Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have reduced numbers of neutrophils in peripheral blood (<500/µl) leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. The majority of patients harbor heterogenous mutations in ELANE, coding for Neutrophil Elastase. 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.
Use of the Internet of Things (IoT) is poised to be the next big advancement in environmental monitoring. We present the high-level software side of a proof-of-concept that demonstrates an end-to-end environmental monitoring system,<br><div>replacing Greater Wellington Regional Council’s expensive data loggers with low-cost, IoT centric embedded devices, and it’s supporting cloud platform. The proof-of-concept includes a Micropython-based software stack running on an ESP32 microcontroller. The device software includes a built-in webserver that hosts a responsive Web App for configuration of the device. Telemetry data is sent over Vodafone’s NB-IoT network and stored in Azure IoT Central, where it can be visualised and exported.</div><br>While future development is required for a production-ready system, the proof-of-concept justifies the use of modern IoT technologies for environmental monitoring. The open source nature of the project means that the knowledge gained can be re-used and modified to suit the use-cases for other organisations.
Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have markedly reduced numbers of neutrophils in peripheral blood (<500/μl), leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. Approximately 7 % of CN patients carry homozygous loss-of-function mutations in the HAX1 gene. 25 % of HAX1-CN patients develop MDS or AML. The only curative therapy for CN patients with overt MDS/AML is hematopoietic stem cell transplantation with its associated risks. A clinical need for gene therapy for CN patients is imminent. 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.
Use of the Internet of Things (IoT) is poised to be the next big advancement in environmental monitoring. We present the high-level software side of a proof-of-concept that demonstrates an end-to-end environmental monitoring system,<br><div>replacing Greater Wellington Regional Council’s expensive data loggers with low-cost, IoT centric embedded devices, and it’s supporting cloud platform. The proof-of-concept includes a Micropython-based software stack running on an ESP32 microcontroller. The device software includes a built-in webserver that hosts a responsive Web App for configuration of the device. Telemetry data is sent over Vodafone’s NB-IoT network and stored in Azure IoT Central, where it can be visualised and exported.</div><br>While future development is required for a production-ready system, the proof-of-concept justifies the use of modern IoT technologies for environmental monitoring. The open source nature of the project means that the knowledge gained can be re-used and modified to suit the use-cases for other organisations.
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