Editing aberrant splice sites efficiently restores ß-globin expression in ß-thalassemia.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.

Metabolic effects of diet containing blue mussel (Mytilus edulis) and blue mussel-fed salmon in a mouse model of obesity.

Alternative feed ingredients for farmed salmon are warranted due to increasing pressure on wild fish stocks. As locally farmed blue mussels may represent an environmentally sustainable substitute with a lower carbon footprint, we aimed to test the potential and safety of substituting fish meal with blue mussel meal in feed for Atlantic salmon. Salmon were fed diets in which fish meal was partially replaced with blue mussel meal in increments, accounting for up to 13.1 % of the ingredients. Fillets from the salmon were subsequently used to prepare obesity-promoting western diets for a 13-weeks mouse feeding trial. In a second mouse trial, we tested the effects of inclusion of up to 8% blue mussel meal directly in a meat-based western diet. Partial replacement of fish meal with blue mussel meal in fish feed preserved the n-3 polyunsaturated fatty acid (PUFA) content in salmon fillets. The observed blue mussel-induced changes in the fatty acid profiles in salmon fillets did not translate into similar changes in the livers of mice that consumed the salmon, and no clear dose-dependent responses were found. The relative levels of the marine n-3 fatty acids, EPA, and DHA were not reduced, and the n-3/n-6 PUFA ratios in livers from all salmon-fed mice were unchanged. The inclusion of blue mussel meal in a meat-based western diet led to a small, but dose-dependent increase in the n-3/n-6 PUFA ratios in mice livers. Diet-induced obesity, glucose intolerance, and hepatic steatosis were unaffected in both mice trials and no blue mussel-induced adverse effects were observed. In conclusion, our results suggest that replacing fish meal with blue mussel meal in salmon feed will not cause adverse effects in those who consume the salmon fillets.

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.

Therapeutic base editing of human hematopoietic stem cells.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe ß-globin disorders sickle cell disease (SCD) and ß-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with ß-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

[AML in Adults - An Update on Diagnosis, Risk Classification and Therapy]. / Akute myeloische Leukämie bei Erwachsenen.

There are several changes in the revised WHO classification for acute leukemia. The latest version of the European Leukemia Network (ELN)-risk classification defines AML with mutations in RUNX1, ASXL1 or TP53 to fall in the unfavorable risk group. Consequently, the somatic molecular genetic testing at the time of initial diagnosis should encompass these before mentioned three genes next to the already routine testing of NPM1, CEBPA and FLT3-ITD. Several new innovative substances have been developed and approved for AML therapy. The multikinase inhibitor midostaurin is a new substance in the induction therapy for patients with FLT-3 mutated AML. CPX-351 (cytarabin and daunorubicin in liposomatic formulation in a defined molar relation) is a new option for therapy associated AML and AML with myelodysplastic changes. Enasidenib is a targeting drug for relapsed or refractory AML with IDH2 mutation. Gemtuzumab, a CD33 + antibody-drug-conjugate is approved for CD33 positive cases.

Activating mutations in the RAS/mitogen-activated protein kinase signaling pathway in sporadic trichoblastoma and syringocystadenoma papilliferum.

Trichoblastoma (TB) and syringocystadenoma papilliferum (SCAP) are both rare adnexal skin lesions occurring either sporadically or as secondary neoplasms in sebaceous nevi. TB and SCAP associated with sebaceous nevi have been shown to carry the same HRAS mutation as the underlying nevus. However, the genetic background of sporadic TB and SCAP has remained unknown. Therefore, we screened 18 sporadic TBs and 23 sporadic syringocystadenoma papillifera from 41 patients for the presence of activating mutations in RAS genes and other oncogenes. Using a RAS SNaPshot assay, HRAS mutations were detected in 2 (11%) of 18 sporadic TB and 6 (26%) of 23 sporadic syringocystadenoma papillifera. A KRAS mutation was identified in 1 sporadic SCAP. High-throughput oncogene mutation profiling furthermore identified BRAF V600E mutations in sporadic syringocystadenoma papillifera, which could be validated in 12 (52%) of 23 lesions using a BRAF SNaPshot assay. BRAF and RAS mutations were mutually exclusive in sporadic syringocystadenoma papillifera. No BRAF mutation could be detected in 3 syringocystadenoma papillifera secondarily arisen from a sebaceous nevus as well as in sporadic TB. In 14 lesions carrying an oncogenic mutation, nonlesional control tissue from the epidermal margin revealed a wild-type sequence, thus proving the somatic character of the mutation. Our results indicate that activation of the RAS-mitogen-activated protein kinase pathway by BRAF and RAS mutations contributes significantly to the tumorigenesis of sporadic SCAP and, less frequently, of sporadic TB.