Abstract
β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.
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Data availability
High-throughput sequencing data generated in the Cas9 off-target activity assessment have been uploaded to the public NCBI data repository: BioProject ID PRJNA691350, available at http://www.ncbi.nlm.nih.gov/bioproject/691350. All other data supporting the findings of this study are either included in the published article and/or Supplementary information files or (if too large to be included therein) are available from the corresponding author on reasonable request. These include, but are not limited to, Sanger sequencing, ddPCR, flow cytometry and HPLC data that were used to generate the figures and conclusions in this study. Source data are provided with this paper.
Code availability
No previously unreported computer code or algorithm was generated during the course of this study.
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Acknowledgements
The authors thank the following funding sources that made this work possible: Doris Duke Charitable Trust (no. 22399) to M.H.P., NIH NHLBI (no. R01-HL135607) to M.H.P. and D.P.D., and NIH NHLBI (no. T32-HL120824) to M.K.C.
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Contributions
D.P.D. and M.H.P. supervised the project. M.K.C., J.C., R.M., V.A.S., J.F.T., D.P.D. and M.H.P. designed experiments. M.K.C., J.C., R.M.M., B.J.L., C.A.V., V.T.L., Y.Z., A.G., F.Z., E.P., W.S., R.O.B. and N.U. carried out experiments. M.K.C., J.C., D.P.D. and M.H.P. wrote the manuscript.
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Competing interests
M.H.P. is a member of the scientific advisory board of Allogene Therapeutics. M.H.P. is on the Board of Directors of Graphite Bio. M.H.P. has equity in CRISPR Tx. C.A.V., N.M.B., G.K., M.A.C., G.R.R. and M.A.B. are employees of Integrated DNA Technologies. D.P.D. is an employee of Graphite Bio.
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Peer review information Nature Medicine thanks the anonymous reviewers for their contribution to the peer review of this work. Kate Gao and Joao Monteiro were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Analysis of Cas9 sgRNAs targeting α-globin loci.
a, Table with guide RNA sequences. PAM shown in grey, and differences between HBA1 and HBA2 are highlighted in red for each guide. b, Schematic depicting locations of all five guide sequences at genomic loci. c, Representative indel spectrum of HBA1-specific sg5 generated by TIDE software.
Extended Data Fig. 2 Off-target analysis of HBA1-specific sg5.
a, Summary of rhAmpSeq targeted sequencing results at on-target and 40 most highly predicted off-target sites by COSMID for HBA1 sg5. Values are indel frequency for RNP treatment after subtraction of indel frequency for Mock treatment at each locus for each experimental replicate. N = 3 biologically independent HSPC donors, though not all values are displayed since some were <0.01% after subtraction of Mock indel frequencies. Bars represent median. b, List of genomic coordinates for forty most highly predicted off-target sites by COSMID for HBA1 sg5.
Extended Data Fig. 3 Targeting α-globin with GFP integration vectors.
a, Timeline for editing and analysis of HSPCs targeted with SFFV-GFP integration vectors. b, Depicted are representative flow cytometry images for human HSPCs 14d post-editing. This indicates that WGR integration yields a greater MFI per GFP+ cell than CS integration at the HBA1 locus. Analysis was performed on BD Accuri C6 platform. Median MFI across all replicates is shown below each flow cytometry image, and schematics of integration vectors are shown above.
Extended Data Fig. 4 Staining and gating scheme used to analyze editing and differentiation rates of RBCs.
a, Representative flow cytometry staining and gating scheme for human HSPCs targeted at HBA1 with HBB-T2A-YFP (HBA1 UTRs) and differentiated into RBCs. This indicates that only RBCs (CD34−/CD45−/CD71+/GPA+) are able to express the promoterless YFP marker. Analysis was performed on BD FACS Aria II platform. b, Representative flow cytometry images of RBCs (CD34−/CD45−/CD71+/GPA+) derived from HSPCs targeted with HBA1 UTRs, HBA2 UTRs, and HBB UTRs vector. AAV only controls were used for each vector to establish gating scheme, leading to slight variation in positive/negative cut-offs across images.
Extended Data Fig. 5 Viability of HSPCs post-editing.
HSPC viability was quantified 2-4d post-editing by flow cytometry. Depicted are the percentage of cells that stained negative for GhostRed viability dye. All cells were edited with our optimized HBB WGR vector using standard conditions (that is electroporation of Cas9 RNP + sg5, 5 K MOI of AAV, and no AAV wash at 24 h). Bars represent median ± interquartile range. WT: N = 5 for mock, N = 3 for RNP only, N = 1 for AAV only, and N = 6 for RNP + AAV treatment group; SCD: N = 2 for each treatment group with exception of RNP + AAV with N = 4; β-thal: N = 3 for mock, N = 1 for RNP only, and N = 7 for RNP + AAV treatment group.
Extended Data Fig. 6 Relationship between % edited alleles and % edited cells.
a, Representative flow cytometry plots of HSPCs simultaneously targeted at HBA1 with GFP (shown in Fig. 1c) and mPlum integration cassettes. b, Table showing % of populations targeted with GFP only, mPlum only, and both colors. Percent of edited cells was then converted to % edited alleles by the following equation: (total % targeted cells + (% dual color)*2)/2 = total % targeted alleles. c, Percent edited cells is plotted against % edited alleles for data shown in panel B. A polynomial regression (R2 = 0.9981) was used to determine an equation to convert between % edited alleles and % edited cells.
Extended Data Fig. 7 Colony-forming ability of edited HSPCs.
a, Distribution of genotypes of methylcellulose colonies displayed in Panels B and D. Numbers of clones corresponding to each category are included in the pie chart. b, In vitro (pre-engraftment) live CD34+ HSPCs from healthy donors were single-cell sorted into 96-well plates containing semisolid methylcellulose media for colony forming assays. 14d post-sorting cells were analyzed for morphology. Depicted are number of colonies formed for each lineage (CFU-E = erythroid lineage; CFU-GEMM = multi-lineage; or CFU-GM = granulocyte/macrophage lineage) divided by the total number of wells available for colonies. Columns represent median ± interquartile range. N = 3 experimental replicates with a minimum of 3 96-well methylcellulose-coated plates for mock, RNP only, and WGR-GFP AAV6 treatment groups; N = 2 for AAV only and HBB-HBA1 AAV6 treatment groups. c, Percent distribution of each lineage among all colonies for each treatment for Panel B. d, As above, in vitro (pre-engraftment) live CD34+ β-thalassemia HSPCs were sorted into 96-well plates for colony forming assays. Depicted are number of colonies formed for each lineage (B = BFU-E and C = CFU-E (erythroid lineage); GE = CFU-GEMM (multi-lineage); or GM = CFU-GM (granulocyte/macrophage lineage)) divided by the total number of wells available for colonies. Columns represent median ± interquartile range. For Mock and RNP + AAV, N = 2 experimental replicates with a minimum of 3 96-well methylcellulose-coated plates for each treatment; N = 1 experimental replicate with 3 plates for RNP only treatment. e, Percent distribution of each lineage among all colonies for each treatment for Panel D.
Extended Data Fig. 8 Engraftment into NSG mice of human HSPCs targeted with GFP at α-globin locus.
a, Timeline for targeting of HSPCs with UbC-GFP integration vector, transplantation into mice (both 1o and 2o engraftment), and subsequent analysis. b, AAV6 DNA repair donor design schematic to introduce a UbC-GFP-BGH integration is depicted at the HBA1 locus. c, 16 weeks after bone marrow transplantation of targeted human CD34+ HSPCs into NSG mice, bone marrow was harvested and rates of engraftment were determined (1°). Depicted is the percentage of mTerr119− cells (non-RBCs) that were hHLA+ from the total number of cells that were either mCd45+ or hHLA+. Bars represent median ± interquartile range. N = 8 biologically independent NSG mouse transplantations. d, Among engrafted human cells, the distribution among CD19+ (B-cell), CD33+ (myeloid), or other (that is HSPC/RBC/T/NK/Pre-B) lineages are indicated. Bars represent median ± interquartile range. N = 8 biologically independent NSG mouse transplantations. e, Percentage of GFP+ cells among pre-transplantation (in vitro, post-sorting) and successfully engrafted populations, both bulk HSPCs and among lineages. Bars represent median ± interquartile range. N = 3 independent HSPC donors from in vitro experiments that were transplanted into N = 6 individual NSG mice, from which N = 4 individual mice were lineage sorted and analyzed. Various green shades correspond to each particular HSPC donor. b, Following primary engraftments, engrafted human cells were transplanted a second time into the bone marrow of NSG mice. 16 weeks post-transplantation, bone marrow was harvested and rates of engraftment were determined (2°). Depicted is the percentage of mTerr119− cells (non−RBCs) that were hHLA+ from the total number of cells that were either mCd45+ or hHLA+. N = 1 NSG mouse transplantation. g, Percentage of GFP+ cells among successfully engrafted population from the secondary transplant depicted in Panel F. N = 1 NSG mouse transplantation.
Extended Data Fig. 9 Characterization of targeted β-thalassemia HSPCs.
a, Following differentiation of targeted HSPCs into RBCs, mRNA was harvested and converted into cDNA. Expression of HBA (does not distinguish between HBA1 and HBA2) and HBB transgene were normalized to HBG expression. Bars represent median ± interquartile range. N = 3 biologically independent editing experiments for all treatment groups with exception of HBA1 UTRs with N = 1. b, Summary of reverse-phase globin chain HPLC results showing % AUC of β-globin and α-globin. Bars represent median ± interquartile range. Bars represent median ± interquartile range. N = 3 biologically independent erythroid differentiation experiments for all treatment groups with exception of RNP only with N = 5. **P < 0.005; ***P < 0.0001 using unpaired two-tailed t test without adjustment for multiple comparisons.
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Cromer, M.K., Camarena, J., Martin, R.M. et al. Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nat Med 27, 677–687 (2021). https://doi.org/10.1038/s41591-021-01284-y
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DOI: https://doi.org/10.1038/s41591-021-01284-y
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