Research ArticleGene Therapy

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1

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Science Translational Medicine  11 Oct 2017:
Vol. 9, Issue 411, eaan0820
DOI: 10.1126/scitranslmed.aan0820
  • Fig. 1. Competitive transplantation of WT and SCID-X1 HSPCs.

    (A) Schematic representation of competitive transplant at different ratios of WT (black) and SCID-X1 Lin HSPCs (red) into lethally irradiated SCID-X1 recipients. HSCT, hematopoietic stem cell transplantation. (B) Chimerism of WT and SCID-X1 cells observed within CD19+ B cells, CD3+ T cells, and CD11b+ myeloid cells 15 weeks after transplant (n = 7 or 8 per group, pooled from two independent experiments). (C) Percent composition of myeloid and lymphoid lineages in PB 18 weeks after transplant. (D) Total counts of CD19+ B cells (left) and CD3+ T cells (right) in PB 18 weeks after transplant (Kruskal-Wallis test, median is plotted). (E) Ratio of CD4 to CD8 T cells in the PB of mice 12 weeks after transplant (Kruskal-Wallis test, median is plotted). (F) Left: Total number of CD8+ T cells measured in PB at indicated times after LCMV Armstrong infection (100% WT, 10% WT, and 1% WT: n = 4; SCID: n = 3). Middle: Percent of CD8+ cells producing IFN-γ measured in PB 8 days after LCMV infection (Kruskal-Wallis test, median is plotted). Right: LCMV titer [log viral genome (VG)/ml] measured in the serum [two-way analysis of variance (ANOVA)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

  • Fig. 2. Efficacy and safety of hematopoietic reconstitution without conditioning.

    (A) Percent composition of myeloid and lymphoid lineages in the PB 12 weeks after transplanting 10% WT cells without irradiation (No irrad; n = 8) or after lethal irradiation (TBI, n = 10) of recipient SCID-X1 mice. (B) Percentage of PB T cells at indicated times after transplant. TBI, n = 15; no irradiation, n = 21 (two-way ANOVA). (C) Total counts of PB T cells measured 12 weeks after transplant. 100% TBI groups (black), n ≥ 7; no-irradiation groups (red), n ≥ 10 (Kruskal-Wallis test, median is plotted). (D) Percentage of lymphoma-free animals in the follow-up of SCID-X1 mice transplanted with WT HSPCs with (TBI, n = 14) or without (n = 32) irradiation (log-rank test). (E) Representative image of a thymic lobe from a mouse with thymic lymphoma and a control C57BL/6 mouse. Scale bar, 1 cm. (F) Hematoxylin and eosin–stained section of thymic lymphoma. Scale bar, 20 μm. (G) Percentage of lymphoma-free mice as in (D) after stratifying the data according to the dose of WT cells transplanted without conditioning (100% WT, n = 15; 10% WT, n = 17) or the extent of PB engraftment with WT cells at 100 days after transplant (above or below 40% threshold). P = 0.14 and P > 0.05, respectively (log-rank test). (H) Percentage of T cells in PB of SCID-X1 mice transplanted with matched doses of WT cells without irradiation (n = 25) or after TBI without (n = 8) or with shielded thymus (TBI + screen, n = 13) (two-way ANOVA). (I) Percentage of lymphoma-free animals in transplanted SCID-X1 mice from (H) (k-sample log-rank test). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3. Hematopoietic reconstitution with clinically relevant conditioning regimens.

    (A) Chimerism of WT and SCID-X1 cells observed within CD11b+ myeloid cells 12 weeks after transplant of 10% WT Lin cells after conditioning the recipients with TBI (n = 5) or treosulfan (n = 5). (B) Percent PB composition of myeloid and lymphoid lineages in mice from (A). (C) Chimerism of WT and SCID-X1 cells observed within CD11b+ myeloid cells 15 weeks after transplant of 10% WT Lin cells in recipients conditioned with TBI (n = 5) or CD45-SAP (n = 5). (D) Percent PB composition of myeloid and lymphoid lineages at the indicated times after transplant in mice from (C). (E) Total counts of CD3+ T cells measured in PB 15 weeks after transplant in mice from (C) (Mann-Whitney test, median is plotted). (F) Left: Total number of CD8+ T cells measured in PB at indicated times after LCMV Armstrong infection of mice from (C). Middle: Percent of CD8+ cells producing IFN-γ measured in PB 8 days after LCMV infection (Mann-Whitney test, median is plotted). Right: LCMV titer (log VG/ml) measured in the serum at the indicated times; nontransplanted WT and SCID-X1 mice were used as controls. **P < 0.01 and ***P < 0.001 (two-way ANOVA).

  • Fig. 4. Functional validation of IL2RG-edited lymphoid cells in transplanted mice.

    (A) Composition of myeloid and lymphoid lineages in PB after transplantation with SCID-X1 cells (n = 7), 1% WT cells (n = 8), or SCID-X1 cells treated with the exon 5 IL2RG electro ZFN (n = 12) or LV gRNA (n = 15) protocols. Significance is shown relative to SCID-X1. (B) PB T cell counts 18 weeks after transplant (n = 8, 12, and 20 for WT, electro ZFN, and LV gRNA, respectively). (C) Percentage of naïve cells within CD8+ (left) and CD4+ (right) splenic T cells (n = 3, 5, 11, and 12 for 1% WT, SCID, electro ZFN, and LV gRNA, respectively; median is plotted). (D) Ratio of CD4 to CD8 T cells in the spleen ≥21 weeks after transplant; untransplanted WT mice reported as controls (n = 8, median is plotted). (E) Percent of the indicated B cell subpopulations within BM B220+ cells ≥21 weeks after transplant [SCID-X1, n = 8; WT, n = 9; EDITED (either of the protocols), n = 15]. (F) Proliferation index (left) and percentage of CD69+ B cells (right) among exon 5 IL2RG-edited B cells from the spleen of transplanted mice (n = 8) or from a WT mouse as control, stimulated or not with unmethylated CpG oligodeoxynucleotides (CpG). Unst, unstimulated. (G) Cell proliferation measured by fluorescent dye dilution of exon 5 IL2RG-edited T cells and SCID-X1 cells, stimulated as indicated. ConA, concanavalin A. (H) Percentage of CD8+ cells expressing IFN-γ from PB 7 days after LCMV infection of transplanted mice (n = 5, 7, and 4 for EDITED, SCID-X1, and 1% WT, respectively; Kruskal-Wallis test, median is plotted). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5. Efficient and scalable IL2RG editing in human CD34+ and T cells.

    (A) Percentage of primary human male T cells edited by HDR (left) or NHEJ (right) at intron 1 IL2RG using WT (intron 1 IL2RG, n = 6) or codon usage optimized (intron 1 IL2RG rec, n = 9) donor template or edited at AAVS1 (n = 3). (B) HDR and NHEJ 3 days after intron 1 IL2RG gene editing of CB CD34+ cells from an SCID-X1 patient (R289X) or WT cells as control. (C) Percentage of GFP+ cells measured within the indicated subpopulations 3 days after intron 1 IL2RG editing of CB-derived CD34+ using the indicated donor template vector and nuclease (n = 5, 7, and 6 for IDLV + ZFN, AAV6 + ZFN, and AAV6 + CRISPR/Cas9, respectively; Mann-Whitney test). (D) Subpopulation composition of treated CD34+ cells from (C). (E) Human CD45+ cell engraftment in PB at indicated times after transplantation of CB CD34+ cells edited at intron 1 IL2RG using IDLV (n = 7) or AAV6 as donor (n = 9) (two-way ANOVA). (F) Percentage of HDR within human cells measured by ddPCR in mice from (E) (n = 9; two-way ANOVA). (G) Percentage of HDR measured by ddPCR in sorted BM CD34+ HSPCs, CD19+ B cells, and CD33+ myeloid cells from mice in (E) (Mann-Whitney test, median is plotted). (H) HDR and NHEJ 3 days after intron 1 IL2RG editing of increasing amounts of CB-derived CD34+ cells in bulk population (left) or within sorted subpopulations (right). (I) Percentage of HDR editing within human cells from mice transplanted with medium-scale (n = 6) or large-scale (n = 12) IL2RG-edited CD34+ cells from (H). (J) Percentage of HDR as in (G) from large-scale transplanted mice at the 23-week end point (n = 6; median is plotted). (K) Percentage of HDR measured in the indicated organs and sorted cells as in (G) 12 weeks upon secondary transplant of human CD34+ cells from mice in (I) (n = 6, median is plotted). (L) Ex vivo growth of IL2RG-edited and unedited T cells (n = 9) from the spleen of NSG mice transplanted with edited CD34+ cells upon stimulation with γ-chain–dependent cytokines (Mann-Whitney test). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Table 1. Off-target quantification for optimized intron 1 IL2RG ZFNs.

    Percentages of indels measured by deep sequencing at the on-target and three off-target sites, as well as cell viability for mobilized human PB CD34+ cells treated with the selected intron 1 IL2RG ZFN lead pair or its optimized version. *P < 0.01 (Bonferroni sigificance). OT, off-target site.

    ZFNDose (μg/ml)OnOT1OT4OT6On/∑OT% Cell viability
    57629 + 577182064.69*6.18*0.030.0810.2776.35
    4065.78*14.88*0.09*0.45*4.2767.85
    57629 improved version +
    57718 improved version
    2074.53*0.050.040.02710.4674.40
    4079.71*0.030.030.05733.9974.75
  • Table 2. On- and off-target analysis in CD34+ cells treated at increasing scales for intron 1 IL2RG editing with optimized ZFNs.

    Percentages of indels at the ZFN on-target and identified off-target sites measured by deep sequencing from samples shown in Fig. 5H. LS, large-scale treated CD34+ cells; MS, medium-scale treated CD34+ cells; SS, small-scale treated CD34+ cells. *P < 0.01 (Bonferroni significance) compared to untreated sample (UT).

    SampleOnOT1OT2OT3OT4OT5OT6OT7
    LS bulk40.84*0.050.100.180.040.040.040.03
    MS bulk66.48*0.030.080.180.030.030.040.01
    SS bulk65.87*0.050.120.140.040.060.030.02
    LS CD3438.09*0.070.080.160.020.020.030.03
    LS CD34+ CD13343.03*0.060.130.170.030.030.040.03
    LS CD34+ CD133+ CD9050.41*0.110.110.130.040.040.050.04
    LS CD34+ CD133+ CD90+50.40*0.100.100.140.030.030.020.05
    UT0.030.050.110.130.020.020.030.04

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/411/eaan0820/DC1

    Materials and Methods

    Fig. S1. Humanized SCID-X1 mice.

    Fig. S2. Phenotypical and functional characterization of humanized SCID-X1 mice.

    Fig. S3. Hematopoietic reconstitution and functional studies of WT/SCID-X1 competitive transplants.

    Fig. S4. Phenotypical characterization of lymphoblastic T lymphomas developing in mice transplanted without irradiation.

    Fig. S5. Molecular and functional characterization of T lymphoma.

    Fig. S6. Depletion of hematopoietic compartments with CD45-SAP in SCID-X1 mice.

    Fig. S7. Development of a gene editing strategy for mouse HSPCs.

    Fig. S8. Functionality of gene-edited lymphoid cells from transplanted mice.

    Fig. S9. Functional validation of IL2RG-edited primary human T cells.

    Fig. S10. Tailoring of gene editing protocol for human HSPCs.

    Table S1. Phenotypical characterization of humanized SCID-X1 mice.

    Table S2. Intron 1 IL2RG ZFNs off-target list.

    Table S3. List of genomic gRNA target sequences.

    Table S4. List of primers and probes.

    Table S5. List of antibodies for flow cytometry.

    Table S6. Raw data for Table 2 (provided as an Excel file).

    References (3743)

  • Supplementary Material for:

    Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1

    Giulia Schiroli, Samuele Ferrari, Anthony Conway, Aurelien Jacob, Valentina Capo, Luisa Albano, Tiziana Plati, Maria C. Castiello, Francesca Sanvito, Andrew R. Gennery, Chiara Bovolenta, Rahul Palchaudhuri, David T. Scadden, Michael C. Holmes, Anna Villa, Giovanni Sitia, Angelo Lombardo, Pietro Genovese,* Luigi Naldini*

    *Corresponding author. Email: genovese.pietro{at}hsr.it (P.G.); naldini.luigi{at}hsr.it (L.N.)

    Published 11 October 2017, Sci. Transl. Med. 9, eaan0820 (2017)
    DOI: 10.1126/scitranslmed.aan0820

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Humanized SCID-X1 mice.
    • Fig. S2. Phenotypical and functional characterization of humanized SCID-X1 mice.
    • Fig. S3. Hematopoietic reconstitution and functional studies of WT/SCID-X1 competitive transplants.
    • Fig. S4. Phenotypical characterization of lymphoblastic T lymphomas developing in mice transplanted without irradiation.
    • Fig. S5. Molecular and functional characterization of T lymphoma.
    • Fig. S6. Depletion of hematopoietic compartments with CD45-SAP in SCID-X1 mice.
    • Fig. S7. Development of a gene editing strategy for mouse HSPCs.
    • Fig. S8. Functionality of gene-edited lymphoid cells from transplanted mice.
    • Fig. S9. Functional validation of IL2RG-edited primary human T cells.
    • Fig. S10. Tailoring of gene editing protocol for human HSPCs.
    • Table S1. Phenotypical characterization of humanized SCID-X1 mice.
    • Table S2. Intron 1 IL2RG ZFNs off-target list.
    • Table S3. List of genomic gRNA target sequences.
    • Table S4. List of primers and probes.
    • Table S5. List of antibodies for flow cytometry.
    • References (3743)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S6. Raw data for Table 2 (provided as an Excel file).