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Several major effectors of adult-stage fetal globin silencing have been identified, including the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening of 1591 transcription factors, we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements, one at its promoter and the other at an enhancer 6 kb upstream, with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. Peaks at these clusters display up to 57- and 77-fold enrichment respectively for ZNF410 binding compared to controls. Footprint analysis shows that the ZNF410 motifs are protected from nuclease cleavage. No other bona fide peaks could be detected across the genome in HUDEP-2 cells or primary CD34+ HSPC-derived erythroid precursors. We demonstrate that the ZNF410-bound elements at CHD4 completely account for ZNF410's effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios (6 homozygous Zfp410Gt/Gt gene-trap allele mice out of 20 live births) with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. Engrafting human erythrocytes induce HbF from ~2.5% in controls to ~17% in ZNF410 edited recipients, by hemoglobin HPLC. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex. FIGURE LEGEND: ZNF410 chromatin occupancy is restricted to CHD4. (A) Dense mutagenesis of ZNF410 shows its five C2H2 zinc-finger domains (red rectangles) are especially critical for HbF repression. (B) Genome-wide ZNF410 binding was identified by CUT&RUN in HUDEP-2 cells. Enrichment for ZNF410 binding was concentrated at just two sites in the genome - at the promoter and at an enhancer ~6 kb upstream of CHD4. (C) Genome-wide ZNF410 motif occurrences (JASPAR motif shown in center) were mapped using a 3 kb sliding window. Clusters of ZNF410 motifs were found only at three locations in the genome, including the CHD4 promoter and CHD4 -6 kb enhancer. (D) CHD4 locus showing ZNF410 binding (red peaks) at the CHD4 promoter and CHD4 -6 kb enhancer regions with anti-HA antibody recognizing epitope-tagged ZNF410 and anti-ZNF410 antibody recognizing endogenous ZNF410 in both HUDEP-2 cells and CD34+ HSPC derived erythroid precursors. CUT&RUN footprints demonstrate ZNF410 motif protection, ATAC-seq chromatin accessibility and phyloP DNA sequence conservation. Disclosures Bauer: Pfizer: Consultancy; Syros Pharmaceuticals: Consultancy; Fulcrum Therapeutics: Consultancy; Sanofi: Research Funding.

Severe congenital neutropenia (SCN) is a life-threatening disorder of insufficient granulocytes. Lifelong granulocyte colony-stimulating factor (G-CSF) injections are the mainstay of treatment, yet there remains a high risk of myelodysplastic syndrome and acute myeloid leukemia. The most common etiology of SCN is germline ELANE mutation. These dominantly acting mutations preserve expression but alter the structure of the neutrophil elastase protein product resulting in altered protein folding and/or trafficking with excess cell death at the promyelocyte/myelocyte stage of maturation. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells (HSCs); nonetheless genetic repair of specific disease-associated mutations remains challenging. We hypothesized that introduction of premature termination codons (PTCs) by nuclease-mediated frameshift mutations within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predicted that the PTCs would trigger nonsense mediated decay (NMD) of the mutant transcript resulting in its loss of expression and thus bypassing neutrophil precursor cell death and consequent neutropenia. The mild phenotype observed in the Papillon-Lefevre syndrome, characterized by combined serine protease deficiency, suggests that isolated neutrophil elastase deficiency would not result in clinically significant immunodeficiency. We delivered 3xNLS-SpCas9 and ELANE targeting sgRNA as ribonucleoprotein (RNP) complexes to primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and conducted in vitro neutrophil maturation culture. Introducing indels at exon 2 of ELANE efficiently triggered NMD. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using three human donors, we found that ELANE exon 2 edited HSPCs produced similar human bone marrow (BM) chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. We found similar lymphoid, erythroid, and myeloid engraftment including similar fraction of human neutrophils (13.4% of total human BM cells in unedited and 13.7% in ELANE exon 2 edited, despite 97.3% on-target indel frequency and 84.3% reduction in ELANE expression in the latter). Using CD34+ HSPCs from four ELANE mutant SCN patient donors, we demonstrated that exon 2 targeting RNPs achieve highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest. In contrast to these ameliorating early exon frameshifts, naturally occurring SCN-associated frameshifts affect late exons of ELANE, suggesting that these mutations might escape NMD. Indeed we found that targeting ELANE exon 5 in HSPCs resulted in robust indels (93.5%), preserving ELANE expression but resulting in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype. To our surprise, we found that only -1 frameshifts and not -2 frameshifts induced by gene editing with NHEJ repair led to the SCN-like phenotype, although we noted that all 23 reported naturally occurring SCN-associated ELANE frameshift mutations result from -1 but not -2 bp frameshifts. Using xenograft of NBSGW recipients, we found that an RNP complex leading to efficient -1 frame indels in ELANE exon 5 produced profound neutrophil maturation block, with reduction from 13.4% neutrophils in controls to 0.5% neutrophils in ELANE exon 5 targeted recipients, with otherwise indistinguishable human monocyte, lymphoid, and erythroid reconstitution as compared to controls. This dramatic phenotype contrasts with mice engineered to express SCN-associated Elane mutations that do not exhibit neutropenia, indicating species differences in granulopoiesis. Together these results support the development of ELANE early exon targeting as a highly efficient universal therapy for ELANE mutant SCN, feasible with existing gene editing technology. Moreover, by late exon ELANE gene editing we have developed a robust new model of SCN using primary human HSPCs that recapitulates neutropenia in vivo following xenotransplant, refines the molecular genetics of mutant ELANE induced neutrophil maturation arrest, and offers opportunities to explore novel therapeutic approaches. Disclosures Newburger: TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.

Base editing with fusions of RNA-guided DNA-binding proteins and nucleotide deaminases represents a promising approach to permanently remedy genetic blood disorders without obligatory double strand breaks, however its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A(N57Q)-BE3 protein for RNP electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We found that sgRNAs targeting for cytidine base editing the core GATA1 binding motif of the BCL11A +58 erythroid enhancer resulted in efficient on-target base edits (81% allele frequency) with low indels. There was similar HbF induction in erythroid progeny as compared to Cas9:sgRNA RNP nuclease mediated modification of the same target sequence (36% median HbF in base edited cells with 81% allele modifications, 39% HbF in 3xNLS-SpCas9:sgRNA#1617 nuclease edited cells with 99% indels, and 5%HbF in unedited cells). A single therapeutic base edit of the BCL11A enhancer was sufficient to ameliorate the pathobiology of both sickle cell disease (SCD) and beta-thalassemia. Base editing of CD34+ HSPCs from plerixafor mobilized PB SCD patient donors resulted in potent HbF induction (24-31% HbF level in bulk cells with 87-90% on-target allele modifications) and reduced sodium metabisulfite induced sickling (from 84% to 29% of erythrocytes). Base editing of non-mobilized PB CD34+ HSPCs from 3 beta-thalassemia patient donors potently induced gamma-globin and improved ineffective erythropoiesis in vitro, with 59-75% on-target allele modifications. Moreover highly efficient multiplex editing could be achieved in HSPCs. Simultaneous disruption of the BCL11A +58 erythroid enhancer core GATA1 motif and correction of the HBB -28A>G promoter mutation in HSPCs resulted in production of at least one therapeutic allele in 35 of 35 erythroid colonies analyzed, with each colony achieving improved alpha-to-non-alpha globin chain balance. Finally we found that base editing could be efficiently produced in engrafting HSCs. Using 3 donors, we observed 62-64% base edits at the BCL11A +58 enhancer GATA1 motif in input HSPCs with one round of electroporation and 73-85% base edits with two rounds of electroporation (separated by 24 hours), albeit with modest reduction of cell viability with tandem electroporation. We observed 70-94% human chimerism and 35-82% base edits in engrafting human cells in primary recipients and 11-61% base edits in secondary recipients as measured 16 weeks following cell infusion. Similar edit frequencies were observed across all assayed hematopoietic lineages including engrafting B-lymphocytes, erythroid precursors, and HSPCs. We observed that compared to non-engrafting progenitors, long-term engrafting HSCs favored C to T (as compared to C to G/A) editing. There was a strong correlation (Spearman r 0.86, P Disclosures No relevant conflicts of interest to declare.