The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.
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D.P.D. was supported by a Stanford Child Health Research Institute (CHRI) Grant and Postdoctoral Award. R.O.B. was supported by an Individual Postdoctoral grant (DFF–1333-00106B) and a Sapere Aude, Research Talent grant (DFF–1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. M.H.P. acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research and NIH grant support PN2EY018244, R01-AI097320 and R01-AI120766. We thank D. Russell for the pDGM6 plasmid, H.-P. Kiem for scAAV6, G. de Alencastro and M. Kay for help with AAV production, the Binns Program for Cord Blood Research at Stanford University for cord-blood-derived CD34+ HSPCs. We also thank Lonza (A. Toell and G. Alberts) for donating the LV unit for performing large-scale genome-editing studies. We further thank members of the Porteus laboratory, D. DiGiusto and M. G. Roncarolo for input, comments and discussion.
M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input into the design, execution, interpretation or publication of the results herein. N.U. was a former employee of Stem Cells, Inc., but they had no input into this manuscript.
Reviewer Information Nature thanks B. Ebert, L. Naldini and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 High tropism of rAAV6 for CD34+ HSPCs, and viability and specificity assessment of gene editing in CD34+ HSPCs.
a, CD34+ HSPCs were transduced with a scAAV6 expressing GFP from an SFFV promoter at multiplicities of infections (MOIs) of 10,000 or 100,000 viral genomes (vg) per cell for 48 h and then analysed for percentage GFP+ expression by flow cytometry using a non-transduced sample to set the GFP+ gate at <0.1% GFP+ cells. scAAV was used because it eliminates second-strand synthesis as a confounder of actual transduction. Results are from two independent experiments from at least two donors and error bars represent s.d. ABM, adult bone marrow; CB, cord blood; mPB: mobilized peripheral blood. b, CD34+ HSPCs were electroporated with the HBB CRISPR system (mRNA or RNP delivery) or without (AAV only), and then transduced with HBB rAAV6 donor vectors at an MOI of 100,000 vg per cell. Day 4 after electroporation, cells were analysed by flow cytometry and live cells were gated in high forward scatter (FSC) and low side scatter (SSC). Percentage of cells in FSC/SSC gate (that is, percentage viability) is shown relative to that of mock-electroporated cells. Each data point represents a unique CD34+ HSPC donor. c, Top, sgRNA target sequences at the HBB on-target site and a highly complementary off-target site (Chr9:101833584–101833606) are shown. PAM sequences are underlined and red sequence highlights the three mismatches of the off-target site. Bottom, HSPCs were electroporated with either the mRNA or RNP-based CRISPR system, and 4 days post electroporation genomic DNA was extracted and analysed for INDEL frequencies using TIDE at the on-target HBB and the off-target site. Results are shown as the ratio of on- to off-target activity highlighting the increased specificity of the RNP system. Averages from three different CD34+ HSPC donors are shown and error bars represent s.e.m. **P < 0.01, unpaired Student’s t-test. d, INDEL frequencies for the data presented in c. *P < 0.05, paired Student’s t-test. e, Representative FACS plots showing stable GFP rates at day 18 after electroporation in donor-nuclease mismatch experiments. Mismatching nuclease and donor (red box) leads to infrequent end-capture events compared to on-target homologous recombination events observed with matched nuclease and homologous rAAV6 donor (green box). HSPCs were electroporated with 15 μg Cas9 mRNA and either HBB or IL2RG 2′-O-methyl-3′-phosphorothioate-modified sgRNA, then transduced with HBB-GFP rAAV6 donor followed by 18 days of culture. f, End-capture experiments were performed in three replicate experiments each in three unique CD34+ HSPC donors. ns (not significant) = P ≥ 0.05, paired Student’s t-test. Activity of the IL2RG CRISPR was confirmed by quantification of INDELs at the IL2RG target site using TIDE analysis.
Extended Data Figure 2 Schematic of targeting rAAV6 Glu6Val homologous donor to the HBB locus.
a, The human HBB locus on chromosome 11 is depicted at the top of the schematic and consists of three exons (black boxes) and two introns. The rAAV6 Glu6Val donor includes the Glu to Val mutation at codon 6, which is the amino acid change causing SCD. Other SNPs (all SNPs are capitalized) were introduced to PAM site (blue) and sgRNA-binding site (bold) to prevent re-cutting following homologous recombination in HSPCs. To analyse targeted integration frequencies in HSPCs, a two-step PCR was performed. First, a 3,400-bp in-out PCR (green) was performed followed by a nested 685-bp PCR (purple) on a gel-purified fragment from the first PCR. This second PCR fragment was cloned into TOPO vectors, which were sequenced to determine the allele genotype (wild type, INDEL or homologous recombination). b, The sequence of a wild-type HBB allele aligned with the sequence of an allele that has undergone homologous recombination. c, Representative INDELs from the data represented in Fig. 1d. The HBB reference sequence is shown in green.
Extended Data Figure 3 Linear regression model shows that the day 4 GFPhigh population is a reliable predictor of targeting frequencies.
Day 4 GFPhigh percentages (x axis) were plotted against day 18 total GFP+ percentages (y axis), and linear regression was performed. Data were generated from experiments including a total of 38 different CD34+ HSPC donors, treated with either 15 μg or 30 μg Cas9 RNP to generate data points with a wider distribution of targeting frequencies.
Extended Data Figure 4 Overview of PCR genotyping of methylcellulose colonies with homologous recombination of the GFP and tNGFR donor at the HBB locus.
a, The HBB locus was targeted by creating a DSB in exon 1 via Cas9 (scissors) and supplying a rAAV6 GFP donor template. Alleles with integrations were identified by PCR (red, 881 bp) on methylcellulose-derived colonies using an in-out primer set. Wild-type alleles were identified by PCR (green, 685 bp) using primers flanking the sgRNA target site. b, Representative genotyping PCRs showing mono- and biallelic clones as well as a clone derived from mock-treated cells. NTC, non-template control (see Supplementary Fig. 1a for uncropped gel). c, Representative Sanger sequence chromatograms for junctions between right homology arm (blue) and insert (green) or genomic locus, highlighting seamless homologous recombination. d, The HBB locus was targeted by creating a DSB in exon 1 via Cas9 (scissors) and supplying a rAAV6 tNGFR donor template. Genotypes were assessed by a three-primer genotyping PCR on methylcellulose-derived colonies using an in-out primer set (red, 793 bp) and a primer set flanking the sgRNA target site (green, 685 bp). Note that the two forward primers are the same. e, Representative genotyping PCRs showing a wild-type/unknown, mono-, and biallelic clone (see Supplementary Fig. 1b for uncropped gel). f, Representative Sanger sequence chromatograms for junctions between left homology arm (in blue) and insert (in green) or genomic locus highlighting seamless homologous recombination.
Extended Data Figure 5 Haematopoietic progenitor CFU assay and targeting in different HSPC subpopulations.
a, GFPhigh HSPCs were single-cell-sorted into 96-well plates containing methylcellulose. Representative images from fluorescence microscopy show lineage-restricted progenitors (BFU-E, CFU-E, CFU-GM) and multipotent progenitors (CFU-GEMM) with GFP expression. b, CFUs were counted 14 days after sorting and shown relative to the total number of cells sorted (percentage colony formation) (n = 2 different HSPC donors). c, Colonies were scored according to their morphology: (1) CFU-erythroid (CFU-E); (2) burst forming unit-erythroid (BFU-E); (3) CFU-granulocyte/macrophage (CFU-GM); and (4) CFU-granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) (n = 2 different HSPC donors). d, Representative FACS plots at day 4 after electroporation of CD34+ HSPCs showing the gating scheme for analysing targeting frequencies in different HSPC subsets (Extended Data Fig. 5f). Cells were immunophenotyped for CD34, CD38, CD90 and CD45RA expression and relevant FACS gates are indicated. e, Representative FACS plots showing GFPhigh cells in the CD34+ CD38− CD90+ CD45RA− population of HSPCs derived from mobilized peripheral blood, bone marrow, or cord blood. f, 500,000 HSPCs isolated from mobilized peripheral blood, adult bone marrow, or cord blood were electroporated with RNP and transduced with GFP rAAV6 donor. At day 4 after electroporation, cells were phenotyped by flow cytometry for the cell surface markers CD34, CD38, CD90 and CD45RA (Extended Data Fig. 5d, e). Percentage GFPhigh cells in the indicated subpopulations are shown (data points represent unique donors, n = 3 per HSPC source), ****P < 0.0001, paired Student’s t-test. g, CD34+ or CD34+ CD38− CD90+ cells were sorted directly from freshly isolated cord blood CD34+ HSPCs, cultured overnight, and then electroporated with RNP and transduced with GFP rAAV6. Bars show average percentage GFP+ cells at day 18 after electroporation. (n = 3 from different HSPC donors), **P < 0.01, paired Student’s t-test. h, Multipotent progenitor (MPP) (CD34+ CD38− CD90− CD45RA−) and HSC (CD34+ CD38− CD90+ CD45RA−) populations were sorted from fresh cord-blood-derived CD34+ HSPCs and immediately after sorting, cells were transduced with scAAV6-SFFV-GFP at an MOI of 100,000 vg per cell along the bulk HSPC population. scAAV6 was used because it eliminates second-strand synthesis as a confounder of actual transduction, although the activity of the SFFV promoter may not be equivalent in each population, thus potentially underestimating the degree of transduction of MPPs and HSCs. Two days later, transduction efficiencies were measured by FACS analysis of GFP expression using non-transduced cells (mock) to set the GFP+ gate. Error bars represent s.e.m., n = 4, two different HSPC donors. **P < 0.01; NS, not significant = P ≥ 0.05, unpaired t-test with Welch’s correction.
Extended Data Figure 6 Analysis of human engraftment.
a, Representative FACS plot from the analysis of the bone marrow of a control mouse not transplanted with human cells. Mice were euthanized and bone marrow was collected from femur, tibia, hips, humerus, sternum and vertebrae. Cells were subject to Ficoll density gradient to isolate mononuclear cells, which were analysed for human engraftment by flow cytometry. Human engraftment was delineated as hCD45/HLA-ABC double positive. From a total of 157,898 cells, 4 were found within the human cell gate of a non-injected control mouse, showing the very limited background human staining. b, Representative FACS plots showing gating scheme for analyses of NSG mice transplanted with human cells and analysed as described in a. Representative plots are from one mouse from the RNP plus rAAV6 experimental group. As above, human engraftment was delineated as hCD45/HLA-ABC double positive. B cells were marked by CD19 expression, and myeloid cells identified by CD33 expression. GFP expression was analysed in total human cells (2.4%), B cells (1.9%) and myeloid cells (2.8%). The GFP brightness in B cells is lower than in myeloid cells, suggesting that the SFFV promoter is not as active in the B-cell lineage compared to the myeloid lineage (see also Fig. 3a). c, Overview of engraftment for RNP plus AAV and RNP plus AAV GFPhigh experimental groups. Average engraftment frequencies and percentage GFP+ human cells ± s.e.m. are shown. Total number of cells transplanted was the same (500,000) for all mice in the RNP group, whereas in the GFPhigh group, one mouse was transplanted with 100,000 cells, two mice with 250,000 cells, and three mice with 500,000 cells. The total number of HSCs transplanted per mouse (±s.e.m.) was calculated based on the frequencies of GFP+ cells in the CD34+ CD38− CD90+ CD45RA− subset analysed by flow cytometry (see Extended Data Fig. 5f) directly before injection. The total number of modified human cells in the bone marrow at week 16 after transplant per mouse (±s.e.m.) was estimated based on calculations presented in the Methods. This shows that the enrichment not only resulted in a higher percentage of edited cells (column 3) but also resulted in an absolute higher number (column 6) of edited cells.
Extended Data Figure 7 Genome-edited human HSPCs in the bone marrow of NSG mice at week 16 after transplantation.
a, b, Representative FACS plots from the analysis of NSG mice from the mock (a) or RNP plus AAV (b) experimental group at week 16 after transplantation. Mice were euthanized and bone marrow was obtained, MNCs were isolated via Ficoll density gradient, after which human CD34+ cells were enriched by magnetic-activated cell sorting (MACS), and finally cells were stained with anti-CD34, anti-CD38 and anti-CD10 antibodies to identify human GFP+ cells in the CD34+ CD10− and CD34+ CD10− CD38− populations (note that CD10 was included as a negative discriminator for immature B cells). c, Collective data from the analysis of GFP+ cells in the human CD34+ CD10− population from the RNP plus AAV (n = 11) and RNP plus AAV GFPhigh (n = 6) experimental groups. For the RNP plus AAV GFPhigh group, cells from all six mice were pooled before analysis and thus, no error bar is available. Error bar on RNP group represents s.e.m.
Extended Data Figure 8 Correction of the sickle cell mutation in patient-derived CD34+ HSPCs.
a, Schematic overview of the sequence of the sickle cell allele aligned with the sequence of an allele that has undergone homologous recombination using the corrective SNP donor. The Glu6Val mutation in patients with SCD (A>T) is highlighted in yellow. The sgRNA recognition sequence, the PAM site and the cut site (scissors) are shown. The donor carries synonymous nucleotide changes between the sickle nucleotide and the cut site to avoid premature crossover during homologous recombination. Synonymous changes are also added to the PAM and an early nucleotide in the sgRNA target site to avoid subsequent re-cutting and potential inactivation of the corrected allele. b, HSPCs from two different patients with SCD were targeted with the corrective SNP donor and seeded in methylcellulose. After 14 days, in-out PCR amplicons from a total of 38 clones were sequenced and genotypes were extracted from sequence chromatograms.
Extended Data Figure 9 Targeting HBB with a cDNA donor and a tNGFR expression cassette.
a, Schematic representation of the AAV6 donor encoding a functional HBB anti-sickling cDNA (Gly16Asp, Glu22Ala, Thr87Gln) followed by an expression cassette for tNGFR. The left homology arm stops just before the sickle mutation (A→T) followed by the remaining HBB cDNA, which has been diverged from the endogenous sequence by introducing synonymous mutations at codon wobble positions. The HBB cDNA expression cassette is followed by an EF1α promoter driving tNGFR expression (HBB cDNA-EF1α-tNGFR), which constitutes a clinically compatible expression cassette enabling enrichment and tracking of modified cells. b, Chromatogram from sequencing of an in-out PCR amplicon on CD34+ cells derived from patients with SCD, electroporated with HBB Cas9 RNP and transduced with rAAV6 HBB cDNA donor. PCR was performed on genomic DNA extracted 4 days after electroporation of a bulk sample. Chromatogram shows the sequence of the full HBB cDNA with start codon, sickle-cell codon position (containing a corrected Glu codon), and stop codon highlighted in red, green and blue boxes, respectively.
Extended Data Figure 10 Edited HSPCs from patients with SCD differentiate into erythrocytes that express glycophorin A.
CD34+ HSPCs derived from patients with SCD were edited with HBB Cas9 RNP and either the corrective SNP donor or the cDNA donor. Four days after electroporation, cells edited with the cDNA donor were sorted for tNGFR+ cells. This population as well as the populations edited with the corrective SNP donor and mock-electroporated cells were subjected to a 21-day erythrocyte differentiation protocol, followed by staining for glycophorin A (GPA). All data points within experimental groups are derived from experiments in cells from different patients with SCD, n = 3 (mock) and n = 2 (SNP and cDNA donor).
This file contains Supplementary Figures 1 and 2, which show the raw data for Extended Data Figure 4b,e (Figure 1) and Figure 3e (Figure 2). (PDF 271 kb)
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Dever, D., Bak, R., Reinisch, A. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). https://doi.org/10.1038/nature20134
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