Neutrophil extracellular traps (NETs) contribute to innate immunity as well as numerous diseases processes such as deep vein thrombosis, myocardial ischemia, and autoimmune disease. To date, most knowledge on NETs formation has been gathered via the qualitative microscopic examination of individual neutrophils in vitro, or aggregate structures in vivo. Here we describe a novel flow cytometry (FLOW)‐based assay to identify and quantify NETs using antibodies against key NETs constituents, specifically DNA, modified histones, and granular enzymes. This method is applicable to both murine and human samples for the assessment of induced NETs in vitro, or detection of NETosis in vivo in blood samples. This FLOW‐based method was validated by comparison with the well‐established microscopy assay using two genetic mouse models previously demonstrated to show defective NETosis. It was then used on healthy human neutrophils for detection of ex vivo induced NETs and on blood samples from patients with sepsis for direct assessment of in vivo NET‐forming neutrophils. This new methodology allows rapid and robust assessment of several thousand cells per sample and is independent of potential observer‐bias, the two main limitations of the microscopic quantification. Using this new technology facilitates the direct detection of in vivo circulating NETs in blood samples and purification of NETting neutrophils by fluorescence‐activated cell sorting (FACS) for further analysis. Am. J. Hematol. 90:1155–1158, 2015.

Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNA^miR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNA^miR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNA^miR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%–70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven microRNA adapted shRNAs (shRNA^miR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4bp shift into the guide strand of shRNA^miRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a microRNA backbone. These findings have important implications in future design of shRNA^miRs for RNAi based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.