabilistic sensitivity analysis. These findings suggest the potential of the CIRDIA to represent a viable alternative organizational model for closed-loop initiation in France, achieving comparable effectiveness at lower cost in our population. Further research with longer follow-up is warranted. From a policy perspective, the resources saved could be at least partly reallocated to support out-of-hospital closed-loop initiation centers.