Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent infections, and an increased leukemia risk. Multiple genetic defects that underlie SCN have been identified, but a genetic diagnosis is still lacking in a significant proportion of patients. In this study, we report 4 independent pedigrees with heterozygous variants in LCP1. Variants c.740-1G>T and c.740-20_744del produced the same alternatively spliced RNA product, causing an in-frame deletion (p.A247_E254del). Variant c.509C>T in the third pedigree produced p.S170L, and variant c.806T>C in the fourth pedigree produced p.L269P. Affected individuals suffered from neutropenia, poor or complete lack of response to granulocyte colony-stimulating factor (G-CSF) treatment, and variable degrees of lymphopenia, hypogammaglobulinemia, and monocytopenia. Patients with A247_E254del and p.L269P presented with tetraploid cells in the bone marrow, indicative of disturbed cytokinesis. In one of these kindreds, 2 individuals developed acute leukemia. G-CSF nonresponsiveness and defective cell cycling were repaired upon correction of the LCP1 A247_E254del variant in patient-derived induced pluripotent stem cells, supporting the monogenic origin of the disease. Indicative of their gain-of-function effect, both the A247_E254del and S170L variants increased F-actin bundling and the formation of abnormal protrusions. Single-cell transcriptome analysis of A247_E254del bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) showed deregulation of signaling pathways that control mitosis in multilineage and lymphoid-primed HSPC subsets. We concluded that activating LCP1 variants cause a new hematopoietic disorder with autosomal dominant inheritance. Depending on the consequences of the LCP1 variants in terms of protein structure, patients may suffer from G-CSF refractory severe neutropenia, lymphopenia, hypogammaglobulinemia, monocytopenia, and defective cytokinesis.

Autosomal dominant mutations in ELANE (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting ELANE expression, either by CRISPR-Cas9-mediated ELANE knockout or promoter targeting using CRISPR-Cas9 nickase, has emerged as a promising gene therapy strategy to restore defective granulocytic differentiation of transplantable hematopoietic stem cells from CN patients. We developed an adenine base editor (ABE)-mediated approach targeting two nucleotides in the ELANE promoter to suppress neutrophil elastase expression, called PRECISE. Analysis of mRNA- and protein-based delivery of ABE revealed that although both platforms were effective in editing hematopoietic stem and progenitor cells from healthy donors with over 80% editing, only protein-based ABE delivery achieved over 68% editing in CN patient cells. Interestingly, 10%-19% editing in CN patients' hematopoietic cells using ABE mRNA restored their granulocytic differentiation in vitro, with a marked expansion and differentiation of ABE ribonucleoprotein (RNP)-edited cells. PRECISE-edited neutrophils retained normal function, including neutrophil extracellular trap formation, oxidative burst, and phagocytosis. Genome integrity analysis showed no genomic alterations or chromosomal aberrations, and only two off-target edits confined to non-coding intronic regions. In conclusion, PRECISE represents a translationally relevant base-editing strategy for ELANE-associated CN and CyN that addresses ELANE mutation heterogeneity.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34+ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.

Neutropenia is a hematological condition characterized by a decrease in absolute neutrophil count (ANC) in peripheral blood, typically classified in adults as mild (1-1.5 × 109/L), moderate (0.5-1 × 109/L), or severe (< 0.5 × 109/L). It can be categorized into two types: congenital and acquired. Congenital severe chronic neutropenia (SCN) arises from mutations in various genes, with different inheritance patterns, including autosomal recessive, autosomal dominant, and X-linked forms, often linked to mitochondrial diseases. The most common genetic cause is alterations in the ELANE gene. Some cases exist as non-syndromic neutropenia within the SCN spectrum, where genetic origins remain unidentified. The clinical consequences of congenital neutropenia depend on granulocyte levels and dysfunction. Infants with this condition often experience recurrent bacterial infections, with approximately half facing severe infections within their first six months of life. These infections commonly affect the respiratory system, digestive tract, and skin, resulting in symptoms like fever, abscesses, and even sepsis. The severity of these symptoms varies, and the specific organs and systems affected depend on the genetic defect. Congenital neutropenia elevates the risk of developing acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS), particularly with certain genetic variants. SCN patients may acquire CSF3R and RUNX1 mutations, which can predict the development of leukemia. It is important to note that high-dose granulocyte colony-stimulating factor (G-CSF) treatment may have the potential to promote leukemogenesis. Treatment for neutropenia involves antibiotics, drugs that boost neutrophil production, or bone marrow transplants. Immediate treatment is essential due to the heightened risk of severe infections. In severe congenital or cyclic neutropenia (CyN), the primary therapy is G-CSF, often combined with antibiotics. The G-CSF dosage is gradually increased to normalize neutrophil counts. Hematopoietic stem cell transplants are considered for non-responders or those at risk of AML/MDS. In cases of WHIM syndrome, CXCR4 inhibitors can be effective. Future treatments may involve gene editing and the use of the diabetes drug empagliflozin to alleviate neutropenia symptoms.