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932 A Universal CRISPR/Cas9n-Mediated Genome Editing Approach for ELANE-Related Severe Congenital Neutropenia

Program: Oral and Poster Abstracts
Type: Oral
Session: 201. Granulocytes, Monocytes, and Macrophages: New Insights into Inherited and Acquired Neutropenias
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, Bone Marrow Failure Syndromes, Inherited Marrow Failure Syndromes, Diseases, Gene Therapy, Therapies
Monday, December 11, 2023: 4:45 PM

Malte Ulrich Ritter1*, Masoud Nasri, PhD2*, Perihan Mir, PhD2*, Benjamin Dannenmann, PhD2*, Masako Monika Kaufmann3*, Yun Xu2*, Natalia Borbaran-Bravo2*, Maksim Klimiankou, PhD2*, Claudia Lengerke, MD4,5, Cornelia Zeidler, MD6*, Toni Cathomen, PhD3*, Karl Welte, MD, PhD2,7 and Julia Skokowa, MD, PhD8,9

1Department of Oncology, Hematology, Immunology, Rheumatology and Pulmonology, University Hospital Tuebingen, Tübingen, BW, Germany
2Department of Oncology, Hematology, Immunology, Rheumatology and Pulmonology, University Hospital Tuebingen, Tübingen, Germany
3Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
4Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tuebingen, Tuebingen, Germany
5University Hospital Tuebingen, Tübingen, Germany
6Hematology / Oncology, SCNIR, Hannover Medical School, Hannover, Germany
7Pediatric Hematology and Oncology, University Children's Hospital, Tuebingen, Baden-Wuerttemberg, Germany
8University Hospital Tuebingen, Tuebingen, Germany
9Gene and RNA Therapy Center (GRTC), University Hospital Tübingen, Tübingen, Germany

Severe congenital neutropenia (CN) is an inherited bone marrow failure syndrome characterized by impaired maturation of neutrophil granulocytes. Due to the lack or very low levels of neutrophils in the peripheral blood, patients experience severe, life-threatening bacterial infections as early as birth. Autosomal-dominant ELANE mutations are the most common cause of CN. Although most patients respond to daily treatment with recombinant human granulocyte colony-stimulating factor, about 15% of patients do not respond to this cytokine, and approximately 20% of patients develop myelodysplasia or acute myeloid leukemia. The only curative treatment available thus far is allogenic hematopoietic stem cell transplantation, which is associated with serious side effects. Autologous transplantation of gene-edited patients` HSPCs offers a novel curative therapy. Considering the wide range of mutations distributed throughout the ELANE gene, A universal, high-safety-profile approach could be advantageous for all ELANE-CN patients. Our group has already published a universal CRISPR/Cas9-mediated knockout approach for the ELANE gene; however, targeting the coding sequence region (CDS) of the ELANE gene could lead to the introduction of new disease-causing variants, so it may be harmful for patients. In order to address this concern, we reformatted our previously published knockout concept into a clinical-grade gene therapy strategy.

We have screened cis-regulatory regions in the ELANE core promoter and, using CRISPR/Cas9n, introduced two nicks on opposing strands of the Goldberg-Hognes box region (TATA-box) to make the transcription starting process inefficient, as transcription factors and RNApol II cannot recognize the modified sequence. The proposed strategy targets the non-coding region of the ELANE gene and thus does not generate new unwanted variants. Also, by replacing Cas9 with Cas9-ncikase, the off-target activity should decrease by up to 1000-fold. We have observed a markedly elevated neutrophil differentiation in gene-edited ELANE-CN patients HSPCs (n = 2), as assessed by the percentage of CD45+CD11b+CD15+, CD45+CD15+CD16+, and CD45+CD16+CD66b+ myeloid/granulocytic cells, compared to the mock electroporated ELANE-CN group, while no disruption of granulopoiesis in healthy donor HSPCs (n = 2) was detected. Results validated by the CFU assay. To have nucleotide-level resolution of on-target events, we performed targeted next-generation sequencing of edited loci on gene-edited healthy donors and ELANE-CN HSPCs. Next-generation sequencing results showed >90% on-target efficiency. CRISPR/Cas9n-mediated targeting of the TATA box rescued defective granulopoiesis in vivo, as gene-edited ELANE-CN HSPCs (n = 3) were transplanted in NSG mice and assessed by the percentage of neutrophils (hCD19-hCD3-hCD66bInt/lowhCD33+hCD16high) after 16 weeks. We have also observed efficient engraftment and preserved the multilineage potential of gene edited HSPCs in immunodeficient NSG mice. The strategy also depicted a safe profile upon applying gene editing to healthy donor CD34+ HSPCs and performing GUIDE-seq and CAST-seq. GUIDE-seq highlighted two potential off-target sites after >=6 mismatches, while no chromosomal translocations were detected by CAST-seq. RNA-sequencing of in vitro-generated neutrophils from gene-edited HSPCs of healthy donors (n = 2) confirmed a >9-fold reduction of ELANE gene expression level. Important to note, none of the other serine proteases or neighboring genes were down-regulated.

Taken together, ex vivo CRISPR-Cas9n-based gene editing of ELANE’s gene promoter in the setting of autologous stem cell transplantation could be a safe therapeutic approach for all ELANE-CN patients.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH