CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

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.

Differential transcriptional control of hematopoiesis in congenital and cyclic neutropenia patients harboring ELANE mutations.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most CyN cases and approximately 25 % of CN cases. In CN and in CyN, a median of 2.8 % of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to G-CSF with a significant upregulation of the hematopoietic stem-cell-specific transcription factors, C/EBP/, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBP//expression in CN restored granulopoiesis.

Generation, expansion, and drug treatment of hematopoietic progenitor cells derived from human iPSCs.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can progress to acute myeloid leukemia (CN/AML). Patient material to study leukemogenesis, especially hematopoietic progenitor cells (HPCs) is limited and hard to access. We have established a protocol for generation of HPCs from iPSCs followed by HPC expansion on Sl/Sl feeder cells expressing FLT3L. We performed drug treatment of iPSC-derived HPCs on feeder cells or under feeder-free conditions. Our protocol is also suitable for primary leukemia blasts. For complete details on the use and execution of this protocol, please refer to Dannenmann et al. (2021), (2020), and (2019).

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.

NAMPT/SIRT2-mediated inhibition of the p53-p21 signaling pathway is indispensable for maintenance and hematopoietic differentiation of human iPS cells.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRT pathway in the regulation of maintenance and differentiation of human-induced pluripotent stem (iPS) cells is not fully elucidated. METHODS: We evaluated the effects of specific inhibitors of NAMPT or SIRT2 on the pluripotency, proliferation, survival, and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells. RESULTS: We demonstrated that NAMPT is indispensable for the maintenance, survival, and hematopoietic differentiation of iPS cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affect hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system. CONCLUSIONS: Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

The chemokine CXCL14 mediates platelet function and migration via direct interaction with CXCR4.

AIMS: Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions. METHODS AND RESULTS: In this study, we showed that CXCL14 deficient platelets reveal reduced thrombus formation under flow compared with wild-type platelets using a standardized flow chamber. Addition of recombinant CXCL14 normalized platelet-dependent thrombus formation on collagen. Furthermore, we found that CXCL14 is a chemoattractant for platelets and mediates migration via CXCR4. CXCL14 promotes platelet migration of platelets through the receptor CXCR4 as evidenced by murine CXCR4-deficient platelets and human iPSC-derived cultured platelets deficient in CXCR4. We found that CXCL14 directly interacts with the CXCR4 as verified by immunoprecipitation and confocal microscopy. CONCLUSIONS: Our results reveal CXCL14 as a novel platelet-derived chemokine that is involved in thrombus formation and platelet migration. Furthermore, we identified CXCR4 as principal receptor for CXCL14, an interaction promoting platelet migration.

CRISPR/Cas9 Genome Editing of Human-Induced Pluripotent Stem Cells Followed by Granulocytic Differentiation.

Research on patient-derived induced pluripotent stem cells (iPSCs) could immensely benefit from the implementation of CRISPR/Cas9 genome editing of iPSCs, creating unique opportunities such as the establishment of isogenic iPSC lines for disease modeling or personalized patient-specific drug screenings. Here we describe a stepwise protocol of safe, efficient, and selection-free CRISPR/Cas9-mediated gene correction or knockout in human iPSCs followed by 3D spin-embryoid body (EB)-based hematopoietic/neutrophilic iPSC-differentiation.

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients.

A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.

Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.

CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible ß (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.

Human iPSC-based model of severe congenital neutropenia reveals elevated UPR and DNA damage in CD34+ cells preceding leukemic transformation.

We describe the establishment of an embryoid-body-based protocol for hematopoietic/myeloid differentiation of human induced pluripotent stem cells that allows the generation of CD34+ cells or mature myeloid cells in vitro. Using this model, we were able to recapitulate the defective granulocytic differentiation in patients with severe congenital neutropenia (CN), an inherited preleukemia bone marrow failure syndrome. Importantly, in vitro maturation arrest of granulopoiesis was associated with an elevated unfolded protein response (UPR) and enhanced expression of the cell cycle inhibitor p21. Consistent with this, we found that CD34+ cells of CN patients were highly susceptible to DNA damage and showed diminished DNA repair. These observations suggest that targeting the UPR pathway or inhibiting DNA damage might protect hematopoietic cells of CN patients from leukemogenic transformation, at least to some extent.

Simultaneous quantification of DNA damage and mitochondrial copy number by long-run DNA-damage quantification (LORD-Q).

DNA damage and changes in the mitochondrial DNA content have been implicated in ageing and cancer development. To prevent genomic instability and tumorigenesis, cells must maintain the integrity of their nuclear and mitochondrial DNA. Advances in the research of DNA damage protection and genomic stability, however, also depend on the availability of techniques that can reliably quantify alterations of mitochondrial DNA copy numbers and DNA lesions in an accurate high-throughput manner. Unfortunately, no such method has been established yet. Here, we describe the high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) and its suitability to simultaneously measure DNA damage rates and mitochondrial DNA copy numbers in cultured cells and tissue samples. Using the LORD-Q multiplex assay, we exemplarily show that the mitochondrial DNA content does not directly affect DNA damage susceptibility, but influences the efficacy of certain anticancer drugs. Hence, LORD-Q provides a fast and precise method to assess DNA lesions, DNA repair and mtDNA replication as well as their role in a variety of pathological settings.

Genome surveillance in pluripotent stem cells: Low apoptosis threshold and efficient antioxidant defense.

Pluripotent stem cells must be endowed with efficient genome surveillance. Here we describe the multiple mechanisms that ensure their genome integrity, including high susceptibility to apoptosis and efficient prevention of DNA lesions. In induced pluripotent stem cells, apoptosis hypersensitivity is mediated by increased expression of proapoptotic BCL-2 protein, whereas DNA damage is prevented by the upregulation of several antioxidant enzymes. Antioxidants might be therefore employed for safer stem cell therapies.

Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.

The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.