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

Abstract

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201⁺CD150⁺CD48^-c-Kit⁺Sca-1⁺Lin^- population of precultured HSCs. Using the Prkdc^scid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Keywords: CRISPR-Cas9; chemically defined culture; clonal expansion; ex vivo expansion; gene editing; gene therapy; hematopoietic stem cell; regenerative medicine; stem cell culture; transplantation.

Graphical abstract

Figure 1

Autologous HSCT gene correction rescues the Prkdc^scid phenotype but introduces on-, off-target indels and large deletions (A) Genomic context of the Prkdc^scid mutation in exon 85. White boxes: exons, gray box, 3′ UTR. ^∗ denotes location of Prkdc^scid 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 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 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 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 5

Autologous HSCT using gene-corrected HSC clones is curative in an immunodeficiency mouse model (A) Schematic of the single clone Prkdc^scid 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). Prkdc^HDR/HDR, homozygous correction; Prkdc^HDR/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 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 HBB^HDR/non-HDR and HBB^HDR/HDR groups (n = 3 each). Right: representative FACS plots. (G) Percentage of HDR alleles in huCD45⁺ cells sorted from bone marrow and spleens of HBB^HDR/non-HDR and HBB^HDR/HDR engrafted mice (n = 3 each). ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001.