Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency

Abstract

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplantation with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

Keywords: FGE; MSD; SUMF1; ex vivo gene therapy; formylglycine-generating enzyme; hematopoietic stem cell transplant; lentiviral gene therapy; lysosomal storage disorder; multiple sulfatase deficiency; sulfatase modifying factor 1.

Graphical abstract

Figure 1

SUMF1 lentiviral vector improves FGE protein expression and sulfatase activities in vitro (A) Lentiviral vector construct encoding human SUMF1 gene, driven by the human elongation factor 1 alpha (EF-1a) promoter and containing ankyrin and foamy insulators. 5′ LTR: 5′ long terminal repeat; 3′ Sin LTR: 3′ self-inactivating long terminal repeat; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; polyA: polyadenylation sequence. (B) Workflow of in vitro experiments. Immortalized MSD patient fibroblasts were transduced with our SUMF1 lentiviral vector before being harvested for downstream analyses. (C) Western blot analysis of FGE protein expression in wild-type (WT) cells, non-transduced MSD cells (MSD), and MSD cells transduced with the SUMF1 lentiviral vector over a range of vector copy numbers (0.4–4.5) shows increasing protein expression with increasing VCNs. Vinculin was used as a loading control. (D) ARSA activity (reported as fold-increase relative to non-transduced MSD cells) of WT cells, non-transduced MSD cells (MSD), and MSD cells transduced with SUMF1 lentiviral vector over a range of vector copy numbers (SUMF1-GT). (E) ARSB activity (reported as fold-increase relative to non-transduced MSD cells) of WT cells, non-transduced MSD cells (MSD), and MSD cells transduced with SUMF1 lentiviral vector over a range of vector copy numbers (SUMF1-GT). (F) SGSH activity (reported as fold-increase relative to non-transduced MSD cells) of WT cells, non-transduced MSD cells (MSD), and MSD cells transduced with SUMF1 lentiviral vector over a range of vector copy numbers (SUMF1-GT). For all sulfatase activity panels, WT and non-transduced MSD values represent the mean of 10 biological replicates for each group. SUMF1-GT individual biological replicate values are shown. For all sulfatase activity panels, the data are fit to an exponential regression line.

Figure 2

SUMF1 lentiviral vector rescues GAG accumulation in vitro (A) Quantification of GAG subspecies, UA-HNAc-UA-1S, associated with MPS I/II. (B) Quantification of GAG subspecies associated with MPS IIIA: HN-UA-1S, HN-UA-HNAc-UA-1S, and HN-UA-HNAc-UA-2S. (C) Quantification of GAG subspecies associated with MPS IIID, GlcNAc-6S. (D) Quantification of GAG subspecies associated with MPS VI, GalNAc-4S. For all panels, the total concentration of GAGs was normalized to micrograms of total protein from cell lysates. Total protein was measured by BCA assay. N = 2–5 biological replicates, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. For all panels: ∗p < 0.05. Data are shown as mean ± SEM. Group sizes: WT N = 2 replicates; MSD N = 4 replicates; SUMF1-GT N = 5 replicates.

Figure 3

Hematopoietic stem cell transplantation with SUMF1 gene therapy is stable and does not affect hematopoiesis in vivo (A) Transplant scheme (upper panel) and time course (lower panel) of in vivo HSCT-GT experiments. For primary transplants, we first harvested bone marrow from donor mice (not pictured) and isolated lineage-negative (lin−) HSCs. Then we transduced the lin− HSCs with our lentiviral vector expressing SUMF1 ex vivo. We transplanted the lin− HSCs transduced with our lentiviral vector into 2-month-old recipient mice. For secondary transplants, we extracted bone marrow cells from 6-month-old, primary-transplanted mice and transplanted them into naive, 2-month-old recipient mice. For both primary and secondary transplants, recipient mice received HSCT-GT at 2 months of age, engraftment and VCN were measured at 4 months of age by retro-orbital bleed, and endpoint analyses were conducted at 6 months of age. (B) Table describing experimental groups including donor mouse genotype, recipient mouse genotype, whether the animals received SUMF1 gene therapy, and transplant type (Primary or Secondary). (C) Percentages of donor cell engraftment for SUMF1 HSCT-GT primary and secondary transplanted mice. (D) Representative flow panels of erythroid populations (CD44+) for untreated WT mice (WT untreated), WT mice receiving SUMF1 HSCT-GT (WT SUMF1-GT), MSD mice receiving primary SUMF1 HSCT-GT (MSD SUMF1-GT [Primary]), and MSD mice receiving secondary SUMF1 HSCT-GT (MSD SUMF1-GT [Secondary]). N = one representative mouse per group. (E) Representative flow panels of lymphoid populations (B220+ or CD3+) for untreated WT mice (WT untreated), WT mice receiving SUMF1 HSCT-GT (WT SUMF1-GT), MSD mice receiving primary SUMF1 HSCT-GT (MSD SUMF1-GT [Primary]), and MSD mice receiving secondary SUMF1 HSCT-GT (MSD SUMF1-GT [Secondary]). N = one representative mouse per group. (F) Quantification of erythroid populations from flow analyses, displayed as percentage of the total live cell population. N = 2–8 mice, one-way ANOVA for each cell population followed by Bonferroni’s multiple comparisons test to compare each group to the WT untreated group. Data are represented as mean ± SEM. Group sizes (N = mice): WT untreated N = 8; WT SUMF1-GT N = 3; MSD SUMF1-GT (Primary) N = 3; MSD SUMF1-GT (Secondary) N = 2. (G) Quantification of lymphoid populations from flow analyses, displayed as percentage of the total live cell population. N = 2–8 mice, one-way ANOVA for each cell population followed by Bonferroni’s multiple comparisons test to compare each group to the WT untreated group. Data are represented as mean ± SEM. Group sizes (N = mice): WT untreated N = 8; WT SUMF1-GT N = 3; MSD SUMF1-GT (Primary) N = 3; MSD SUMF1-GT (Secondary) N = 2. (H) Vector copy numbers of SUMF1 HSCT-GT primary and secondary transplanted mice. For all panels: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

SUMF1 ex vivo gene therapy rescues ARSA activity in the spleen ARSA activity quantifications (nmol/h/mg total protein) for (A) brain, (B) heart, (C) lung, (D) liver, and (E) spleen tissue homogenates from WT mice (WT), untreated MSD mice (MSD), or MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT). N = 4–7 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. Group sizes (N = mice): WT N = 7; MSD N = 6; SUMF1-GT N = 4. (F) Quantification of vector copy numbers (VCNs) in whole-tissue homogenates of SUMF1-GT mice indicates that VCNs in the spleens of treated mice (mean = 0.69) were significantly greater than other tissues with lower sulfatase activities (mean[s] = 0.01–0.23). N = 3 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each tissue to the spleen. For all panels: ∗p < 0.05, ∗∗∗p < 0.001. Data are represented as mean ± SEM.

Figure 5

SUMF1 ex vivo gene therapy reduces GAG accumulation, while transplantation alone exacerbates it in vivo Quantification of GAG subspecies associated with MPS II, IIIA, IIID, and IVA/VI of untreated WT mice (WT), untreated MSD mice (MSD), MSD mice receiving non-transduced MSD bone marrow (MSD BM), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) for (A) spleen, (B) brain, (C) liver, (D) heart, and (E) lung tissue homogenates. Concentrations of GAGs (fmol/μg protein) were normalized to μg of total protein. N = 2–7 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to untreated MSD mice. Data are represented as mean ± SEM. For all panels: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Group sizes (N = mice): WT N = 7; MSD N = 6; MSD BM N = 2; SUMF1-GT N = 4.

Figure 6

SUMF1 ex vivo gene therapy rescues microgliosis in the cortex (A) Representative immunofluorescence images of Iba1+ cells in cortical brain sections of WT mice (WT), untreated MSD mice (MSD), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) reveal decreased microgliosis after treatment. Labeling with anti-Iba1 antibody (red fluorescence) and DAPI (nuclei, blue). Scale bars, 100 μm. (B) Quantification of Iba1 immunofluorescence intensity confirms significant decrease in SUMF1 HSCT-GT animals as compared with untreated MSD animals. (C) Quantification of Iba1+ cells in cortical sections validates decreased microgliosis in SUMF1 HSCT-GT animals as compared with untreated MSD animals. For all quantifications, N = three biological replicates were analyzed with five images per replicate, two-way ANOVA by genotype/treatment group and mouse, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. For all panels: ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM. Group sizes (N = mice): WT N = 3; MSD N = 3; SUMF1-GT N = 3.

Figure 7

SUMF1 ex vivo gene therapy improves cognitive function and neurodegeneration, but not motor phenotypes (A) Quantification of rotarod assay for WT mice (WT), untreated MSD mice (MSD), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) suggests partial improvement in motor coordination phenotypes after treatment. The latency (seconds) of the mouse to fall off the rotarod was measured over 4 days. N = 7–18 mice, one-way ANOVA at each testing day, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. WT mice stay on the rotarod significantly longer on all four days of testing than untreated MSD mice (day 1: p = 0.042, day 2: p = 0.002, day 3: p = 0.021, day 4: p = 0.004). The SUMF1-GT group was not statistically significant from the untreated MSD group at any time point. Data are represented as mean ± SEM. Group sizes (N = mice) and sex distribution: WT N = 18 (11 female, 7 male); MSD N = 17 (2 female, 15 male); SUMF1-GT N = 7 (3 female, 4 male). (B) Quantification of grip strength assay for WT mice (WT), untreated MSD mice (MSD), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) reveals no muscular improvement after treatment. The force (KgF) generated by the mouse forelimb is reported. N = 7–20 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. Data are represented as mean ± SEM. Group sizes (N = mice) and sex distribution: WT N = 20 (11 female, 9 male); MSD N = 17 (2 female, 15 male); SUMF1-GT N = 7 (3 female, 4 male). (C) Quantification of Barnes maze assay for WT mice (WT), untreated MSD mice (MSD), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) shows improved neurocognitive function after SUMF1 HSCT-GT. Percentage (%) of total nose pokes is the number of times an animal checked the target hole divided by the total number of times it checked any hole. N = 7–20 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. Data are represented as mean ± SEM. Group sizes (N = mice) and sex distribution: WT N = 20 (11 female, 9 male); MSD N = 12 (2 female, 10 male); SUMF1-GT N = 7 (3 female, 4 male). (D) Quantification of hindlimb clasping scores for WT mice (WT), untreated MSD mice (MSD), and MSD mice receiving SUMF1 HSCT-GT (SUMF1-GT) reveals rescue of neurodegenerative phenotype after SUMF1 HSCT-GT. N = 7–20 mice, one-way ANOVA, followed by Bonferroni’s multiple comparisons test to compare each group to the untreated MSD control group. Data are represented as mean ± SEM. Group sizes (N = mice) and sex distribution: WT N = 20 (11 female, 9 male); MSD N = 12 (2 female, 10 male); SUMF1-GT N = 7 (3 female, 4 male). For all panels: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.