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Co-acquisition of CSF3R and RUNX1 mutations is the most frequent combination of acquired genetic abnormalities found in AML and MDS cases associated with severe congenital neutropenia (CN). Using the iPSC-based model for CN-associated leukemia, we compared the outcomes of RUNX1 missense mutations and RUNX1 haploinsufficiency by means of hematopoietic differentiation and transcriptome analysis. We generated iPSC lines from 2 CN/AML patients harboring ELANE mutations p.C151Y or p.G214R (CN1 and CN2, accordingly). CN/AML iPSC clones of CN patient 1 were generated from leukaemia cells with CSF3R p.Q741X, missense RUNX1 p.R139G mutation, and trisomy 21 (CN/AML1.1 and 1.2). CN/AML iPSC clones of CN patient 2 (CN/AML2) have CSF3R p.Q743X mutation and truncated RUNX1 mutation p.E175fs introduced by CRISPR/Cas9 gene-editing. The iPSC clones from CN (n = 4) and CN/AML stage (n = 6) were subjected for embryoid body (EB)-based differentiation into hematopoietic stem and mature myeloid cells. We observed dramatically increased proliferation of CN/AML iPSC-derived hematopoietic stem cell progenitors (HSPCs) collected at day 14 of EB differentiation and further cultured on the FLT3-L-secreting feeder cells, as compared to CN - iPSC derived HSPCs. Moreover, myeloid differentiation was severely diminished in CN/AML-iPSC group compared with CN iPSC group. To elucidate the mechanism of leukemogenic transformation in CN, we performed RNA-sequencing of CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from CN and CN/AML iPSC clones. Differential gene expression analyses of RNA-sequencing data of iPSC-derived HSPCs using DESeq2 R package identified 132 up- and 570 down-regulated (log2FC > 1 or < -1, adj. P-value < 0.05), as well as 579 up- and 1422 down-regulated (log2FC > 1 or < -1, adj. P-value < 0.05) genes between CN/AML and CN stages for CN patient 1 and CN patient 2, respectively. There were 189 differentially expressed genes shared between CN/AML1 vs CN1 and CN/AML2 vs CN2 comparisons. Among the top significantly upregulated pathways in the gene set enrichment analysis (GSEA) of CN/AML1 iPSCs with missense RUNX1 mutation were "Hallmark E2F Targets", "Hallmark Oxidative Phosphorylation", "Hallmark MYC Targets V1", whereas genes corresponding to the gene set "Platelet Specific Genes" were significantly downregulated. In CN/AML2 iPSCs with truncated RUNX1 mutation, we detected enrichment of the gene sets "Hallmark G2M Checkpoint", "Hallmark E2F Targets" as well as "Hallmark TGFb signaling". In contrast, the gene set "GO:Structural Constituent of Ribosomes" was enriched in CN2 derived HSPCs. Intriguingly, transcription factor enrichment analysis (TFEA) using the lists of differentially expressed genes (log2FC > 1 or < -1, adj. P-value < 0.05) demonstrated similarities between significantly enriched TFs motifs sets (P-value < 0.05) from both CN/AML vs CN comparisons. Among the transcription factor binding motifs significantly enriched in CN/AML cells (P-value < 0.05) with missense RUNX1 mutation and RUNX1 haploinsufficiency, we found motifs for GATA1, GATA2, and RUNX1, as well as for AML-associated factors such as SUZ12 and EZH2. The vast majority of enriched kinases (HIPK2, MAPK1/3/14, CSNK2A1, ERK1, AKT1, etc.) at the selected threshold (P-value Taken together, we found a dramatic difference in the gene expression signature between CN- and CN/AML-iPSCs derived HSPCs. The vast majority of significantly enriched transcription factors and kinases were overlapped in HSPCs carrying different types of RUNX1 mutations. These data will help to better understand the mechanistic outcomes of different types of the endogenously expressed mutated RUNX1 proteins in leukemogenesis. This info is crucial for the identification of specific drug targets for CN/AML and de novo AML with mutant RUNX1. Disclosures No relevant conflicts of interest to declare.

To the editor: Congenital neutropenia (CN) and cyclic neutropenia (CyN) are rare genetic disorders of hematopoiesis predominantly caused by ELANE mutations.[1][1][⇓][2]-[3][3] Due to overlaps in their genetic profiles, CyN can be distinguished from CN by cycling neutrophil counts, usually at 21

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have markedly reduced numbers of neutrophils in peripheral blood ( Here, we describe for the first time the application of CRISPR/Cas9 gene-editing in combination with recombinant adeno associated virus 6 (rAAV6)-based delivery of the template for homology-directed repair (HDR) for the mutated HAX1 gene in primary bone marrow mononuclear CD34+ cells (HSPCs) of HAX1-CN patients. We selected HAX1 mutation p.W44X as the most frequently described mutation in HAX1-CN. We established the delivery of the chemically modified sgRNA in combination with SpCas9 V3 in primary HSPCs using electroporation. The HDR template was generated by PCR from healthy donor HSPCs and cloned into pRC6 vector for the production of high titer rAAV6 (>12x1012 viral copies per ml). Our gene-editing protocol produced on average 79,7 % (± 8,62 %) of total editing (TE) in healthy donor HSPCs (n=6). When we transduced healthy donor HSPCs with rAAV6 containing the template at MOI 105 after electroporation with CRISPR/Cas9 RNP, we achieved 38,1 % (± 1,3 %) knock-in (KI) efficiency and 82,3 % (± 8,2 %) TE (n=2). We further applied this approach to primary HSPCs from 5 CN patients harboring the p.W44X HAX1 mutation. We achieved 84,4 % (± 4,2 %) TE and 65,8 % (± 7,12 %) KI. Too proof, that our editing reintroduced HAX1 protein expression, we performed Western Blot analysis of edited cells (n=2) and were able to detect relevant amounts of HAX1 protein. To assess the effect of HAX1 correction on the neutropenic phenotype in vitro, we performed a liquid culture differentiation assay of edited HSPCs to neutrophils. HSPCs from the same patients that were edited in the AAVS1 safe harbor were used as isogenic controls. In the AAVS1 locus the editing efficiency was 76,74 % (± 17,07 %) total indels. By morphological assessment of Wright-Giemsa stained cytospins of edited cells derived on day 14 of differentiation revealed significant (p = 0,005) increases of mature neutrophils for all five edited HAX1-CN patient samples, as compared to the respective controls. This phenotype correction was also observed in flow cytometry by a significant (p = 0,011) increase of mature CD34-CD45+ CD15+CD16+ neutrophils (n=5). To investigate if the HAX1 mutation correction and reinforced expression of HAX1 protein improved the sensitivity of HSPCs to oxidative stress as described by Klein et al. 2007, we performed live-cell imaging of caspase3/7 activation. Live-cell imaging revealed a substantial reduction of H2O2-induced apoptosis in corrected HAX1-CN patients derived HSPCs (n=3). Furthermore, the corrected differentiated cells were investigated for functional hallmarks of granulocytes. We could observe that HAX1 gene-edited HSPCs showed comparable chemotaxis, phagocytosis and no defects in ROS production to isogenic control edited cells. Taken together, we established a protocol for efficient selection-free correction of HAX1 p.W44X mutation in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. Our gene-editing reintroduced HAX1 protein expression in primary HSPCs from HAX1-CN patients. Neutrophils derived from corrected cells showed functional improvements in survival to oxidative stress and general neutrophil functions. We believe that these results are enticing to be investigated further for potential clinical translation as an autologous stem cell therapy for HAX1-CN patients. Disclosures No relevant conflicts of interest to declare.

The transcription factor lymphoid enhancer-binding factor 1 (LEF-1), which plays a definitive role in granulocyte colony-stimulating factor (G-CSF) receptor-triggered granulopoiesis, is downregulated in granulocytic progenitors of severe congenital neutropenia (CN) patients. However, the exact mechanism of LEF-1 downregulation is unclear. CN patients are responsive to therapeutically high doses of G-CSF and are at increased risk of developing acute myeloid leukemia. The normal expression of LEF-1 in monocytes and lymphocytes, whose differentiation is unaffected in CN, suggests the presence of a granulopoiesis-specific mechanism downstream of G-CSF receptor signaling that leads to LEF-1 downregulation. Signal transducer and activator of transcription 5 (STAT5) is activated by G-CSF and is hyperactivated in acute myeloid leukemia. Here, we investigated the effects of activated STAT5 on LEF-1 expression and functions in hematopoietic progenitor cells. We demonstrated that constitutively active STAT5a (caSTAT5a) inhibited LEF-1-dependent autoregulation of the LEF-1 gene promoter by binding to the LEF-1 protein, recruiting Nemo-like kinase and the E3 ubiquitin-ligase NARF to LEF-1, leading to LEF-1 ubiquitination and a reduction in LEF-1 protein levels. The proteasome inhibitor bortezomib reversed the defective G-CSF-triggered granulocytic differentiation of CD34(+) cells from CN patients in vitro, an effect that was accompanied by restoration of LEF-1 protein levels and LEF-1 messenger RNA autoregulation. Taken together, our data define a novel mechanism of LEF-1 downregulation in CN patients via enhanced ubiquitination and degradation of LEF-1 protein by hyperactivated STAT5.

Congenital neutropenia (CN) is a bone marrow failure syndrome which is characterized by a maturation arrest of myeloid progenitors in the bone marrow. The pathomechanism leading to the defective granulopoiesis in CN patients is not fully elucidated. We recently reported the inability of G-CSF to induce GADD45β (Growth arrest and DNA damage-inducible 45, beta) expression in myeloid progenitors of CN patients harboring ELANE or HAX1 mutations. GADD45β functions as stress sensor downstream of myeloid cytokines and is important in murine stress-induced hematopoiesis. Moreover, we could show that C/EBPα, which is severely diminished in CN cells, binds and activates the GADD45β promoter. Importantly, rescue of the GADD45β expression in ELANE-CN HSPCs by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation compared to control transduced cells. Transduction of mouse HSCs with ectopic Gadd45β resulted in drastic decrease of immature and increase of mature myeloid cells over time. Colony forming unit (CFU) and CFU re-plating assay showed that Gadd45β overexpressing mouse HSCs exhibit lower proliferation capacity. To determine the role of GADD45β in vivo, we performed CRISPR/Cas9 knockout of gadd45bb in wild type and Tg(mpo:gfp) zebrafish embryos, in which GFP expression is activated by the mpo promoter. We found that gadd45bb-deficiency lead to dramatic reduction in neutrophil numbers as assessed by counting mpo+ cells. Ectopic expression of G-CSF using a heat-inducible system increased the number of mpo+ cells in gadd45bb crispants but not to a level similar to wild type zebrafish expressing G-CSF. GADD45 proteins are implicated in active DNA demethylation by recruiting the DNA repair machinery to specific sites in the genome to convert 5-methylcytosine into unmethylated cytosine. Nothing is known about active DNA demethylation downstream of G-CSF and the role of GADD45β in this process. We sought to unveil whether GADD45β mediates G-CSF triggered myeloid differentiation by specific gene demethylation. To address this, we performed RNA sequencing and DNA methylation analysis of wild type (WT) and GADD45β knockout (KO) CD34+ HSPCs treated or untreated with G-CSF for 72h. The RNA-seq analysis demonstrated a GADD45β-dependent upregulation of genes which play a role in neutrophil differentiation and function as for instance RXRA, ITGAM (CD11b), FPR1/2, MEFV and BATF upon G-CSF treatment. Strikingly, these genes were also upregulated in promyelocytes of healthy controls upon G-CSF treatment, but not in CN patient promyelocytes. Genomatix-based gene ontology analysis of significantly upregulated genes in the G-CSF-treated WT but not GADD45β KO group displayed that diseases associated with this expression signature included neutropenia, leukocyte disorders and AML. Motif enrichment analysis of GADD45β-responsive genes predicted mainly motifs for myeloid-specific transcription factors (e.g. RXRA, SPI1 and C/EBPβ) to be enriched. Finally, DNA methylation analysis revealed significant differences in methylation pattern between GADD45β WT and KO groups upon G-CSF treatment. In WT cells G-CSF caused the hypomethylation of 13.516 CpGs and in GADD45β KO cells, only 8.440 CpGs were hypomethylated. Methylation changes between WT and KO groups mainly occurred in myeloid-specific genes, e.g. ELANE, MPO, CSF3R and AZU1. We also identified myeloid differentiation-related genes of which mRNA expression was regulated by GADD45β-mediated DNA demethylation, as for instance RXRA, CXCR1, MEFV, FPR2 and SERPINA1. Importantly, diseases associated with the defective gene demethylation downstream of G-CSF were neutropenia, immune system diseases, pre-leukemia and AML, as assessed by Genomatix analysis. Taken together, we report for the first time the importance of GADD45β-dependent DNA demethylation for human and zebrafish G-CSF-triggered neutrophil differentiation. The failure of G-CSF to induce GADD45β in CN patients might be a reason for the maturation arrest of granulopoiesis. Disclosures No relevant conflicts of interest to declare.

Background: Congenital neutropenia (CN) is a group of rare inborn genetic disorders of hematopoiesis including a variety of different gene mutations and different patterns of inheritance. Independent of the underlying genetic subtypes, CN patients benefit from G-CSF maintenance treatment, which improved the life expectancy and quality of life significantly. The majority of CN patients documented by the SCNIR Europe respond well to G-CSF treatment and achieve and maintain neutrophil response with an anticipated ANC of greater 1000/µL with a median G-CSF dose of 4.77 µg/kg/day. However, the G-CSF dose used is very heterogeneous and dose increase is dependent on the decision of each treating physician. Here we report on the incidence, clinical characteristics and outcome of patients receiving G-CSF doses of 20µg/kg/d or higher. Methods: In the European database of the SCNIR we have identified 420 CN patients with different genetic subtypes (including SDS and GSD1b) who are treated with G-CSF. Non-response is defined as median neutrophil counts below 500/µL upon G-CSF doses of 20µg/kg/day or higher. Partial responders have median ANCs of 500 to 999/µL with these G-CSF doses or severe infections despite ANCs of greater 1000/µL. Eighty of the 420 pts received G-CSF doses of 20µg/kg/d or higher. Results: According to the above described response criteria we identified 26 non- responders (6.2%), 18 partial responders (4.3%) and 36 patients reaching ANCs above 1000 with G-CSF doses of 20µg/kg/d or higher (32 patients receiving 20 to 39µg/kg/d; 4 patients receiving more than 50µg/kg/d) out of the 420 CN patients treated with G-CSF. Although patients with different genotypes receive long-term G-CSF treatment, only ELANE or JAGN1 mutations were identified in both non- and partial responders. Analysing ELANE patients separately, 9.3% were non-responders and 5.3 % partial responders to G-CSF. While gender ratio male/female is almost even in the CN cohort (1.08 including 6 males with x-linked TAZ mutations) as well as in the ELANE subgroup (0.87), the gender ratio for ELANE non-responders is 0.2 and 0.4 in ELANE partial responders. In 50% of the identified non- and partial responders the genotype is still unclassified. In 3 patients who were not included in the analysis G-CSF non-response was due to a homozygous CSF3R mutation. These patients respond well to GM-CSF but not to G-CSF. Conclusions: Non-response or partial response to high doses of G-CSF treatment is correlated with distinct genotypes (ELANE, JAGN1, SRP54 and so far unclassified). However, requirement of high G-CSF doses (20µg/kg/d or higher) does not per se indicate non-response, since a substantial number of patients reach sufficient ANCs with these G-CSF doses, especially in patients with ELANE and HAX1 genotype. Treating physicians often do not increase G-CSF doses to >20µg/kg/d to rule out response to high doses. Patients with CSF3R homozygous mutations cannot respond to G-CSF, but respond well to GM-CSF, while ELANE or JAGN1 patients do not respond to GM-CSF. While patients with HAX1 mutations may require high G-CSF doses, but then respond well, patients with SBDS or other mutations (e.g. G6PC3, CXCR4, GFI-1, TAZ etc.) did not require high G-CSF doses for a longer period or were non-responsive to G-CSF treatment. Our data support the dosing recommendation of the SCNIR not to stop G-CSF at 20µg/kg/day, but to increase further, since a substantial number of patients are still responsive. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (CN) is a monogenic bone marrow failure syndrome with the frequency of 1:200,000 and is characterized by an absolute neutrophil count below 500 cells per microliter. Patients with CN suffer from severe life-threatening bacterial infections starting early after birth due to the absent or very low numbers of neutrophils in peripheral blood. While CN is a heterogeneous disease caused by many different gene mutations, autosomal-dominant ELANE mutations are the most common cause of CN. Although the majority of CN patients respond to daily treatment with granulocyte colony-stimulating factor (G-CSF), approximately 15 % do not respond at doses up to 20 μg/kg/day and approximately 20 % of G-CSF treated patients develop myelodysplasia (MDS) or acute myeloid leukemia (AML). In the present study, we first established an efficient gene-editing platform for induced pluripotent stem cells (iPSC) of CN patients using CRISPR/Cas9 technology. The platform uses ribonucleoprotein form of CRISPR/Cas9 making the editing approach safer as it is virus- or DNA free. Also, any further selection step or introducing extra modifications in the genome of edited cells such as silent mutation are not required. We generated and characterized iPSCs from ELANE-CN patients harboring p.A57V, p.C151Y, and p.G214R mutations, that are more severe hot-spot mutations associated with G-CSF non-response or MDS/AML. We corrected each mutation followed by EB-based hematopoietic differentiation, to evaluate and compare granulocytic differentiation of CN-patient specific iPSCs, with or without ELANE mutation, in an isogenic model. To study granulocytic differentiation, we performed live cell counts, flow cytometry analysis of myeloid-specific surface marker expression, CFU assay, cell morphology of cytospin preparations and neutrophil functional tests. Our isogenic model showed that correction of ELANE mutations led to fully normalized granulocytic differentiation. We have recently shown that CRISPR/Cas9 mediated ELANE knockout (KO) enables neutrophilic maturation of primary HSPCs and iPSCs of CN patients. We observed that granulocytic differentiation of ELANE KO iPSCs and primary HSPCs were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO neutrophils were also normal. To model CN in silico and to reveal the key driving pathomechanisms, we designed an isogenic patient-specific disease modeling system by comparing RNA-sequencing results of CN-ELANE corrected- or CN-ELANE KO hematopoietic stem and progenitor cells (HSPCs) to the original CN-ELANE patient cells. HSPCs were derived from iPSC lines. Our analysis showed a degree of similarity in enriched pathways upon ELANE correction or ELANE KO in a patient-specific manner. Thus, upon correction of p.C151Y mutation, TNF, IL4 and IL13 signaling pathways as well as MAPK signaling, PD-1 signaling and IL10 signaling were down-regulated. Interestingly, the same pathways were down-regulated upon ELANE KO in HSPCs of the same CN patient. Correction of p.A57V mutation led to down-regulation of IL12 expression which activates STAT family. Upon ELANE KO in the cells from the same patient, IL12, IL18, and IL1-beta expression were down-regulated. We also identified common pathways enriched in most of the isogenic samples upon ELANE correction or ELANE KO like down-regulation of MAPK or IFN α/β signaling as well as down-regulation of the Rap-1 signaling pathway leading to the Erk pathway activation. Analysis of putative transcription factor binding sites (TFBSs) that are enriched in the differentially expressed gene list upon ELANE mutation correction or ELANE KO showed that transcription factors GKLF (KLF4), MAZ, Kaiso (ZBTB33) and CHURCHILL are highly enriched in UP-regulated genes, for both, correction and KO samples. Taken together, we established a safe and efficient CRISPR/Cas9-RNP based ELANE gene-correction/knockout platform of iPSCs of ELANE-CN patients that may be used to establish an isogenic disease modeling system or provide novel stem cell-based therapy for CN patients with a high risk of leukemia development as well as for G-CSF-non-responsive patients. This platform could be also applied for other monogenic bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

Background: Primary Autoimmune Neutropenia (AIN) is the most frequent type of neutropenia in children with a prevalence of 1/100,000 between infancy and 10 years of age. Primary AIN is caused by anti-neutrophil antibodies binding to neutrophil-specific antigens, resulting in a decrease of circulating neutrophils in the blood, but normal numbers of mature neutrophils in the bone marrow. Typically, primary AIN is present from infancy on until spontaneous remission in early childhood, when anti-neutrophil antibodies disappear. Patients with primary AIN show severe to moderate neutropenia but only rarely suffer from serious infections. Patients remain in the registry for follow up after normalization of blood counts to evaluate late secondary events. Aims: Here we describe the cohort of AIN pts with positive anti-neutrophil antibody testing documented by the European Branch of the SCNIR. We analyzed the course of neutropenia, the frequency of G-CSF treatment for AIN, the incidence of severe bacterial infections and administration of AB prophylaxis. Methods: We identified 102 primary AIN patients within the neutropenia cohort documented by the European Branch of the SCNIR since 1994. We classified primary AIN by positive anti-neutrophil antibody testing (95 pts) or severe neutropenia in peripheral blood with normal bone marrow morphology in patients with age under 5 years (7 pts). Results: Primary AIN has been identified in 102 (61 female; 41 male) pts. The median age of the cohort is 5.18 years (range 1.37-22.71 years), with 630.28 pt years under observation. Median age at diagnosis was 12.07 months (range 0.9-70 months). All pts are currently alive, 40 patients already resolved from primary AIN at a median age of 3.02 years (range 0.83-9.08 years). Median follow-up time after neutropenia had resolved was 2.25 years (range 0-9.27 years). Sixteen of 102 pts (15.7%) received intermittent G-CSF treatment with a median dose of 4.5 µg/kg/day compared to 4.77 µg/kg/day for the congenital neutropenia cohort of the SCNIR Europe. Analysis of infections (tab.1) showed less minor and severe infections comparing to congenital neutropenia (CN) pts. Life-threatening infections like liver abscesses were not seen in primary AIN patients but in 1.8% of CN pts. Twelve AIN pts (11.7%) have received antibiotic prophylaxis for prevention of infection, 6 pts intermittent and 6 pts continuously. However, antibiotic prophylaxis was usually stopped before termination of AIN. Due to the milder course of infections most AIN pts were able to go to Kindergarten and to live a normal life. In 3 pts additional auto-immune related diseases were identified (autoimmune thrombocytopenia, allergic colitis and Kawasaki syndrome) during AIN. Sixteen of 102 AIN patients received genetic analysis, with no mutation being detected. In addition to the 102 AIN pts we identified another 31 CN pts who have initially been classified as AIN due to positive anti-neutrophil antibodies, but who were later genetically confirmed as CN (15 ELANE+, 8 HAX1+, 2 SBDS+, 2 CXCR4+, 2 CSF3R+, 1 G6PC3+). This proportion of pts showed more and more severe infections compared to primary AIN. Genetic testing has been performed in these pts due to ongoing infections and prolonged neutropenia until school age. Conclusions: Pts suffering from primary AIN present with severe to moderate neutropenia. A minority of pts might require G-CSF treatment on demand/interventionally due to antibiotic resistant infections, but long-term G-CSF treatment is regularly not indicated. Primary AIN is a self-limiting condition and in most pts neutropenia resolves until early childhood. Accumulation of secondary diagnoses like autoimmune related diseases, though postulated, has not been confirmed for AIN pts by our data. In AIN pts with severe infections, or prolonged neutropenia CN should be ruled out by genetic analysis and/or bone marrow morphology. Registries are needed to document long-term data on primary AIN pts to analyse potential additional features of primary AIN, possibly other accompanying autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood ( Up to now, the only curative therapy for CN patients that do not respond to G-CSF or with overt AML remains hematopoietic stem cells transplantation with its associated risks. A clinical need for gene therapy for these patients is imminent. We recently described the CRISPR/Cas9 mediated ELANE knockout as a possible gene therapy approach for CN patients with ELANE mutations (ELANE-CN) (Nasri et al. 2019). As an alternative, we wanted to test if specific target therapy for individual ELANE-CN patients could be an option. Here we describe the correction of ELANE mutations using CRISPR/Cas9 to edit the ELANE gene and recombinant adeno-associated virus 6 (rAAV6) to deliver a template for homology directed repair (HDR). We selected ELANE mutations p.A57V or p.A57T in exon 2, and p.G214R or p.G214RV in exon 5, both known hot spot mutations observed in G-CSF non-responders or in CN/AML patients (Makaryan et al. 2015). We used SpCas9 V3 and chemically modified sgRNA. For exon 2, we choose the highly efficient sgRNA (Nasri et al. 2019) yielding the benefit, that double-strand breaks (DB) that do not result in HDR correction are producing ELANE knockout. For exon 5, we established a sgRNA that produced average 87% (± 6%) editing in healthy donor cells. Two HDR donor template backbones (DTB) were generated. DTB1 is spanning exons 1-3 and DTB2 exons 4-5 of ELANE. Silent mutations were introduced in the repair templates for both ELANE mutations between the cut site and mutation to enhance HDR. To test the knock-in efficacy, we electroporated healthy donor CD34+cells with CRISPR/Cas9 RNP and transduced them with rAAV6 containing the templates at MOI 105. We achieved 34,5% (± 4,5%) knock-in (KI) and 35,6% (± 2,5%) indels for exon 2, or 39,2% KI (± 12,8%) and 18,85% indels (± 4,25%) for exon 5. Edited cells showed high viability, expanded and differentiated well into neutrophils in vitro. We further applied this approach to primary HSPCs from 4 CN patients harboring selected ELANE mutations. For p.A57, we achieved 14% (±2,3%) KI and 44,7% (±1,9%) indels. For p.G214, the KI was 59,9% (± 0,1%) and indels 28,8% (± 0,6%). To assess the effect of ELANE correction on the neutropenic phenotype in vitro, we performed CFU and liquid culture neutrophilic differentiation assays. We compared the corrected cells to cells from the same patient that were edited in the AAVS1 safe harbor, as isogenic controls. We observed a significant (p < 0,05) increase in number of CFU-GMs for CRISPR/Cas9 edited HSPCs from two CN patients with p.A57V/T mutations and of CFU-G or CFU-GM for two CN patients with p.G214R/V ELANE mutation. Morphological assessment of Wright-Giemsa stained cytospins of cells derived on day 14 of differentiation revealed significant increases of mature neutrophils for all four edited patient samples ascompared to the respective controls. Further we performed live cell imaging of neutrophil extracellular trap (NET) formation after PMA stimulation and chemotaxis. NET formation was either improved or comparable between control- and ELANE- edited cells. Chemotaxis showed no difference between control- and ELANE-edited cells. For a patient with p.G214V ELANE mutation, we were able to evaluate chemotaxis and phagocytosis in vivo in zebrafish embryos at 48hpf, as described in Nasri et al 2019. This showed a qualitative improvement of ELANE- corrected cells ascompared to control AAVS1 edited cells. This indicates that our manipulation does not alter the functionality of produced neutrophils while increasing the number of mature cells being produced. Taken together, we established a protocol for efficient correction of ELANE mutations in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. We reached high enough editing to correct the dominant negative effects of mutations, as assessed by markedly improved neutrophilic differentiation in vitro. Generated repair constructs allow fast adaptation to patient-specific mutations in all exons of ELANE. This approach is enticing to be investigated further for clinical translation. Disclosures No relevant conflicts of interest to declare.

To the editor: Severe congenital neutropenia (CN) is a heterogeneous disease characterized by an absolute neutrophil count (ANC) below 500 cells per microliter and recurrent, life-threatening bacterial infections. Treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) was

The mechanism of maturation arrest of bone marrow myeloid progenitors in severe congenital neutropenia (CN) patients is not fully elucidated. We found, that treatment of healthy individuals with G-CSF induces mRNA expression of GADD45b (Growth Arrest and DNA-Damage-inducible, beta) in CD33+ bone marrow myeloid progenitors. However, the expression of GADD45b was not activated in CD33+ cells of G-CSF treated CN patients. GADD45b functions as stress sensor downstream of G-CSF signaling and is essential in stress-induced murine myelopoiesis. Less is known about the function of GADD45b in the myeloid differentiation of human HSPCs. We hypothesized, that the inability of G-CSF to induce GADD45b expression might be a cause of diminished granulopoiesis in CN patients. To test this hypothesis, we inhibited GADD45b expression in CD34+ cells and iPSCs of healthy donors by introducing indels in exon 1 of the GADD45B gene using specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). We evaluated G-CSF-triggered myeloid differentiation of GADD45b-deficient iPSCs using embryoid body (EB)-based method and found that iPSCs cells present with severely diminished granulocytic differentiation upon GADD45b knockout, as assessed by FACS, CFU assay and morphological examination of cytospin slides. We also observed reduced G-CSF-mediated granulocytic differentiation of GADD45b-deficient CD34+ cells of healthy individuals in colony-forming units (CFU) assay and liquid culture differentiation followed by FACS analysis on day 7 and day 14. Importantly, rescue of GADD45b in HSPCs of one CN patient by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation, as compared to control transduced cells. These data strongly support the essential role of GADD45b in G-CSF-mediated granulocytic differentiation. GADD45b rescue analysis of additional CN patients cells is ongoing. To study the mechanism of GADD45b activation upon G-CSF stimulation of hematopoietic cells, we performed in silico analysis of GADD45B promoter and found putative binding sites for G-CSF responsive hematopoietic transcription factors, including CEBPA, CEBPB, KLF4, STAT3 and STAT5. Using the dual luciferase reporter assay with 1.6 kb region of the GADD45B gene promoter, we found that KLF4, STAT5, CEBPA and CEBPB activate GADD45b expression in a dose-dependent manner. Intriguingly, CEBPA expression is severely diminished in myeloid cells of CN patients (Skokowa et al., 2006) and we assumed that G-CSF is not able to activate GADD45b expression in CN patients because of defective CEBPA. To study the mechanism by which GADD45b mediates myeloid differentiation, we performed RNA sequencing of WT or GADD45b-deficient CD34+ HSPCs treated or not with G-CSF. Interestingly, in GADD45b-deficient cells, G-CSF failed to induce mRNA expression of several genes essential for granulocytic differentiation and granulocyte functions including GLI1, CAMP/LL37, MMP8, CD16, LCN2, OLFM4, CX3CR1, SIGLEC5, as compared to WT cells. Reactome and Gene Set Enrichment Analysis (GSEA) of RNA-Seq data sets also revealed deregulation of the "myeloid CEBPA network", "GLI proteins pathway" and "neutrophil degranulation pathway" in G-CSF-treated GADD45b-deficient CD34+ cells, as compared to control G-CSF-exposed cells. Of note, severely diminished expression of plasma CAMP/LL37 levels is a unique feature of CN patients (Y. Ye et al. 2015). In summary, our data suggest that GADD45b plays an essential role in granulocytic differentiation of human hematopoietic cells and inability of G-CSF to induce GADD45b expression in myeloid cells of CN patients may be a reason for the defective granulopoiesis. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: A promising rich pipeline of combination therapies targeting checkpoint molecules expressed on T cells and/or tumor cells is currently being developed to abrogate tumor-induced immunosuppression. Novel in vivo models suitable for validating these immunotherapies and predict safety issues are warranted to accelerate their translation to patients. AIM: Epstein Barr virus (EBV) is a type 1 carcinogen that is directly associated with the development of human B cell neoplasms. We modelled EBV infection and tumor progression in long-term humanized mice and investigated the activation of T cells with PD-1 expression. Further, we performed studies evaluating the effects of an anti-PD-1 antibody (pembrolizumab/ keytruda) in on EBV infections and/or tumor growth. METHODS: Humanized mice transplanted with human cord-blood CD34+ stem cells and showing long-term (15 weeks) human T cell reconstitution were infected with an oncogenic recombinant Epstein Barr Virus (EBV), encoding enhanced firefly luciferase (fLuc) and green fluorescent protein (GFP). EBV infections were monitored by optical imaging analyses and PCR. CD8+ and CD4+ T cell subtypes (PD-1+, naïve, central memory, effector memory and terminal effector) were sequentially monitored in blood by longitudinal flow cytometry analyses and in organs at experimental endpoint. Histopathological analyses were performed to characterize EBV infection (EBER+) and PD-1+ T cell-rich infiltrates in tissues and tumors. We used the model to evaluate the effects of pembrolizumab administered after EBV challenge at low dose (first dose 1.65mg/kg and then 3.30 mg/kg, every other week, n=3) or high dose (first dose 5.00 mg/kg and then 10.00 mg/kg every other week, n=3) in respect to EBV infected controls (n=2). RESULTS: EBV-fLuc was detectable one week after infection by non-invasive optical imaging in the spleen, from where it spread rapidly and systemically. Among the EBV-infected mice, 8/18 (=44%) developed macroscopically visible tumors in the spleen. For further analyses of the data, we then compared EBV-infected mice with ("EBV-Tumor") or without ("EBV") macroscopic tumors. At 6 weeks post-infection, the relative CD8+ T cell frequencies increased significantly and constantly (control Vs. EBV p=0.0021, control Vs. EBV-Tumor p= CONCLUSIONS: We are currently performing additional experiments in order to elucidate this mechanism of EBV rebound. This humanized mouse model contributes to risk assessment prior to clinical trials of the use of checkpoint inhibitors in patients after transplantations at high risk of EBV infections. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

Objectives: Epstein-Barr virus (EBV) is associated with lymphoproliferative disease in immunocompromised hosts and with human B-cell lymphomas and carcinomas. Adoptive transfer of virus-specific T cells is not practical when memory T cells from HLA-matched donors are not available. Hence, we designed T cells expressing EBV-specific chimeric antigen receptors (CARs) to bypass the need of matched memory T cells. The surface-bound glycoprotein 350 (gp350) was used as target, because it is abundantly expressed during lytic EBV replication and it can also be detected in EBV-immortalized cells. Methods: gp350-CARs were constructed by fusion of single-chain variable fragments of two high affinity gp530-specific human mABs (7A1 and 6G4) to CAR-backbones containing the CD28/CD3ζ domains. Transduction of human T cells from PBMC and cord blood with γ-retroviral vectors showed higher expression levels of 7A1-gp350-CAR than 6G4-gp350-CAR. Results: We used 293T cells expressing gp350 and B95-8 immortalized cells from a tamarin monkey (6-10% gp350+) and human B cells immortalized with the EBV laboratory strain M81 (30% gp350+) in order to compare the potency of gp350-CAR T cells in vitro. Both 7A1-gp350-CAR and 6G4-gp350-CAR were activated and proliferated in the presence of gp350+ cells, inducing cytotoxicity of the target cells. Pilot experiments in a preclinical humanized mouse model consisting of Nod.Rag mice transplanted with human cord-blood (CB) stem cells and infected with an EBV/fLUC strain, we could confirm persistence of CB-matched 7A1-gp350-CAR T cells in spleen, bone marrow and lung for up to 6 weeks. In some animals, this was correlated with lower EBV dissemination measured by optical imaging and PCR. Conclusions: We showed that EBV-specific CARs can reprogram naïve or memory T cells from PBMC or CB to react against EBV infected cells in an HLA-independent manner. This approach can be translated in the future for the generation of anti-EBV-CAR T cells for patients in Need. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

Patients with pre-leukemic bone marrow failure syndrome, severe congenital neutropenia (CN) have ~ 20% risk of developing acute myeloid leukemia (AML) (CN-AML). More than 70 % of CN-AML patients co-acquire CSF3R and RUNX1 mutations as shown by our group (Skokowa et al 2014), indicating a cooperative role of the mutations in these two genes in the development of AML in CN patients. In order to investigate the interaction between these mutations we conducted in vitro experiments on lineage negative (lin-) bone marrow mononuclear cells (BMCs) from C57BL/6-d715csf3r mice (d715-mice). These mice carry homozygous d715G CSF3R mutations, but do not develop AML. We isolated lin- BMCs from d715 mice and transduced these cells with four different lentivirus vectors carrying BFP only (CTRL), RUNX1-Wild type BFP (RUNX1-WT), RUNX1-R139G BFP (RUNX1-MUT1) and RUNX1-R174L BFP (RUNX1-MUT2). These RUNX1 mutations where found in CN-AML patients. 72 hours after transduction, we sorted BFP+ cells and compared G-CSF triggered myeloid differentiation in vitro. We found that cells transduced with each RUNX1 mutants exhibited reduced percentages of myeloid CD11b+, Gr-1+ and double positive cells compared to RUNX1-WT. We also conducted CFU re-plating experiments with transduced cells and found that cell transduced with each of RUNX1 mutants showed 7- (RUNX1-MUT1) and 8- (RUNX1-MUT2) times higher re-plating capacity than RUNX1-WT and CTRL transduced cells. To identify signaling pathways that are deregulated in G-CSFR-mutated HSCs clones after co-acquisition of RUNX1 mutations, we performed microarray study. We starved transduced and sorted lin- BMCs for 24 hours and treated cells with G-CSF for 48 hours before mRNA was collected. Expression profiles where generated by microarray (GeneChip Mouse Gene 2.0 ST Array). Pathway Analysis was conducted using IPA Software and Motif activity response analysis (MARA) was performed using ISMARA web tool. Reported Transcription factors and targets have Z-value ≥ 2 or ≤-2 and p ≤ 0,05 and are thus considered statistically significant. Interestingly, ISMARA analysis showed, that the highest active motif in RUNX1-Mutants was Irf2_Irf1_Irf8_Irf9_Irf7 motif which is essential for the regulation of the interferon pathway genes. The corresponding transcription factors are amongst others regulated by Sp1 and Stat2 that were also active. Correspondingly, IPA Pathway analysis showed, that Interferon Signaling was highly upregulated in cells transduced with each of two RUNX1 mutants, compared to overexpressed RUNX1-WT (Z = 2). Additionally, pathway analysis showed the upregulation and activation of IL-6, IL-8-, Toll like Receptor- and TREM1 signaling pathways. This data suggests that the mutated RUNX1 may cause activation of the pro-inflammatory cell state propagating proliferation, which may be emerging as a cause of clonal hematopoiesis (CH) and consequently may lead to MDS/AML (Hemmati et al 2017). Another active motif is Spi1/PU.1 mainly known as an essential transcription factor for monocytic differentiation, but also as a maintenance factor of the pre-leukemia initiating cells (pre-LICs) or even leukemia initiating cells (LICs) (Staber et al 2014). Interestingly, we recently described elevated expression of PU.1 in hematopoietic cells of CN patients. These data together with our in vitro finding indicating that RUNX1 mutation causes differentiation block and clonal proliferation of HSCs, supporting the hypothesis, that the RUNX1 mutations are the driving factor in leukemic transformation in CN. Additionally, ISMARA revealed an upregulation of the Metyl-CpG Binding Protein 2 (Mecp2) motif. Mecp2 is a proto-oncogene that represses transcription through interaction with the corepressor SIN3A and histone deacetylases. Thus, remodeling the transcriptional profile and inhibiting differentiation. Taken together, our data shows that RUNX1 mutations in combination with CSF3R mutations may cause (1) increased proliferation through the induction of a proinflammatory cell state, (2) induce self-renewal through expression of essential proteins for LIC maintenance and (3) dimineshed myeloid differentiation through demethylation inhibition and down regulation of hematopoietic differentiation pathways. We are currently validating the model of the leukemogenic transformation in CN patients using functional studies in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. We recently reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. To study the mechanism of leukemia development in CN, we established a model for step-wise leukemia progression in CN using iPSC-based hematopoietic differentiation in combination with CRISPR/Cas9-mediated gene editing of iPSCs. Using this model, we confirmed that co-acquisition of CSF3R and RUNX1 mutations is necessary and sufficient to induce leukemia in CN. We also identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event downstream of RUNX1 and CSF3R mutations. BAALC mRNA was upregulated in CN/AML blasts (n = 5) and in CD34+ HSPCs generated from CN/AML iPSCs of two patients. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs reversed defective myeloid differentiation of CN/AML blasts to the levels observed in healthy donor hematopoietic stem cells. We further investigate the mechanism of BAALC up-regulation. In silico analysis of the BAALC gene promoter in combination to publicly available ChIP-Seq data revealed three putative RUNX1 binding sites that were validated using ChIP assay in lysates of NB4 cells. Interestingly, transduction of healthy donor CD34+ cells with lentiviral constructs expressing WT RUNX1 led to inhibition of BAALC mRNA expression, whereas transduction with two RUNX1 RUNT domain mutants resulted in the marked BAALC up-regulation, as compared to the control BFP transduced cells. These data suggest that mutated RUNX1 failed to inhibit BAALC expression in CD34+ HSPCs. To evaluate the mechanism of leukemogenic transformation in CN, we performed RNA-Seq analysis of CD34+ cells derived from CN and CN/AML iPSC clones. GSEA revealed that changes in gene expression between CN- and CN/AML-HSPCs were strongly correlated with gene expression signatures of "Wierenga STAT5 targets" and "reactome ATF4 targets", an observation in line with the markedly elevated levels of STAT5 and ATF4 in CN/AML-HSPCs. Importantly, gene expression differences between CN/AML-HSPCs and CN-HSPCs were correlated to "Valk AML" targets in GSEA, suggesting that HSPCs generated from CN/AML-iPSCs possess characteristics of AML cells. Strong support for the leukemogenic role of upregulated BAALC in CN/AML was provided by further GSEA analysis of the BAALC KO CN/AML-HSPCs. We observed a reversal in the expression of a majority of genes in the studied leukemia-associated pathways in CN/AML-HSPCs after BAALC knockout compared with CN/AML-HSPCs. Since there are no direct inhibitors for BAALC available and protein structure is not solved yet, BAALC effects can be targeted only indirectly. Morita et al., Leukemia, 2015 showed that BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and Klf4. We treated CD45+ cells generated from CN/AML or healthy donor (HD) iPSCs with MEK1/2- or MEK1-specific inhibitors or vehicle control and evaluated cell proliferation and differentiation (CFU assay). We were able to induce ~ 40-60 % cell death of CN/AML cells upon treatment with each of inhibitors, whereas HD CD45+ cells were unaffected. Moreover, treatment of CN/AML cells with MEK1/2 inhibitor led to an increase in CFU-G formation, as compared to vehicle control cells. Using connectivity Map analysis of RNA-Seq data of CD34+ cells generated from CN/AML iPSCs vs CN/AML BAALC KO iPSCs, we identified small molecule p38/MAPK14 inhibitor that could possibly reverse BAALC-mediated leukemogenic gene expression signature. We treated CN/AML iPSC-generated CD34+ cells for 7 days with this inhibitor and subsequently performed CFU assay. We found an increase in CFU-GM formation. In summary, using CN/AML-iPSC-model, we confirmed the major role of BAALC in leukemia development downstream of CSF3R and RUNX1 mutations in CN. Inhibition of MAPK/ERK-pathway downstream of BAALC reduced proliferation and partially induced myeloid differentiation of CN/AML-derived hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Like most other bone marrow failure syndromes, it is associated with a marked propensity to transform into a myelodysplastic syndrome (MDS) or acute leukemia, with a cumulative rate of transformation to MDS/leukemia that exceeds 20%. The genetic (and/or epigenetic) changes that contribute to malignant transformation in SCN are largely unknown. In this study, we performed mutational profiling of 14 genes previously implicated in leukemogenesis using 14 MDS/leukemia samples from patients with SCN. We used high-throughput exon-based resequencing of whole-genome–amplified genomic DNA with a semiautomated method to detect mutations. The sensitivity and specificity of the sequencing pipeline was validated by determining the frequency of mutations in these 14 genes using 188 de novo AML samples. As expected, mutations of tyrosine kinase genes (FLT3, KIT, and JAK2) were common in de novo AML, with a cumulative frequency of 30%. In contrast, no mutations in these genes were detected in the SCN samples; instead, mutations of CSF3R, encoding the G-CSF receptor, were common. These data support the hypothesis that mutations of CSF3R may provide the “activated tyrosine kinase signal” that is thought to be important for leukemogenesis.