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Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

Becker, Hans Jiro, Ishida, Reiko, Wilkinson, Adam C., Kimura, Takaharu, Lee, Michelle Sue Jann, Coban, Cevayir, Ota, Yasunori, Tanaka, Yosuke, Roskamp, Meike, Sano, Tsubasa, Tojo, Arinobu, Kent, David G., Yamazaki, Satoshi·2023·Cell Stem Cell
DOI 10.1016/j.stem.2023.06.002PMC PMC10338855
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Graphical abstract CC BY · PubMed Central
Figure 1 Autologous HSCT gene correction rescues the Prkdcscid phenotype but introduces on-, off-target indels and large deletions (A) Genomic context of the Prkdcscid mutation in exon 85. White boxes: exons, gray box, 3′ UTR. ∗ denotes location of Prkdcscid mutation. (B) Experimental scheme of the gene editing and HSC expansion model. (C) Post-editing allele distribution at the Prkdc locus, assessed by inference of CRISPR edit (ICE) (n = 3 cultures). (D) Fractions of immunophenotypically defined HSPC populations within cultures on day 10 of culture, 7 days post-editing. Percentage of all live cells (n = 3 cultures). (E) Absolute cell numbers (left panel) and fold-change expansion (right panel) of cultured HSPCs, day 10 of culture. (F) Left: frequencies of peripheral blood (PB) leukocytes as percentage of all live leukocytes (n = 3 groups, 3–4 mice per group). Plot next to dashed line shows frequencies 12 weeks post-secondary SCT (n = 5 mice). Right: representative fluorescence-activated cell sorting (FACS) plots 20 weeks post-transplant. (G) Frequencies of Prkdc alleles in sorted PB cells 20 weeks post-SCT (n = 3 experiments, 3–4 mice per group). (H) On- and off-target (OT) activity of the Prkdc-specific gRNA, assessed with tracking of indels by decomposition (TIDE). The seven highest scoring off-target sites, as predicted by COSMID, were interrogated. See Table S1 for detailed information about the off-target sites. (I) Copy-number analysis of Prkdc probes against reference gene (n = 3). Two-way ANOVA with Sidak’s multiple comparison test. Error bars represent SD. ∗p < 0.05, ∗∗∗∗p < 0.0001.
Figure 1 Autologous HSCT gene correction rescues the Prkdcscid phenotype but introduces on-, off-target indels and large deletions (A) Genomic context of the Prkdcscid mutation in exon 85. White boxes: exons, gray box, 3′ UTR. ∗ denotes location of Prkdcscid mutation. (B) Experimental scheme of the gene editing and HSC expansion model. (C) Post-editing allele distribution at the Prkdc locus, assessed by inference of CRISPR edit (ICE) (n = 3 cultures). (D) Fractions of immunophenotypically defined HSPC populations within cultures on day 10 of culture, 7 days post-editing. Percentage of all live cells (n = 3 cultures). (E) Absolute cell numbers (left panel) and fold-change expansion (right panel) of cultured HSPCs, day 10 of culture. (F) Left: frequencies of peripheral blood (PB) leukocytes as percentage of all live leukocytes (n = 3 groups, 3–4 mice per group). Plot next to dashed line shows frequencies 12 weeks post-secondary SCT (n = 5 mice). Right: representative fluorescence-activated cell sorting (FACS) plots 20 weeks post-transplant. (G) Frequencies of Prkdc alleles in sorted PB cells 20 weeks post-SCT (n = 3 experiments, 3–4 mice per group). (H) On- and off-target (OT) activity of the Prkdc-specific gRNA, assessed with tracking of indels by decomposition (TIDE). The seven highest scoring off-target sites, as predicted by COSMID, were interrogated. See Table S1 for detailed information about the off-target sites. (I) Copy-number analysis of Prkdc probes against reference gene (n = 3). Two-way ANOVA with Sidak’s multiple comparison test. Error bars represent SD. ∗p < 0.05, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Figure 2 Identification of a surface marker combination for long-term (LT) expanding HSC clones (A) Experimental setup. (B) Uniform manifold approximation and projection (UMAP) representation of sorted KSL clones with overlay of panel 1 (upper) and 2 (lower) surface markers. Expansion colony-forming clones are indicated in red. (C and D) Quantification of markers associated with colony expansion. Left: fluorescence intensity (FI) measured at index sorting. Data presented as log-transformed and normalized to mean. Boxplots with whiskers showing minimum and maximum. Center: fraction of clones of the indicated phenotype showing LT expansion. Right: representative FACS sorting plots, LT-expanding clones indicated in red. (C) Panel 1 (n = 110 clones); (D) panel 2 (n = 117 clones). Multiple Mann-Whitney tests with FDR correction. (E) RNA-seq expression profiles of select HSC- and progenitor-associated genes. Error bars represent SD. One-way ANOVA with Tukey’s post-test. (F and G) Gene set enrichment analysis (GSEA) of differentially expressed genes in CD201+CD150+CD48−KSL (F) and CD201−CD150+KSL (G) cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 2 Identification of a surface marker combination for long-term (LT) expanding HSC clones (A) Experimental setup. (B) Uniform manifold approximation and projection (UMAP) representation of sorted KSL clones with overlay of panel 1 (upper) and 2 (lower) surface markers. Expansion colony-forming clones are indicated in red. (C and D) Quantification of markers associated with colony expansion. Left: fluorescence intensity (FI) measured at index sorting. Data presented as log-transformed and normalized to mean. Boxplots with whiskers showing minimum and maximum. Center: fraction of clones of the indicated phenotype showing LT expansion. Right: representative FACS sorting plots, LT-expanding clones indicated in red. (C) Panel 1 (n = 110 clones); (D) panel 2 (n = 117 clones). Multiple Mann-Whitney tests with FDR correction. (E) RNA-seq expression profiles of select HSC- and progenitor-associated genes. Error bars represent SD. One-way ANOVA with Tukey’s post-test. (F and G) Gene set enrichment analysis (GSEA) of differentially expressed genes in CD201+CD150+CD48−KSL (F) and CD201−CD150+KSL (G) cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Figure 3 Optimization of polymer-based cultures for single-cell HSC expansion (A) Scheme of experimental setup. (B) Percentage of colonies with ≥20% live cells (n = 5 experiments). Unpaired, two-tailed t test. (C) Percentage of phenotypic HSC populations in live colonies cultured in PVA (n = 94)- and Soluplus (n = 155)-based media. Multiple Mann-Whitney tests with FDR correction. (D) Schematic of split-clone transplantation. (E and F) Donor PB chimerism (E) and lineage distribution (F) in 3 recipient groups transplanted with split clones. Numbers over graphs in (E) represent percentage of CD201+CD150+KSL cells in the transplanted clone (%) and the number of recipients (n). Secondary SCT was performed with the group showing highest chimerism, data shown in graph with gray axis. (G) Left: ELDA output of HSC frequency calculation. Right: boxplot represents calculated reciprocal mean, upper, and lower limits of HSC frequency. See also Figure S3 and Table S2. Error bars represent SD. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 3 Optimization of polymer-based cultures for single-cell HSC expansion (A) Scheme of experimental setup. (B) Percentage of colonies with ≥20% live cells (n = 5 experiments). Unpaired, two-tailed t test. (C) Percentage of phenotypic HSC populations in live colonies cultured in PVA (n = 94)- and Soluplus (n = 155)-based media. Multiple Mann-Whitney tests with FDR correction. (D) Schematic of split-clone transplantation. (E and F) Donor PB chimerism (E) and lineage distribution (F) in 3 recipient groups transplanted with split clones. Numbers over graphs in (E) represent percentage of CD201+CD150+KSL cells in the transplanted clone (%) and the number of recipients (n). Secondary SCT was performed with the group showing highest chimerism, data shown in graph with gray axis. (G) Left: ELDA output of HSC frequency calculation. Right: boxplot represents calculated reciprocal mean, upper, and lower limits of HSC frequency. See also Figure S3 and Table S2. Error bars represent SD. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Figure 4 Single-cell cloning of gene-edited functional HSCs (A) Schematic showing the extracellular domain of CD45 with allele-specific antibody clones 104 and A20 and the epitope-defining amino acid. (B) Experimental setup of the single-cell editing and expansion experiment. (C) Left: fractions of CD201+CD150+KSL cells in single-cell-derived cultures 14 days after cloning (n = 261 clones). Right: Histogram of CD201+CD150+KSL cell frequency. Zoomed-in region shows clones with >10% CD201+CD150+KSL cells. (D and E) CD45.1+ donor PB chimerism (D) and lineage distribution (E) in single recipients with long-term (LT) engraftment ≥5% and multilineage reconstitution (n = 8). Numbers over graphs in (D) represent percentage of CD201+CD150+KSL cells in the transplanted clone (%). (F) Linear correlation plots of CD201+CD150+KSL cell frequency and 16-week donor chimerism. Red dots indicate LT repopulating and multilineage clones. Pearson correlation. (G) CD45.1+ PB chimerism and lineage distribution in secondary recipients (n = 5). See also Figure S4 and Table S3. Error bars represent SD.
Figure 4 Single-cell cloning of gene-edited functional HSCs (A) Schematic showing the extracellular domain of CD45 with allele-specific antibody clones 104 and A20 and the epitope-defining amino acid. (B) Experimental setup of the single-cell editing and expansion experiment. (C) Left: fractions of CD201+CD150+KSL cells in single-cell-derived cultures 14 days after cloning (n = 261 clones). Right: Histogram of CD201+CD150+KSL cell frequency. Zoomed-in region shows clones with >10% CD201+CD150+KSL cells. (D and E) CD45.1+ donor PB chimerism (D) and lineage distribution (E) in single recipients with long-term (LT) engraftment ≥5% and multilineage reconstitution (n = 8). Numbers over graphs in (D) represent percentage of CD201+CD150+KSL cells in the transplanted clone (%). (F) Linear correlation plots of CD201+CD150+KSL cell frequency and 16-week donor chimerism. Red dots indicate LT repopulating and multilineage clones. Pearson correlation. (G) CD45.1+ PB chimerism and lineage distribution in secondary recipients (n = 5). See also Figure S4 and Table S3. Error bars represent SD.CC BY · PubMed Central
Figure 5 Autologous HSCT using gene-corrected HSC clones is curative in an immunodeficiency mouse model (A) Schematic of the single clone Prkdcscid correction model. (B) Single-cell SCID HSC expansion outcomes. Left: frequencies of phenotypic HSC populations in screened colonies (n = 384 from 3 experiments). Right: histogram of CD201+CD150+KL cell frequency. Enlarged region shows clones with ≥10% CD201+CD150+KL cells. (C) Genotyping of candidate clones (Sanger sequencing) (n = 96 clones, 3 experiments). Only clones with at least one HDR-corrected allele were sequenced at the off-target loci. (D) Allelic composition of the combined cell mixture at the edited Prkdc locus (n = 3). (E) Frequencies of PB leukocytes in CB17/SCID recipients. Left: lineage distributions in treated mice (n = 3) and in recipients receiving only 2 × 105 CB17/SCID whole bone marrow cells (neg. ctrl., n = 3). Right: representative FACS plots at 16 weeks post-SCT. (F) Allele frequencies in sorted PB cells 20 weeks post-SCT (n = 3 mice from 3 experiments). (G) Xenograft transplantation assay. A549 cells expressing the luminescent reporter Akaluc were injected subcutaneously (s.c.) and tumor growth was tracked by in vivo imaging. Left: representative images from CB17/WT, transplanted CB17/SCID, and untreated CB17/SCID mice 3 and 14 days after inoculation. Right: quantification of luminescence over a 14-day period (CB17/WT: n = 4, CB17/SCID − SCT: n = 3, CB17/SCID + SCT: n = 3). Two-way ANOVA and Tukey’s multiple comparison test. (H) Genotyping of candidate clones (Sanger sequencing) (n = 173 clones, 2 experiments). Clones producing a single, HDR-corrected sequencing trace were checked for LD (n = 29). PrkdcHDR/HDR, homozygous correction; PrkdcHDR/Δ, hemizygosity. (I) Prkdc copy-number analysis using 0.0 kb Prkdc (around cutsite) probe, quantified against reference gene. See also Figure S5. Error bars represent SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001.
Figure 5 Autologous HSCT using gene-corrected HSC clones is curative in an immunodeficiency mouse model (A) Schematic of the single clone Prkdcscid correction model. (B) Single-cell SCID HSC expansion outcomes. Left: frequencies of phenotypic HSC populations in screened colonies (n = 384 from 3 experiments). Right: histogram of CD201+CD150+KL cell frequency. Enlarged region shows clones with ≥10% CD201+CD150+KL cells. (C) Genotyping of candidate clones (Sanger sequencing) (n = 96 clones, 3 experiments). Only clones with at least one HDR-corrected allele were sequenced at the off-target loci. (D) Allelic composition of the combined cell mixture at the edited Prkdc locus (n = 3). (E) Frequencies of PB leukocytes in CB17/SCID recipients. Left: lineage distributions in treated mice (n = 3) and in recipients receiving only 2 × 105 CB17/SCID whole bone marrow cells (neg. ctrl., n = 3). Right: representative FACS plots at 16 weeks post-SCT. (F) Allele frequencies in sorted PB cells 20 weeks post-SCT (n = 3 mice from 3 experiments). (G) Xenograft transplantation assay. A549 cells expressing the luminescent reporter Akaluc were injected subcutaneously (s.c.) and tumor growth was tracked by in vivo imaging. Left: representative images from CB17/WT, transplanted CB17/SCID, and untreated CB17/SCID mice 3 and 14 days after inoculation. Right: quantification of luminescence over a 14-day period (CB17/WT: n = 4, CB17/SCID − SCT: n = 3, CB17/SCID + SCT: n = 3). Two-way ANOVA and Tukey’s multiple comparison test. (H) Genotyping of candidate clones (Sanger sequencing) (n = 173 clones, 2 experiments). Clones producing a single, HDR-corrected sequencing trace were checked for LD (n = 29). PrkdcHDR/HDR, homozygous correction; PrkdcHDR/Δ, hemizygosity. (I) Prkdc copy-number analysis using 0.0 kb Prkdc (around cutsite) probe, quantified against reference gene. See also Figure S5. Error bars represent SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001.CC BY · PubMed Central
Figure 6 Controlling genetic heterogeneity in gene-edited human HSCs (A) Scheme of experimental setup. (B) Post-editing allele distribution at the HBB locus, assessed by ICE (n = 3 cultures). (C) Copy-number analysis of HBB probes against reference gene (n = 3). Two-way ANOVA with Sidak’s multiple comparison test. (D) Genotyping of candidate clones (Sanger sequencing) (n = 133 clones, 2 experiments). Heterozygous clones were combined for SCT. Clones producing a single, HDR-edited sequencing trace were checked for LD (n = 16). (E) Copy-number analysis of HBB probe 0.0 kb against reference gene. (F) Left: spleen huCD45+ chimerism in HBBHDR/non-HDR and HBBHDR/HDR groups (n = 3 each). Right: representative FACS plots. (G) Percentage of HDR alleles in huCD45+ cells sorted from bone marrow and spleens of HBBHDR/non-HDR and HBBHDR/HDR engrafted mice (n = 3 each). ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
Figure 6 Controlling genetic heterogeneity in gene-edited human HSCs (A) Scheme of experimental setup. (B) Post-editing allele distribution at the HBB locus, assessed by ICE (n = 3 cultures). (C) Copy-number analysis of HBB probes against reference gene (n = 3). Two-way ANOVA with Sidak’s multiple comparison test. (D) Genotyping of candidate clones (Sanger sequencing) (n = 133 clones, 2 experiments). Heterozygous clones were combined for SCT. Clones producing a single, HDR-edited sequencing trace were checked for LD (n = 16). (E) Copy-number analysis of HBB probe 0.0 kb against reference gene. (F) Left: spleen huCD45+ chimerism in HBBHDR/non-HDR and HBBHDR/HDR groups (n = 3 each). Right: representative FACS plots. (G) Percentage of HDR alleles in huCD45+ cells sorted from bone marrow and spleens of HBBHDR/non-HDR and HBBHDR/HDR engrafted mice (n = 3 each). ∗∗p < 0.01, ∗∗∗∗p < 0.0001.CC BY · PubMed Central

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Metadata

Authors
Becker, H. J., Ishida, R., Wilkinson, A. C., Kimura, T., Lee, M. S. J., Coban, C., Ota, Y., Tanaka, Y., Roskamp, M., Sano, T., Tojo, A., Kent, D. G., & Yamazaki, S.
ORCID
Becker, Hans Jiro
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2023-07
Volume
30
Issue
7
Pages
987-1000.e8
ISSN
1934-5909
DOI
10.1016/j.stem.2023.06.002
PMC
PMC10338855
Cited by
32
References
7
Language
en
Licence
Funders
  • Blood Cancer UK (15008)
  • German Research Foundation (BE 6847/1-1)
  • Nihon Gakujutsu Shinkokai (21F21108, 20K21612, 20K16234, 23K15315)
  • Institute of Medical Science, University of Tokyo
  • Medical Research Council (MC_PC_21043, MR/V005502/1)
  • BASF
  • Kay Kendall Leukaemia Fund
  • AMED (21bm0404077h0001, 21bm0704055h0002)
  • European Research Council
  • Japan Science and Technology Agency (18071245)
  • Cancer Research UK (DCRPGF\u2216100008)

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