Development of a gene therapy for congenital neutropenia caused by ELANE mutations

Abstract

The monogenetic disease congenital neutropenia (CN) is a pre-leukemia bone marrow failure syndrome. In approximately 50 % of CN patients, the disease is caused by mutations in the ELANE gene. This gene encodes the protein neutrophil elastase (NE). Patients suffer from an insufficient number of neutrophils, resulting in frequent severe and life-threatening bacterial infections and sepsis. The current standard of care is regular injections with the recombinant human granulocyte-colony stimulating factor (rhG-CSF), a cytokine produced naturally in the body. This treatment is not curative and does not prevent leukemia development, for which the patients have a 15 – 20 % lifetime risk. If the patient does not respond to rhG-CSF or develops leukemia, the only chance of survival is a bone marrow transplantation, which is associated with a risk of graft-vs-host disease, graft failure, infections, and a mortality risk of about 20 %. These circumstances create a clear unmet medical need for therapy improvements. The recent development of CRISPR/Cas9 nucleases for gene editing has created a versatile tool ideally suited for genetic manipulation for gene therapy. Here, I present preclinical investigations into the application of CRISPR/Cas9 gene editing tools for the purpose of gene therapy development for patients with CN caused by ELANE mutations. Specifically, the development of a knockout strategy for the ELANE gene that restores neutrophil development and preserves neutrophil functions. And a novel knockdown strategy that reduces ELANE expression by genetically modifying the ELANE promotor region using two CRISPR/Cas9 nickases. The therapeutic potential of this approach has been demonstrated in vitro and in vivo. This approach has been shown to have a favorable genotoxic profile with no detectable off-target gene editing and no chromosomal translocations. Finally, work on establishing an in vitro model with mouse hematopoietic stem and progenitor cells (HSPCs) with the mutations most frequently involved in MDS and AML development in CN patients has provided new insights into the dysregulated pathways and mechanisms underlying the progression from pre-leukemic to leukemic stages. These are mainly driven by increased innate immune activation in HSPCs. These observations are also crucial for disease-specific safety considerations in gene therapy development. My work has contributed to understanding CN's pathophysiological mechanisms and offers novel gene therapeutic approaches for ELANE mutations that cause CN.