Research ArticleLysosomal Storage Diseases

Tolerance induction and microglial engraftment after fetal therapy without conditioning in mice with mucopolysaccharidosis type VII

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Science Translational Medicine  26 Feb 2020:
Vol. 12, Issue 532, eaay8980
DOI: 10.1126/scitranslmed.aay8980
  • Fig. 1 In utero enzyme replacement improves overall survival to weaning in MPS7 mice, and activity is enriched in monocytes.

    (A) Heterozygous MPS+/− mice were mated, and their fetuses underwent intrahepatic injection at E14.5 of either PBS control or recombinant human β-glucuronidase (rhGUS). Surviving mice underwent intravenous booster injections every other week starting at 3 weeks until harvest. Mice underwent behavioral testing at 6 weeks and at harvest, at which point they also underwent biochemical enzyme activity testing, histology, and computed tomography (CT) imaging. (B) Overall survival to birth (percentage of injected) in MPS7 or B6 litters (B6, n = 141; IUPBS, n = 121; IUERT, n = 240; ****P < 0.001, χ2 test). (C) Survival to weaning (percentage of injected) in MPS7 litters after injection of PBS (n = 121) or rhGUS (IUERT, n = 240; **P < 0.01, χ2 test). (D) Survival to weaning graphed as deviation from expected 25% survival in individual MPS7−/− pups within the litter (total mice at wean: uninjected, n = 236; PBS, n = 27; IUERT, n = 86; *P < 0.05 and ****P < 0.0001, Fisher’s exact test). (E and F) Peripheral blood was taken at multiple time points after systemic injection of rhGUS in adult mice, and GUS activity in individual leukocyte populations (representative flow cytometry histogram at day 3 after injection) (E) was measured over time (F). n ≥ 3 per group and time point. FITC, fluorescein isothiocyanate.

  • Fig. 2 In utero ERT results in detectable enzyme within the microglia of treated mice, but postnatal therapy does not.

    (A) Biochemical activity of enzyme in the brain 4 to 7 days after in utero injection or 4 to 7 days after adult injection. n ≥ 5 per group. Data are means ± SEM. ****P < 0.0001 (ANOVA with Tukey’s multiple comparisons test). (B) Representative flow cytometry plots of GUS staining in microglia and nonmicroglia 4 days after in utero injection. Graphs showing (C) percentages of cells that are positive for GUS and (D) MFI of GUS in individual cells. n ≥ 3 per group. Data are means ± SEM. *P < 0.05 and **P < 0.01 (Kruskal-Wallis with Dunn’s multiple comparisons test). MG, microglia. (E) Representative images of CD68 immunohistochemical staining (scale bars, 200 μm). (F) Integrated density graph of CD68 staining in mice harvested at 8 to 10 weeks. n = 3 per group. Data are means ± SEM. *P < 0.05 and ***P < 0.001 (ANOVA with Tukey’s multiple comparisons test). (G) Grip strength in unaffected and MPS7−/− mice with and without treatment. n ≥ 5 per group. Data are means ± SEM. *P < 0.05 (ANOVA with Tukey’s multiple comparisons test).

  • Fig. 3 Combination of in utero and postnatal ERT improves pathologic lysosomal accumulations of GAGs as well as bone length.

    Mice were harvested at 8 weeks of age, and their livers, spleens, and kidneys were examined with a PAS stain. (A) Representative images from the liver, spleen, and kidney (scale bars, 20 μm) demonstrating intracellular accumulations (vacuolated cells, solid arrows) of GAGs. Quantification is shown to the right of the histology images; n ≥ 5 per group. Data are means ± SEM. ****P < 0.0001 (ANOVA with Tukey’s multiple comparisons test). (B) Representative CT images of femurs in unaffected, MPS7−/− untreated, and MPS7−/−-treated mice harvested at 8 to 10 weeks of age. Compiled data for (C) femurs and (D) tibias. n ≥ 5 per group. *P < 0.05, ***P < 0.001, and ****P < 0.0001 (ANOVA with Tukey’s multiple comparisons test).

  • Fig. 4 In utero ERT results in tolerance to rhGUS.

    (A) Mice underwent in utero injection with either rhGUS enzyme or PBS followed by postnatal boosting starting at 3 weeks (arrows) and continuing every other week. At 6 weeks of age, mice underwent intraperitoneal (IP) injection of rhGUS with complete Freund’s adjuvant (CFA). Plasma concentrations of antibodies against rhGUS were measured by ELISA at 8 weeks. (B) Amounts of IgG1 (left graph) and IgG3 (right graph) antibodies against rhGUS. n ≥ 10 per group. Data are means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 (Kruskal-Wallis with Dunn’s multiple comparisons test).

  • Fig. 5 In utero hematopoietic cell transplantation resulted in multilineage blood chimerism and bone marrow HSC engraftment.

    (A) Experimental design. (B) Flow cytometry gating strategy for blood and bone marrow. (C) Peripheral blood flow cytometry showing composition of CD45+ cells in donor and host compartments (n ≥ 27 per group). Data are means ± SEM. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (D) Bone marrow flow cytometry showing donor cell correlation between bone marrow KLS and blood CD45 cells (n = 15).

  • Fig. 6 IUHCT in MPS7 mice resulted in multilineage chimerism in bone and blood and some evidence of long-term tissue cross-correction.

    (A) Gating strategy to identify host liver Kupffer cells (left) and quantify GUS expression by Kupffer cells (right); GUS expression by host cells is secondary cross-correction by donor cells. (B) Compiled data for the percentage of host Kupffer cells expressing GUS and its relationship to chimerism (top) and the mean fluorescence intensity (MFI) of GUS in Kupffer cells and its relationship to chimerism (bottom) (n ≥ 4 per group). Data are means ± SEM. *P < 0.05 and **P < 0.01 (Mann-Whitney test). (C) Tissue histology representative images from liver, kidney, and spleen and (D) area of lysosomal GAG accumulation measured in wild-type (WT) controls, MPS7−/− chimeras, and untreated MPS7−/− controls (scale bars, 20 μm). n ≥ 4 per group. Data are means ± SEM. ****P < 0.0001 (Mann-Whitney test). (E) Correlation between the area of lysosomal GAG accumulation and peripheral blood chimerism rate in liver, kidney, and spleen (n ≥ 11 per graph, Spearman correlation test P values shown).

  • Fig. 7 Donor-derived microglia were found in the brain after IUHCT.

    (A) Representative brain slices examined for microglial engraftment. (B) Confocal imaging revealed clusters of microglial engraftment in chimeric animals. (C) Correlation between brain chimerism and peripheral blood chimerism (n = 14). PCA, principal components analysis. Bulk RNA sequencing of donor versus host-derived microglia and donor-derived monocytes isolated from brains of chimeric animals 5 weeks after IUHCT, shown as (D) principal components analysis, (E) heatmap of top 1000 genes by variance, and (F) heatmap of “signature” microglial genes (n ≥ 7 per group).

  • Fig. 8 Immunofluorescence staining of MPS−/− brain sections revealed evidence of decreased inflammation near engrafted donor cells.

    (A) Representative image showing CD68 staining (microglial inflammation) in relation to areas of engraftment with donor cells (GFP+). (B) Representative images showing GFP+ donor cells, Iba1 staining (microglia), and CD68 staining (microglial activation) (scale bars, 50 μm). Subsequent graphs represent (C) CD68 intensity per host cell, (D) host cell diameter, and (E) host cell number compared to number of donor cells per confocal image (n ≥ 7 images per graph). (F) CD68 expression (raw integrated density) per host cell graphed against number of donor cells, combining all chimeric MPS7 brains.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/532/eaay8980/DC1

    Fig. S1. Dose comparison with in utero enzyme replacement reveals higher tissue enzyme activity after 20 mg/kg compared to the 4 mg/kg dose.

    Fig. S2. In utero enzyme replacement results in detectable enzyme in all tissues.

    Fig. S3. Behavioral testing for open-field basic movement and rearing.

    Fig. S4. Additional ELISA experiments.

    Fig. S5. Peripheral blood chimerism and survival rates after IUHCT.

    Fig. S6. Peripheral blood monocyte cross-correction.

    Fig. S7. RNA sequencing studies and further data.

    Data file S1. Original data.

  • The PDF file includes:

    • Fig. S1. Dose comparison with in utero enzyme replacement reveals higher tissue enzyme activity after 20 mg/kg compared to the 4 mg/kg dose.
    • Fig. S2. In utero enzyme replacement results in detectable enzyme in all tissues.
    • Fig. S3. Behavioral testing for open-field basic movement and rearing.
    • Fig. S4. Additional ELISA experiments.
    • Fig. S5. Peripheral blood chimerism and survival rates after IUHCT.
    • Fig. S6. Peripheral blood monocyte cross-correction.
    • Fig. S7. RNA sequencing studies and further data.

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    Other Supplementary Material for this manuscript includes the following:

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