Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Abstract

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-βE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.

Keywords: HPFH mutation; MT: RNA/DNA Editing; beta-thalassemia; deletional HPFH; fetal hemoglobin; gene editing; gene therapy; hematopoietic stem cells; large deletions; locus control region.; sickle cell diseases.

Graphical abstract

Figure 1

Genomic deletion encompassing PRR to βE1 is sufficient to reproduce deletional HPFH phenotype (A) Diagrammatic representation of β-globin cluster and the break points of naturally occurring HPFH deletions (green). These deletions are introduced in the experiments shown in (B)–(D). Break points of deletions (red) introduced in the experiment shown in (F)–(K). All these deletions were generated in HUDEP-2 cell lines by CRISPR-Cas9 dual gRNA approach. (B) Percentage of gene editing in HUDEP-2 cell lines, gene edited for HPFH deletions. Type of HPFH deletions are indicated at the x axis. Indels (cut site A and cut site B) measured by Sanger sequencing and ICE analysis. Deletion + Inversion (Del+Inv) (red checker box) quantified by ddPCR. n = 2. (C) Percentage of HbF^+ve cells upon introducing HPFH deletions indicated on the x axis. The edited cells were differentiated into erythroblasts and analyzed for HbF by flow cytometry. n = 6. (D) γ-Globin chain synthesis as measured by γ/γ+β ratio in the HUDEP-2 erythroblasts as measured by HPLC chain analysis. n = 5. (E) Magnified image of β-globin locus showing the binding sites of the key gRNA employed in this study to create various deletions mentioned in (F)–(K). (F) Percentage of gene editing in HUDEP-2 cell lines gene edited for various deletions as indicated in the x axis. Indels (cut site A and cut site B) measured by Sanger sequencing and ICE analysis. Deletion + Inversion (Del+Inv) (red checker box) quantified by ddPCR. n = 2. (G) Representative flow cytometry plot of HbF^+ve cells. HUDEP-2 cell lines gene edited for Sicilian HPFH deletion and deletion of its encompassing region in the β-globin cluster, were differentiated into erythroblasts and analyzed for HbF^+ve cells. Inset shows percentage of HbF^+ve cells. (H) Percentage of HbF^+ve cells upon introducing deletions in the region encompassing Sicilian HPFH. n = 4. (I) Representative globin chain HPLC chromatograms. (J) γ-Globin chain synthesis as measured by γ/γ+β ratio in the HUDEP-2 cell lines as measured by HPLC chain analysis. n = 4. (K) Percentage of HbF tetramer in erythroid differentiated HUDEP-2 cells gene edited for introducing deletions in the region encompassing Sicilian HPFH as measured by variant HPLC analysis. n = 2. Error bars represent mean ± SEM, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparisons test).

Figure 2

Robust γ-globin induction and β-globin silencing in the erythroblasts differentiated from PRR-βE1-edited HSPCs (A) Percentage of gene editing in PRR, βE1, and PRR-βE1 gene-edited healthy donor HSPCs. Indels measured by Sanger sequencing and ICE analysis. Deletion + Inversion (Del+Inv) (red checker box) in PRR-βE1 quantified by ddPCR. The PRR-βE1-edited cells had deletion, indels at PRR region and βE1. Donor = 5, n = 11. (B) FACS analysis of percentage of HbF^+ve cells in erythroblasts generated from gene-edited HSPCs. Gene-editing targets are indicated at the bottom. Control refers to unedited cells. Each dot indicates an individual experiment. Donor = 5, n = 11. (C) Percentage of fetal hemoglobin (HbF) tetramer as measured by variant HPLC for HSPCs gene edited for PRR, βE1, and PRR-βE1 and differentiated into erythroblasts. Donor = 3, n = 4. (D) Representative western blot image showing the band intensity of globin chains for erythroblasts derived from control, PRR, βE1, and PRR-βE1 gene-edited HSPCs. The editing in PRR and βE1 indicates the percentage of indels by ICE analysis and for PRR- βE1 edited, the percentage of editing includes the deletion + inversion quantified by ddPCR, cut site A and cut site B indels by ICE analysis. Donor = 1, n = 3. (E) Percentage of reticulocytes generated on erythroid differentiation of HSPCs gene edited for PRR, βE1, and PRR-βE1. Flow cytometric analysis of reticulocytes percentage was quantified on day 20 of three-phase erythroid differentiation. Donor = 5, n = 11. (F) Ratio of erythroid (E) to granulocyte-monocyte (GM) CFU colonies. HSPCs were gene edited for AAVS1, PRR, βE1, and PRR-βE1 and plated in methocult medium. Both BFU-E and CFU-E colonies were considered as erythroid (E) colonies. Donor = 2, n = 10. (G) Percentage of gene manipulation as measured by ddPCR for quantifying deletions in PRR-βE1 and Sicilian HPFH. Indel analysis of AAVS1, BCL11A enhancer, and HBG promoter by ICE analysis. Donor = 2, n = 4. (H) FACS analysis of percentage of HbF^+ve cells in erythroblasts generated from PRR-βE1, Sicilian HPFH, BCL11A enhancer, and HBG promoter. Donor = 2, n = 4. (I) Representative hemoglobin variant HPLC chromatograms showing HbF and HbA tetramers in gene-edited cells. (J) Percentage of HbF tetramers. HSPCs were gene edited for PRR-βE1, Sicilian HPFH, BCL11A enhancer, and HBG promoter, differentiated into erythroblasts and analyzed by variant HPLC. Donor = 2, n = 4. (K) Percentage of HbA tetramers. HSPCs were gene edited for PRR-βE1, Sicilian HPFH, BCL11A enhancer, and HBG promoter, differentiated into erythroblasts and analyzed by variant HPLC. Donor = 2, n = 4. (L) Representative western blot image showing the band intensity of globin chains for erythroblasts derived from PRR-βE1, Sicilian HPFH, BCL11A enhancer, and HBG promoter gene edited HSPCs. The editing in AAVS1, BCL11A enhancer, and HBG promoter indicates the indels quantified by ICE analysis. For PRR-βE1 and Sicilian HPFH, editing indicates the percentage of deletion and inversion quantified by ddPCR excluding the cut site indels. Donor = 1, n = 2. Error bars represent mean ± SEM, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparisons test).

Figure 3

PRR-βE1 gene-edited HSPCs repopulate for long-term and generate HbF^+ve cells in vivo Control and PRR-βE1 gene-edited healthy donor HSPCs were transplanted into NBSGW mice and analyzed 16 weeks post transplantation (A–G). Each dot indicates a single mouse. Donor = 2. Each cohort indicates an independent experiment infused with HSPCs gene edited for PRR-βE1. Error bars represent mean ± SEM, ns, nonsignificant. ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001 (two-way ANOVA followed by Dunnett’s test). (A) Percentage of engraftment in the bone marrow, peripheral blood, and spleen calculated flow cytometrically using hCD45 and mCD45.1 markers. (B) Percentage of HSPC and lineage markers in bone marrow (BM) – CD3 (T cells), CD13 (monocyte), CD19 (B cells), CD235a (erythroid), and CD34 (HSPCs) in engrafted cells. CD235a⁺ cells were analyzed form CD45⁻ cells. (C) Percentage of PRR-βE1 deletion+inversion (Del+Inv), PRR cut site indels, and βE1 cut site indels in PRR-βE1 gene-edited HSPCs in infused fraction and in engrafted cells. (D) Percentage of HbF^+ve cells in hCD235a^+ve cells obtained from mouse BM. (E) Percentage of HbF^+ve cells generated by erythroid differentiation of engrafted cells in the BM. (F) Ratio of γ/γ+β chains. Mouse BM was collected, in vitro differentiated into erythroblasts and analyzed by chain HPLC. (G) Percentage of engraftment in BM of secondary recipients analyzed 14 weeks post transplantation. AAVS1 and PRR-βE1 gene-edited healthy donor HSPCs were gene edited immediately after CD34 purification (day 0) and 48 h post CD34 purification (day 2) and transplanted into NBSGW mice and analyzed 16 weeks post transplantation (G)–(J). Each dot indicates a single mouse. Donor = 1. Error bars represent mean ± SEM, ns, nonsignificant. ^∗p ≤ 0.05 (two-way ANOVA followed by Dunnett’s test). (H) Percentage of PRR-βE1 deletion+inversion (Del+Inv), PRR cut site indels, and βE1 cut site indels in PRR-βE1 gene-edited HSPCs in Day 0 and Day 2 edited input fraction and in engrafted cells. (I) Percentage of HbF^+ve cells in hCD235a^+ve cells obtained from mouse BM. (J) Ratio of γ/γ+β chains. Mouse BM was collected, in vitro differentiated into erythroblasts, and analyzed by chain HPLC.

Figure 4

PRR-βE1 gene-edited patient HSPCs reverses sickle cell disease phenotype Plerixafor-mobilized HSPCs from sickle cell patients of genotype HbS/CD41/42(-TCTT) and HbS/IVS1-5 were gene edited for AAVS1, PRR, βE1, and PRR-βE1. Error bars represent mean ± SEM. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001 (two-way ANOVA followed by Dunnett’s test). (A) Percentage of gene editing. Indels measured by Sanger sequencing and ICE analysis. Deletion/inversion (Del+Inv) (red checker box) in PRR-βE1 quantified by ddPCR. The indels in the PRR-βE1 edited cells (gray checker box) were assessed using ICE analysis. Donor = 2, n = 4. (B) Relative globin mRNA expression. The patient HSPCs were gene edited for PRR, βE1, and PRR-βE1 and differentiated into erythroblasts. Real-time PCR analysis was used for mRNA quantification and the globin chain expression was normalized with β-actin. The patient genotype is indicated at the bottom. Donor = 2, n = 4. (C) Percentage of HbF^+ve cells. The gene-edited patient HSPCs were differentiated into erythroblasts and intracellular HbF positive cells were analyzed by FACS. Donor = 2, n = 4. (D) Percentage of sickle cells. Gene-edited patient HSPCs were differentiated into erythroblasts in hypoxia (5% O2) and the FACS sorted reticulocytes were treated with 1.5% sodium metabisulfite. Cells were scored from random fields using EVOS FL Auto Imaging System microscope. At least eight fields were analyzed. Each field contained a minimum of 150 cells. Donor = 2, n = 4. (E) Representative image of sickle cells (red arrow) and non-sickled cells. (F) Representative variant HPLC chromatogram showing HbA, HbF, and HbS. Donor = 2, n = 4. (G) Proportion of hemoglobin tetramer. The gene-edited patient HSPCs were differentiated into erythroblasts and the hemoglobin tetramers were analyzed by variant HPLC. Donor = 2, n = 4.

Figure 5

PRR-βE1 gene-edited patient HSPCs reverse β-thalassemia phenotype The G-CSF mobilized HSPCs from β-thalassemia patients of genotype IVS1-5 (G>C), and CD30 (G>A) were gene edited for AAVS1, βE1, and PRR-βE1. For HbE (G>A)/IVS1-5 (G>C), the PBMNCs were differentiated into erythroblasts and gene edited for AAVS1, βE1, and PRR-βE1. Error bars represent mean ± SEM. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001 (two-way ANOVA followed by Dunnett’s test). Donor = 3, n = 5. (A) Percentage of gene editing in HSPCs. Deletion/Inversion (Del+Inv) in PRR-βE1 as quantified by ddPCR. Indels of the cut sites PRR and βE1 were measured by ICE analysis. (B) Representative globin chain HPLC chromatograms. (C) α/non-α ratio in the erythroblasts generated from gene-edited HSPCs. (D) Representative western blot image showing the band intensity of globin chain erythroblasts derived from AAVS1, PRR, βE1, and PRR-βE1 gene-edited CD30 (G>A) patient HSPCs. Donor = 1, n = 1. The editing in PRR and βE1 indicates the percentage of indels in ICE analysis and for PRR-βE1 edited, the percentage of editing includes the deletion + inversion quantified by ddPCR, cut site A and cut site B indels by ICE analysis. Donor = 1, n = 1. (E) Representative flow cytometry image of Annexin V staining. (F) Percentage of Annexin V in the erythroblasts generated from gene-edited HSPCs. (G) Representative flow cytometry plots of reticulocytes marked by CD235a⁺/Hoechst⁻. (H) Percentage of reticulocytes generated from gene-edited HSPCs.

Figure 6

PRR-βE1 gene-edited HSPCs have intact genome integrity (A) Percentage of gene editing on PRR-βE1 editing in healthy donor HSPCs using HiFi Cas9. Indels measured by Sanger sequencing and ICE analysis. Deletion/inversion (Del+Inv) (red checker box) in PRR-βE1 quantified by ddPCR. Donor = 1, n = 2. (B) Percentage of micronucleus (MN) in Mitomycin C treated, unedited, and PRR-βE1 gene-edited HSPCs scored 48 h post nucleofection after staining with Giemsa. Donor = 1, n = 2. (C) KaryoStat analysis of healthy donor HSPCs gene edited for PRR-βE1 deletion. Donor = 1, n = 2. (D) Circos plot showing off-target mediated translocation between the PRR-βE1 on-target site and βE1 off-target site in PRR-βE1 edited samples present in chr16 identified by CAST-Seq. Donor = 1, n = 4. Error bars represent mean ± SEM. ns, nonsignificant, ∗∗p ≤ 0.01 (one-way ANOVA followed by Dunnett’s multiple comparisons test).

Figure 7

PRR-βE1 gene editing reconfigures chromosome looping and alters globin expression (A) 4C analysis of single-cell sorted control and two PRR-βE1 biallelic gene-edited HUDEP-2 clones using HBG2 promoter as a viewpoint. n = 4. (B) Heatmap of the top differentially expressed genes of erythroblasts derived from PRR-βE1 and βE1 gene-edited HSPC indicating the relative gene expression pattern of genes up- and downregulated compared with control. Donor = 1, n = 2. (C) Cluster per million (cpm) values for the globin transcripts obtained from RNA sequencing. (D) Relative HBBP1 mRNA expression in erythroblasts derived from βE1 and PRR-βE1 gene-edited HSPCs compared with AAVS1. The globin chain expression is normalized with β-actin. Donor = 1, n = 4. (E) Relative BGLT3 mRNA expression in erythroblasts derived from βE1, a PRR-βE1 gene-edited HSPC compared with AAVS1. The globin chain expression is normalized with β-actin. Donor = 2, n = 8. Error bars represent mean ± SEM.