Erythroid Enhancer Research Articles

Zinc finger nuclease-mediated gene editing in hematopoietic stem cells results in reactivation of fetal hemoglobin in sickle cell disease

BIVV003 is a gene-edited autologous cell therapy in clinical development for the potential treatment of sickle cell disease (SCD). Hematopoietic stem cells (HSC) are genetically modified with mRNA encoding zinc finger nucleases (ZFN) that target and disrupt a specific regulatory GATAA motif in the BCL11A erythroid enhancer to reactivate fetal hemoglobin (HbF). We characterized ZFN-edited HSC from healthy donors and donors with SCD. Results of preclinical studies show that ZFN-mediated editing is highly efficient, with enriched biallelic editing and high frequency of on-target indels, producing HSC capable of long-term multilineage engraftment in vivo, and express HbF in erythroid progeny. Interim results from the Phase 1/2 PRECIZN-1 study demonstrated that BIVV003 was well-tolerated in seven participants with SCD, of whom five of the six with more than 3 months of follow-up displayed increased total hemoglobin and HbF, and no severe vaso-occlusive crises. Our data suggest BIVV003 represents a compelling and novel cell therapy for the potential treatment of SCD.

Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching

AbstractThe switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (β-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A–mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA–mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Identification of a Novel Gene Regulatory Element in Human Erythroid Progenitor Cells

Early erythropoiesis involves commitment of multi-lineage progenitors to erythroid progenitor cells and differentiation into erythroid burst-forming unit (BFU-E) cells, then erythroid colony-forming unit (CFU-E) cells. In contrast to terminal erythroid differentiation, regulation of gene expression in early erythropoiesis is poorly defined. To address this knowledge gap, we identified active enhancers in erythroid progenitor cells linked to known disease genes and genes associated with erythrocyte traits in genome wide association studies. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from mobilized peripheral blood were cultured towards the erythroid lineage. From these cultures, FACS-based methods were utilized to purify four stages of erythroid progenitor cells, termed EP1 to EP4, and five populations of terminally differentiated erythroblasts, from proerythroblasts to orthochromatic erythroblasts. ChIP-seq was performed using antibodies to H3K4me1, H3K27Ac, H3K27me3, and H3K9me2/3. Chromatin accessibility was determined by ATAC-seq. Patterns of gene expression were determined by RNA-seq. Active enhancers were identified using the ModHMM algorithm. Numerous erythroid progenitor cell-specific active enhancers were identified near genes linked to known disease genes and GWAS-associated erythrocyte traits. rs162375, a single nucleotide polymorphism (SNP) linked to mean corpuscular hemoglobin, was in a 475bp Long Terminal Repeat Retrotransposon (LTR) element upstream of the RRP1B gene. The LTR enhancer near the RRP1B gene is a member of the LTR15 family. Transposable elements are a source of novelty in primate gene regulation when they acquire binding sites for critical tissue-specific transcription factors, and then these tissue-specific regulatory regions direct tissue-specific expression of nearby genes. The SNP-containing LTR15 erythroid progenitor-specific enhancer upstream of RRP1B contains 2 conserved GATA motifs and a conserved RUNX motif. The enhancer region had a peak of open chromatin determined by ATAC-seq, and H3K27 acetylation and strong GATA1 binding determined by ChIP-seq. These findings were present only in erythroid progenitor cells, not in earlier hematopoietic cells, terminally differentiating erythroid cells, or other non-erythroid cells. CRISPR-based gene editing was utilized to delete the LTR15 enhancer in CD34+ HSPCs cultured towards the erythroid lineage into erythroid progenitor cells. mRNA expression of RRP1B was decreased to 70.2% of wild type. Luciferase-based reporter gene studies demonstrated the enhancer increased expression of a minimal promoter 3.2-fold over background. Mutation of the GATA and RUNX sites individually or together decreased reporter gene activity to background levels. Bioinformatic analyses identified 291 additional erythroid progenitor cell specific LTR15 element active enhancers, with most containing two GATA motifs and a RUNX motif. The majority of the novel enhancer regions were also marked by progenitor cell-specific ATAC peaks and strong GATA1 binding in ChIP-seq. GATA1 and RUNX, both present in early erythropoiesis, are required to activate the regulatory elements in erythroid progenitor cells. Decreasing levels of RUNX in terminal erythroid differentiation parallels loss of enhancer activity. Since LTR15 first appeared in Catarrhine, old-world monkeys, these data indicate this novel regulatory element has recently evolved to contribute to regulation of gene expression in early erythropoiesis in primates.

Epitope Edited Hematopoietic Stem Cells Allow Immune-Based In Vivo Selection of Genome-Engineered Cells

Hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative options for patients affected by conditions for which the substitution of endogenous hematopoiesis with genetically corrected or healthy donor-derived cells can halt the pathogenic process. Nonetheless, the short and long-term adverse effects of genotoxic conditioning remain a substantial barrier to a wider application of HSCT and gene therapy. HSCT survivors suffer from life-long irreversible consequences, such as chronic graft-versus-host-disease (GvHD), infertility, susceptibility to viral infections, organ damage (including renal, liver and lung) and increased risk of secondary malignancies. Whereas monoclonal and toxin-conjugated antibodies (Abs) targeting HSPCs have been proposed as an alternative to chemo/radiotherapy, the mechanism of action and pharmacokinetics of these agents hamper their safe clinical use due to the risk of on target killing of transplanted HSPCs. To avoid this, the cell product is not administered at peak myeloablation to allow for washout of the conditioning agent, further impairing engraftment and therapeutic potential. Here, we show that precise editing of the targeted epitope in HSPCs can endow hematopoietic lineages with selective resistance to monoclonal Abs (mAbs) without affecting stem cell function. This strategy allows improved myeloablation - which can continue after HSCT - and progressive enrichment of gene-modified cells. We have identified amino-acid changes in the extracellular domain of KIT (CD117) that abrogate the binding of therapeutic mAbs without affecting surface expression, ligand affinity, kinase phosphorylation, downstream signaling, transcriptional and proliferative response (Fig.A). We exploited adenine base editing (BE) to introduce these mutations with high efficiency (~80%) in CD34+ HSPCs, with preservation of long-term repopulating and multilineage differentiation capacity in primary and secondary recipient mice. To ensure full preservation of KIT functionality, we performed growth analysis, RNA sequencing, and phospho-proteomic analyses of epitope-edited HSPCs stimulated with KIT ligand (stem cell factor, SCF) and observed no difference in the proliferation, transcriptional, and phosphorylation response compared to controls. In vitro differentiation of edited HSPCs toward myeloid, macrophage, classical dendritic, granulocytic and megakaryocytic lineages was similar to controls and did not result in counterselection of edited cells. Lineage-differentiated cells showed preserved functionality, including: reactive oxygen species production by myeloid cells, E.coli phagocytosis by macrophages, M1/M2-like macrophage polarization, phospho-flow profiling of IL4-, PMA/ionomycin-, GM-CSF-, IFN type-I-, IL-6- and LPS-stimulated myeloid cells, HLA class-II/CD86 upregulation by DCs, induction of granulocyte NETosis and generation of hyperdiploid megakaryocytes. By plating edited HSPCs with increasing concentrations of the mAb, we observed preserved expansion of edited HSPCs in response to SCF while control cells were inhibited in a dose-dependent manner. To improve HSPCs therapies for hemoglobinopathies, such as sickle cell disease, we multiplexed BE of the KIT epitope and two BCL11A erythroid enhancer motifs. We obtained editing efficiencies comparable to single editing and the erythroid progeny of triple-edited cells showed upregulation of HbF, as expected (~40%). In a competitive xeno-transplantation setting, with co-injection of multiplex BE and AAVS1-BE control CD34+ cells, mice treated with KIT mAb showed a 2-fold co-enrichment of both KIT and BCL11A editing when compared to mock treated controls, confirming the possibility to select gene-modified cells up to therapeutic thresholds. Similarly, mAb conditioning combined with epitope-edited grafts allowed human hematopoietic substitution in mice pre-humanized with unedited CD34+ HSPCs (89% substitution with 7.3-fold higher absolute cell counts, Fig.B), compared to AAVS1-edited controls that failed to engraft. We envision a new paradigm for non-genotoxic conditioning based on transplantation of epitope engineered HSPCs resistant to immunotherapeutic agents to achieve engraftment and in vivo selection of gene corrected HSPC with substantial advantages over alternatives in terms of safety and tolerability.

Association of DDX5/p68 protein with the upstream erythroid enhancer element (EHS1) of the gene encoding the KLF1 transcription factor

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. Regulation of KLF1 is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the DDX5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We further show that this protein associates with DEK and CTCF. We postulate that the range of interactions of DDX5/p68 with these and other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1 and enables the formation of a proper chromatin configuration at the Klf1 locus. These individual interactions provide quantitative contributions that, in sum, establish the high-level activity of the Klf1 promoter and suggest they can be selectively manipulated for clinical benefit.

Efficacy and Safety of Brl-101, CRISPR-Cas9-Mediated Gene Editing of the BCL11A Enhancer in Transfusion-Dependent β-Thalassemia

BACKGROUND: β-Thalassemia is an inherited hemolytic disease that is prevalent worldwide. Over 200 mutations in the HBB gene, which encodes adult hemoglobin (HbA), result in β-thalassemia. Hereditary persistence of fetal hemoglobin (HbF) can alleviate the symptoms of anemia. CRISPR-Cas9-mediated disruption of the BCL11A erythroid enhancer results in the reduction of BCL11A expression and the induction of fetal γ-globin, which is a practicable therapeutic strategy for treating transfusion-dependent β-thalassemia (TDT). METHODS: We obtained mobilized autologous CD34+ cells from 4 TDT patients sponsor initiated in phase I/II clinical trial (NCT05577312) and 6 TDT patients in investigator initiated clinical study（NCT04211480, NCT04205435）respectively. These cells were edited with CRISPR-Cas9 RNP at the +58 erythroid specific enhancer region of the BCL11A gene and then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, neutrophil and platelet engraftment. Efficacy assessments included proportion of transfusion-independent (TI) subjects, levels of total hemoglobin and HbF, and proportion of red blood cells expressing HbF in peripheral blood. TI was evaluated for elimination of transfusions starting 42 days after the last transfusion and Hb above 90 g/L. RESULTS: Between October 15, 2019 and November 20, 2022, 10 patients with TDT were enrolled and received BRL-101 with a median age of 12.4 years (6-26). Among all the treated patients, 5 patients were β 0/β 0 phenotype, 4 patients were β0/β+ phenotype and 1 patient were β +/β + phenotype. As of July 20, 2023, the median follow- up was 24.6 months (4.3-39.2 m) (Tab.1). All of 10 patients achieved TI. The median duration of TI was 22.1 months (1.3 - 37.2 m). The longest duration of TI was 37.2 months (Fig.1). The levels of HbF ranged from 2.3 to 140.7g/L and the levels of total hemoglobin ranged from 105.7 to 149 g/L (Fig.2). At 6 months after BRL-101 infusion, the proportion of HbF-expressing red blood cells in peripheral blood had reached 96.5%, and then continued to rise and remained around 98-99% (Fig.3). Treatment-related adverse events were typical of those associated with myeloablation and autologous stem-cell transplantation, mainly manifested as hematological toxicities (Tab.2, Tab.3). 3 patients experienced adverse events (AEs) grade ≥ 3 which related to BRL-101. 4 patients experienced serious AEs (SAEs), including decreased platelet count, shock, febrile infection, soft tissue infection, and veno-occlusive liver disease. Only decreased platelet count may related to BRL-101, the others were due to busulfan treatment. All the SAEs were resolved. No study drug-related withdrawals or deaths occurred during treatment. CONCLUSIONS: Whether genotype was β 0/β 0 or non-β 0/β 0, BRL-101 demonstrated clinically meaningful increases in total Hb and HbF which occurred early and have been maintained over time, the safety profile of BRL-101 is generally consistent with that of myeloablative conditioning and autologous hematopoietic stem cell transplant. The updated data with 10 patients reported here are consistent with previous reports and support continued investigation of BRL-101 as a potential functional cure for patients with TDT.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

Precision Editing as a Therapeutic Approach for β-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

5612617 EFFICACY AND SAFETY OF A SINGLE DOSE OF EXAGAMGLOGENE AUTOTEMCEL FOR TRANSFUSION-DEPENDENT-THALASSEMIA AND SEVERE SICKLE CELL DISEASE

Background: Elevated fetal hemoglobin(HbF) is associated with improved outcomes in patients(pts) with transfusion-dependent β-thalassemia(TDT) and sickle cell disease(SCD). Exagamglogene autotemcel(exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells(HSPCs). Early data from the pivotal trials CLIMB THAL-111(NCT03655678) and CLIMB SCD-121(NCT03745287) showed that a single dose of exa-cel increased HbF and total Hb sufficiently to eliminate red blood cell(RBC) transfusions(TDT) and vaso-occlusive crises(VOCs; SCD). Aims: To report efficacy and safety data from the first 75 pts dosed with exa-cel in the ongoing CLIMB THAL-111 and CLIMB SCD-121 trials. Methods: Following pharmacokinetic-adjusted busulfan myeloablation and exa-cel infusion, pts(12-35y) are monitored for engraftment, total Hb, HbF, BCL11A edited alleles, transfusions, VOCs(SCD only), and adverse events(AEs). Data presented as mean(min-max) unless noted. Results: 44 pts with TDT(age 21.3 [12-35] y) and 31 pts with SCD(age 22.5 [12-34} y) had been infused with exa-cel at data cut(follow-up 12.3 [1.2-37.2] mo and 9.6 [2.0-32.3] mo, respectively). 26/44 pts with TDT(59.1%) had β0/β0 or a β0/β0-like genotype(β0/IVS-I-110, IVS-I-110/IVS-I-110). In the 2-yr period before screening, pts with TDT received 36.0(15.0-71.0) units RBC/yr and pts with SCD had 3.9(2.0-9.5) severe VOCs/yr. After exa-cel infusion, all pts engrafted neutrophils and platelets. Median time to neutrophil and platelet engraftment was 29 and 43 days in pts with TDT and 27 and 32 days in those with SCD, respectively. Overall, 42 of 44 pts with TDT stopped RBC transfusions(duration 0.8 to 36.2 mo); 2 pts had not yet stopped transfusions but had 75% and 89% reductions in transfusion volume. By Month 3, increases in HbF and mean total Hb levels(>9 g/dL) were achieved, with mean total Hb levels increasing to >11 g/dL thereafter and were maintained. All pts with SCD(n=31) no longer had severe VOCs after exa-cel infusion(duration 2.0 to 32.3 mo). The mean proportion of HbF was >20% by Month 3, increasing to ~40% at Month 4 and was stable thereafter, with mean total Hb levels >11 g/dL after Month 3. Pts with TDT and SCD with ≥1 yr follow-up had stable proportions of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells. Two pts with TDT had serious AEs(SAEs) considered related to exa-cel. The first pt was previously reported. The second pt had delayed neutrophil engraftment and thrombocytopenia, which were considered related to both exa-cel and busulfan. All SAEs resolved. No pts with SCD had SAEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to elimination of transfusions in almost all patients with TDT and elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles remained stable after more than 1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with busulfan myeloablation and autologous transplant. These results indicate exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for TDT and severe SCD. Reference 1. Frangoul et al, N Engl J Med 2021; 384:252-260.

Increased Potency and Uniformity of Fetal Hemoglobin Induction from Base Editing Compared to Cas9 Nuclease

Genetic manipulations to increase fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs) can alleviate β-thalassemia and sickle cell disease (SCD). We and others demonstrated that CRIPSR/Cas9 disruption of γ-globin promoter binding motifs for the repressors BCL11A or ZBTB7A raises erythroid HbF expression to potentially therapeutic levels. Moreover, disruption of an erythroid BCL11A gene enhancer to induce HbF produced clinical improvement in humans with SCD. However, Cas9-induced double-stranded breaks (DSBs) can cause potentially deleterious chromosomal abnormalities. Base editing represents a promising approach to install precise nucleotide alterations through DSB-independent mechanisms. In contrast to Cas9 generated indels, base editors can recreate naturally occurring base pair substitutions that cause hereditary persistence of fetal hemoglobin (HPFH), including those that create de novo binding motifs for erythroid transcriptional activators at the γ-globin promoter. Base editing and Cas9 nuclease-based approaches to induce HbF have not been compared directly and the best therapeutic strategy is not known. We compared two Cas9-based strategies and three base editing strategies for HbF induction by modifying CD34+ hematopoietic stem and progenitor cells (HSPCs) and examining HbF expression in erythroid progeny generated by in vitro differentiation. We used Cas9 nuclease to disrupt the BCL11A gene erythroid enhancer or the BCL11A binding motif in the γ-globin promoter. Alternatively, we used the adenine base editor ABE7.10 to create base pair substitutions at γ-globin promoter positions -198, -175, and -113, to generate de-novo binding motifs for transcriptional activators KLF1, TAL1, and GATA1, respectively. The -175 A>G edit was most potent, producing an approximately 1% rise in HbF per % editing. In contrast, Cas9 disruption of the BCL11A enhancer or the BCL11A binding motif in the γ-globin promoter caused 0.33% and 0.30% increases in HbF per % editing, respectively. Cas9 nuclease but not base editing induced a TP53-mediated DNA damage response, as reflected by increased expression of CDKN1(P21) mRNA. To compare further the different edits, we analyzed burst forming unit-erythroid (BFU-E) colonies derived from modified CD34+ cells. Erythroid colonies with -175 A>G edit in all four γ-globin genes expressed 81±7% HbF compared to 14±11% in unedited controls. In contrast, HbF in BFU-E colonies was 33±19% after Cas9 disruption of the BCL11A repressor binding motif in the promoters of all γ-globin alleles and 50±14% after Cas9 disruption of the erythroid enhancer in the BCL11A gene. Among Cas9 mutations disrupting the γ-globin BCL11A binding motif, significantly increased HbF occurred with a 13 bp deletion but not with -1, -2, -3 or +1 indels. This finding is unexpected because elimination of BCL11A binding is believed to be sufficient for γ-globin induction. All high frequency indels targeting the BCL11A enhancer induced HbF similarly, albeit with greater variability than in colonies with the -175 A>G base edit. In normal donor CD34+ cells, ABE7.10 installed γ-globin -175 A>G at efficiencies of 43±6% (n=6). The newer, more active ABE8e produced a 1.4-fold improvement in editing efficiency and a 1.3-fold increase in HbF induction relative to ABE7.10. In preliminary studies, we achieved on-target editing of 62% with ABE8e in SCD patient CD34+ HSPCs. At 16 weeks after xenotransplantation of the modified HSPCs into NBSGW mice, on-target editing of donor-derived cells was 53±11%. Base edited erythroid cells isolated from recipient mouse bone marrow expressed 53±5% HbF, versus 2±1% in unedited cells. After xenotransplantation of the same donor CD34+ cells that were treated with Cas9 nuclease to disrupt the γ-globin promoter BCL11A binding motif, editing retention was 92±4%, with 29±12% HbF in donor derived erythroblasts. Thus, the -175 A>G edit produces superior HbF induction. Our data identifies a base editing strategy for potent, uniform induction of HbF and provides insights into γ-globin gene regulation by showing that disruption of the promoter BCL11A binding motif is not sufficient for transcriptional activation. More generally, we demonstrate that diverse indels generated by Cas9 nuclease cause unexpected variation in biological outcomes that can be circumvented by base editing, with important implications for therapeutic gene editing efforts.

Preliminary Safety and Efficacy Results of EDI001: An Investigator Initiated Trial on CRISPR/Cas9-Modified Autologous CD34+ Hematopoietic Stem and Progenitor Cells for Patients with Transfusion Dependent β-Thalassemia

Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1). Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints. Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation. One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion. The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B). The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively. Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Safety of a Single Dose of Exagamglogene Autotemcel for Severe Sickle Cell Disease

Background: Elevated fetal hemoglobin (HbF) is associated with improved outcomes in patients with sickle cell disease (SCD). Exagamglogene autotemcel (exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). Recent data from the pivotal CLIMB SCD-121 (NCT03745287) trial showed that a single dose of exa-cel increased HbF and total hemoglobin (Hb) sufficiently to eliminate vaso-occlusive crises (VOCs) in patients with severe SCD. Here, we report efficacy and safety data from the first 31 patients dosed with exa-cel in the ongoing CLIMB SCD-121 trial. Methods: Patients aged 12 to 35 years with severe SCD and a history of ≥2 VOCs per year in the previous 2 years before screening were eligible. Following pharmacokinetic-adjusted, busulfan myeloablation and infusion of exa-cel, patients are monitored for engraftment, total Hb, HbF, BCL11A-edited alleles, transfusions, VOCs, and adverse events (AEs). The primary endpoint is the proportion of patients who have not experienced a severe VOC for at least 12 months after the infusion of exa-cel, starting 60 days after their last RBC transfusion. Updated efficacy and safety results on all dosed patients will be included in the presentation. Data are reported as mean (min-max), unless noted. Results: At the most recent data cut in February 2022, 31 patients with SCD (age 22.5 [12-34] years) had been infused with exa-cel (follow-up 9.6 [2.0-32.3] months), of whom 6 (19.4%) were between the ages of 12 and <18 years and 29 (93.5%) had the βs/βs genotype. In the 2-year period before screening, patients experienced 3.9 (2.0-9.5) severe VOCs per year. After exa-cel infusion, all patients engrafted neutrophils and platelets with a median time of 27 and 32 days, respectively. All patients were VOC-free at the time of the data cut (duration of follow-up 2.0-32.3 months after exa-cel infusion; Figure). Median time from exa-cel infusion to last RBC transfusion was 19 (11-52) days. The mean proportion of HbF was >20% by Month 3, with mean total Hb levels >11 g/dL on and after Month 3. All 11 patients who have at least 12 months of follow-up after exa-cel infusion have maintained HbF levels >20% while experiencing no VOCs. At Month 6, the mean proportion of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells was 86.6% and 76.0%, respectively. These proportions remained stable in all patients who had ≥1 year of follow-up (Figure). There were no patients who had serious AEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to the elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles have remained stable after ≥1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with that of busulfan myeloablation and autologous transplant. Exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for SCD. Figure. Clinical data for patients with SCD (N=31) infused with exa-cel. Patients were VOC-free after exa-cel infusion. Time (months) since exa-cel infusion is indicated by the dark blue bar. Inset depicts the percentage of BCL11A-edited alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells of patients with SCD infused with exa-cel over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic Gene Editing of HSCs Ex Vivo without in Vitro Culture Avoids Genotoxicity, Simplifies Procedures, and Preserves Efficiency and Stemness

Therapeutic gene editing of HSCs promises to cure inherited blood disorders. However, current ex vivo gene editing protocols may be associated with genotoxic potential and complex in vitro cell manufacturing procedures. Base and prime editing reduce double strand breaks (DSBs) but are generally less efficient and might have unique genotoxic potential (e.g. untargeted deaminase activity). Here we test the hypothesis that therapeutic gene editing with targeted endonucleases disrupting HbF-repressing elements for b-hemoglobinopathies may be made safer, simpler, and at least as effective by focusing the editing on nondividing cells. Genotoxic risks of DSBs appear to be concentrated in cycling cells. For example, Cas9-induced DSBs can result in long deletions at the on-target site that appear to be mainly the result of unidirectional extensive resection selectively active in S/G2 cell cycle phases1. In addition, DSBs must be resolved prior to mitosis to prevent chromosome mis-segregation leading to copy number variation and cytoplasmic micronuclei2,3. We measured the cell cycle state of mobilized PB CD34+ HSPCs immediately after collection, or after 24 or 48 hours of culture with cytokines (SCF, TPO, and FLT3L each at 100 ng/ml). 100% of cells were in G0/G1 at 0 hours, 94.6%±1.5% at 24 hours, and 76.7%±2.6% at 48 hours. We electroporated CD34+ HSPCs with 0, 24, or 48 hours of cytokine culture before performing 3xNLS-HiFi-SpCas9:sgRNA ribonucleoprotein (RNP) electroporation targeting the BCL11A erythroid enhancer. We employed a flow cytometry assay to quantify micronuclei in edited HSPCs. The fraction of micronuclei was increased in cells electroporated with Cas9:sgRNA RNP following 24 or 48 hours of cytokine stimulation but not in unstimulated cells. We performed ddPCR on sorted nuclei and micronuclei using assays specific to distal chromosome 2p (telomeric to the BCL11A cleavage site) and to chr1 as a reference autosome. We found ~9-fold enrichment of chr2p in micronuclei after 24 or 48 hours of pre-stimulation cytokine culture prior to gene editing, but strikingly did not observe any enrichment of chr2p in micronuclei in cells edited without pre-stimulation culture, suggesting that chromosome mis-segregation and unwanted downstream consequences could be avoided by editing quiescent HSCs (Fig. 1A). In addition, we developed ddPCR assays to measure genome editing outcomes after editing the BCL11A enhancer with paired cleavages (at the +58 and +55 enhancers) separated by 3-kb. These assays measure: 1) programmed 3-kb deletions, 2) programmed 3-kb inversions, or 3) short indels or unedited alleles. Any missing signals (genomic copies fewer than found on a reference nontargeted autosome) may represent long deletions or rearrangements. Editing cells without pre-stimulation culture was associated with undetectable missing signals (inferred long deletions and rearrangements) as compared to editing after 24 or 48 hours of pre-stimulation culture. We compared our standard protocol of 24 hours of pre-stimulation cytokine culture and 24 hours of post-electroporation cytokine culture to a protocol in which CD34+ HSPCs were electroporated immediately after thawing and then cryopreserved 15 minutes after electroporation, without any cytokine culture, before infusion to NBSGW mice. After 16 weeks, HbF levels were potently induced to similar levels after gene editing in recipients of HSPCs without and with cytokine culture (Fig. 1B). Overall human engraftment was similar for all groups, as was multilineage repopulation. Gene edit frequencies were similar for recipients of HSPCs without and with cytokine culture. We observed comparable editing and engraftment potency of HSPCs without as compared to with cytokine culture across mobilization type (G-CSF or plerixafor), donor disease status (healthy and SCD), and sgRNA target sequence. Together, these results suggest that editing HSPCs ex vivo without in vitro culture: avoids unintended on-target genotoxicity potential, including production of micronuclei, long deletions, and rearrangements; simplifies protocols, eliminating time, cost, and complexity; and preserves gene editing efficiency and HbF induction and HSC repopulation potency. Editing nondividing cells selectively could be a straightforward modification to improve ex vivo editing as well as to advance future efforts for safe and potent in vivo editing of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

KLF1 Acts As a Pioneer Transcription Factor Via SMARCA4 to Open Chromatin and Facilitate Redeployment of an Enhancer Complex Containing GATA1 and SCL

Introduction: Sequence specific transcription factors (TFs) bind to short DNA motifs in vitro; but, only a tiny fraction of possible binding sites within the genome are bound. Factors that influence in vivo binding are relative affinity for the specific motif at the site, co-operative interactions with additional TFs that bind to adjacent motifs, and physical impediments provided by chromatin. Pioneer TFs are a special class of TF that can interact with 'silent' chromatin, increasing local accessibility. This can facilitate the binding of other TFs that do not possess pioneering activity (settler TFs). KLF1 is an essential erythroid-specific TF that binds to ~1000 enhancers, often in co-operation with other essential TFs such as GATA1 and SCL/TAL1. Aim: To determine whether KLF1 acts as a pioneer TF and, if so, address the mechanism. Metods: We employed the erythroid cell line, K1ER1, to study the immediate effects of KLF1 on chromatin re-organization following addition of 4-OHT. This erythroid cell line is null for Klf1 and stably expresses an inducible KLF1-ERTM transgene. We performed ATAC-seq, and ChIP-seq for KLF1, GATA1, TAL1, NF-E2 and SMARCA4 in the presence or absence of 4-OHT. We generated a deletion series of KLF1 in stably expressing K1ER clones to map the domains responsible for chromatin remodeling and we examined the neo-morphic DNA-binding mutants, nan and CDA-IV, to determine whether ectopic binding led to ectopic pioneering. We validated the cell line results by ATAC-seq and ChIP-seq in Klf1-/- versus wild type fetal liver erythroid cells. We employed to SMARCA4 inhibitor, BRM014, to test its requirement for erythroid gene expression. Results: Using ATAC-seq and ChIP-seq we show KLF1 binding induces an increase in local chromatin accessibility that facilitates assembly of a complex of settler TFs including GATA1 and SCL/TAL1, but not p45-NF-E2. Interestingly, KLF1 rapidly induces relocation of GATA1 in local neighborhoods, including GATA1 'desertion' of certain sites. The pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such R2 at -26kb in the a-globin LCR, an important E2f2 intronic enhancer, and dematin/Epb4.9 gene enhancers. We confirmed these findings in primary erythroid cells from Klf1-/- fetal liver. We show neo-morphic mutations in the DNA-binding domain of KLF1 (Nan and CDA-IV mutations) lead to ectopic pioneering in some but not all cases of ectopic binding. We show a N-terminal domain of KLF1 that is distinct from the transactivation domains (TADs) is responsible for SMARCA4/Brg1 recruitment (Fig 1), the likely effector of chromatin remodeling. Inhibition of SMARCA4 using specific inhibitors2 leads to inhibition of induction of some KLF1 target genes such as dematin/Ebp4.9, but not others such as beta-globin. Conclusions: KLF1 is a pioneer TF that recruits GATA1, TAL1 and likely an enhancer complex that includes LMO2 and LDB1 to key erythroid enhancers and super enhancers. KLF1 can induce GATA1 to abandon certain bound sites within some genome neighborhoods but not globally. Pioneering occurs in part by recruitment of SMARCA4 via an N-terminal domain that is distinct from the TADs, and this is essential for expression of subset of KLF1 target genes. This work has implications for how KLF1 works to direct erythroid versus lineage trajectories from bi-potential progenitors, and how KLFs reprogram cells generally.

Base Editing for Therapeutic Induction of Fetal Hemoglobin in β-Thalassemia

β-thalassemia, one of the most common inherited genetic disorders worldwide, is caused by more than 300 different mutations in the β-globin locus (HBB), all of which result in quantitative deficiency of β-globin protein with severe anemia and reduced or absent HbA (α2β2). Additionally, free α-globin protein forms toxic intracellular inclusions resulting in hemolysis of mature red blood cells, maturation arrest and apoptosis of erythroid precursors. The only approved cure for severe β-thalassemia is allogeneic hematopoietic stem cell transplantation. However, many patients lack ideal histocompatible donors and immune complications such as graft rejection and graft-versus-host disease can occur. These problems can be avoided by "gene therapies” in which the patient's own hematopoietic stem cells (HSCs) are isolated, genetically modified ex vivo and reinfused after administration of bone marrow conditioning to facilitate engraftment of the modified cells. Most β-thalassemia mutations can be treated by genetic manipulations that de-repress fetal-expressed γ-globin genes (HBG1 and HBG2) to induce fetal hemoglobin production (HbF, α2γ2) postnatally. Thus, Cas9 disruption of an erythroid enhancer in the γ-globin repressor gene BCL11A shows promising results in an ongoing clinical trial. However, multiple other strategies to induce HbF exist and the best one is not known. Compared to conventional Cas9 nuclease, base editors offer theoretical advantages, including the ability to create precise base pair substitutions without requiring genotoxic double strand DNA breaks. We used the adenine base editor ABE8e to generate the naturally occurring γ-globin -175 A>G variant, which causes hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates co-inherited β-thalassemia. We edited human CD34+ hematopoietic stem and progenitor cells (HSPCs) from two individuals with severe transfusion dependent β-thalassemia by electroporating ribonucleoprotein (RNP) complexes of ABE8e-NG protein + single guide RNA (sgRNA) designed to install the γ-globin -175 A>G variant, followed by in vitro erythroid differentiation. On-target editing rates were 48-53% for donor 1 (n=3 replicates) and 22.9% for donor 2 (n=1). Analysis of burst forming unit erythroid (BFU-E) colonies revealed at least one on-target edit in 78% of donor 1 HSPCs and 63% of those from donor 2. Reverse phase HPLC of erythroid progeny showed increased β-like/α globin protein ratios: 0.92 ± 0.04 vs 0.53 ± 0.03 in unedited controls (p<0.0001) for donor 1, and 0.8 vs 0.5 in unedited controls for donor 2. Quantitative RT-PCR showed 6-8-fold increased γ-globin mRNA in the ABE-treated β-thalassemia cells. Ion exchange HPLC analysis of erythroid cell lysates showed reductions in free α-globin levels after ABE treatment: from 21.5 ± 2% of total globin protein in unedited cells to undetectable levels after editing in donor 1 and from 18% to 2.5% for donor 2. Maturation to the reticulocyte stage was also increased in base edited cells: 14.6 ± 2% vs 3.3 ± 1% in unedited controls for donor 1 (P<0.0001); and 26% vs 6.9% in unedited controls for donor 2. Reduction in apoptosis (37.4 ± 2.2% vs 23.6 ± 1.5% in unedited control cells, p=0.0003) and reactive oxygen species measured by DCFDA (MFI: 690 ± 97 vs 266 ± 9 in unedited control cells, p=0.009) was also seen in the ABE edited groups. To study editing outcomes in vivo, CD34+ HSPCs from donor 2 were treated with RNP and transplanted into immunodeficient NBSGW mice. After 16-18 weeks, human donor-derived cells were isolated from mouse recipient bone marrow and analyzed (n=4 mice per group). On-target editing frequencies in CD235a+ erythroblasts and CD34+ hematopoietic stem/progenitors were 17.5 ± 5% and 19.5 ±3%, respectively, compared to 22.9% in the input CD34+ cells. Similar to what we observed after in vitro differentiation, base edited erythroid cells generated in vivo exhibited improved globin chain balance with β-like/α globin protein ratios of 0.86 ± 0.12 vs 0.69 ± 0.04 in unedited controls, (p=0.04), reduced apoptosis (16.8 ± 4.2 % vs 26.6 ± 2.9 % in unedited control cells, p=0.004) and enhanced maturation into enucleated reticulocytes (14.4 ± 4.7 % vs 2.87 ± 1.4 % in unedited control cells, p=0.005). Our study provides proof of concept that ABE8e generation of the γ-globin -175 A>G HPFH variant in β-thalassemic HSCs represents a new therapeutic approach for β-thalassemia.

HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Reactivation of γ-globin expression using a minicircle DNA system to treat β-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for β-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of β-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of β-thalassemia.

Hemogen/BRG1 cooperativity modulates promoter and enhancer activation during erythropoiesis

AbstractHemogen is a hematopoietic tissue–specific gene that regulates the proliferation and differentiation of hematopoietic cells; however, the mechanism underlying its function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role for hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of corepressors nucleosome remodeling and histone deacetylase (NuRD) complex and facilitated LDB1 complex–mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a coactivator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To determine whether hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen-knockout/knockin mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in embryonic days 12.5 to 16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. Chromatin immunoprecipitation sequencing in wild-type and hemogen-knockout animals revealed that BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, the hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.

Combined +58 and +55 BCL11A enhancer Editing Yields Exceptional Efficiency, Specificity and HbF Induction in Human and NHP Preclinical Models

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed.Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. DisclosuresNo relevant conflicts of interest to declare.

Hemogen/BRG1 Cooperativity Is Required for Erythrocyte Maturation and Hemoglobin Production during Mammalian Erythropoiesis

Hemogen, also known as EDAG, is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells. However, the mechanism underlying hemogen function in erythropoiesis is unclear. We found that depletion of hemogen in human CD34 + erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role of hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of co-repressors NuRD complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a co-activator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To ask if hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen KO/KI mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in E12.5-E16.5 impeded erythroid differentiation through reducing the production of mature erythroblasts. ChIP-seq in WT and hemogen KO animal revealed BRG1 is largely dependent on hemogen to occupy chromatin at erythroid gene promoters and enhancers. In summary, hemogen/BRG1 interaction in mammals is essential for erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization. DisclosuresNo relevant conflicts of interest to declare.