Development of Base Editing for Gene Therapy of Elane-Mutated Severe Congenital Neutropenia

We propose to develop a novel approach to gene therapy for severe congenital neutropenia (SCN), a life-threatening bone marrow failure syndrome. Patients present early in life with recurrent infections and sepsis; without treatment, most die by age 2 years. Current treatment with lifelong filgrastim injections has dramatically improved survival, but at the cost of progression to myelodysplasia and acute myeloid leukemia. Allogeneic hematopoietic stem cell transplant is the only currently curative treatment, but is limited by donor availability, transplant-related morbidity and mortality, and risk for graft-versus-host disease. Thus there is an unmet need for safe and effective therapy for SCN.Approximately 40% of cases derive from autosomal dominant monoallelic mutations in ELANE, the gene encoding neutrophil elastase. All known pathogenic mutations preserve expression but alter the structure of the protein product resulting in an unfolded protein response or abnormal intracellular trafficking leading to excess apoptosis.Our specific aims are unchanged from the original proposal:Aim 1: We will define optimal sgRNAs and ABE8e constructs to maximize ELANE inactivation and minimize off-target editing in transformed cell lines. Aim 1a. We will define the optimal promoter target sites for ABE8e inactivation of ELANE expression in highly permissive cell lines such as HEK293T for target optimization and NE-expressing myeloid cell lines for ELANE expression. Aim 1b. We will maximize base editing efficiency and specificity by assessment of both on- and off-target editing in highly permissive cells.Aim 2: We will optimize therapeutic base editing conditions in human CD34+ cells and establish the consequences of editing on ELANE expression. Aim 2a. We will optimize base editing efficiency and specificity in CD34+ HSPCs, starting from and further adapting conditions from Aim 1. 2b.