P1357: milestone (modifying elane goldberg–hognes box to inhibit expression), a universal, safe and effective crispr/cas9n-mediated genome editing strategy for elane related severe congenital neutropenia

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Severe congenital neutropenia (CN) is an inherited bone marrow failure syndrome. Autosomal-dominant ELANE mutations are the most common cause of CN. Although most patients respond to daily treatment with granulocyte colony-stimulating factor, a group of patients does not respond to this cytokine and approximately 20 % of patients develop myelodysplasia or acute myeloid leukemia. Hematopoietic stem cell transplantation would be a treatment of choice for CN patients non-responding to GCSF, but it is associated with severe adverse events. Gene editing of patients` HSPCs ex vivo, followed by autologous transplantation offers a novel curative therapy. Correction of mutation is a fascinating approach to treat ELANE-related CN (Tran et al., Sci Adv. 2022), However, considering the wide range of reported mutations, it could not benefit all ELANE-CN patients. Our group has already developed a universal strategy for ELANE-CN by CRISPR/Cas9-mediated ELANE knockout (Nasri et al., Haematologica 2019). Aims: CRISPR/Cas9 gene-editing in the coding sequence region of the ELANE gene might be source of potentially harmful events due to the possible introduction of new ELANE mutations as an unwanted editing outcome. We aimed to reformat the potential therapeutic ELANE knockout concept into a universal, safe, and clinical-grade gene therapy approach. We have screened cis-regulatory regions in the ELANE core promoter and, using CRISPR/Cas9n, introduced two nicks on opposing strands of Goldberg–Hognes box region (TATA-box) to make the transcription starting process inefficient, as TFs/RNApol II cannot recognize the modified sequence. We called it MILESTONE. MILESTONE does not generate new unwanted variants of ELANE as the non-coding region is targeted. Methods: rhAmpSeq system and CRISPRESSO package (Clement et al., Nature Biotechnology 2019) were used to amplify the ELANE gene promoter region and analyze the on-target editing efficiency. GUIDE-seq and CAST-seq were performed for safety profiling. Reactive oxygen species (ROS) assay was measured by ROS-Glo Assay kit (Promega, #G8820). Assessment of phagocytosis kinetics was performed using the IncuCyte ZOOM system. Results: Using the MILESTONE gene-editing strategy, we observed no disruption of granulopoiesis in healthy donor HSPCs (n = 2), while there was a markedly elevated neutrophil differentiation in gene-edited ELANE-CN HSPCs (n = 2), as assessed by the percentage of CD45+CD11b+CD15+, CD45+CD15+CD16+, and CD45+CD16+CD66b+ myeloid/granulocytic cells and compared to mock electroporated ELANE-CN group. Similar results were achieved by CFU-assay, showing significantly more CFU-G colonies after gene editing compared to mock electroporated ELANE-CN group. Moreover, the in vitro generated neutrophils were functional, being able to perform phagocytosis and produce reactive oxygen species (ROS) upon activation. Transcriptomic profiling and qRT-PCR analysis of in vitro generated neutrophils confirmed a reduction of ELANE gene expression in gene-edited cells of up to 6-fold, while, none of the other serine proteases were down-regulated. Applying MILESTONE in ELANE-CN HSPCs restored defective granulopoiesis in vivo, as assessed by the percentage of neutrophils (hCD19-hCD3-hCD66bInt/lowhCD33+hCD16high) after transplantation of gene-edited ELANE-CN HSPCs in NSG mice. CRISPResso analysis showed >90% on target efficiency. Off-target analysis in healthy donor CD34+ HSPCs depicted a safe off-target profile. Summary/Conclusion:Ex vivo CRISPR-Cas9n-based gene editing of the ELANE gene using MILESTONE in the setting of autologous stem cell transplantation is a feasible, safe, and universal therapeutic approach for ELANE-CN patients. Keywords: Severe congenital neutropenia, Gene therapy