Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice

Abstract

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.

Figure 1. Enzymatic reconstitution and correction of neurological defects in Arsa^–/– mice.

(A) ARSA activity in PBMC (left y axis) and LV content in BM (right y axis) of untreated (Arsa^–/–), mock-treated (GFP), and GT-treated (pool and groups A and B) Arsa^–/– mice and WT controls. ARSA activity is expressed as fold increase compared with WT levels and LV content in CpC. (B) ARSA activity (left y axis) and LV CpC (right y axis) from liver samples from the same groups as in A. (C) ARSA activity of brain extracts is expressed as percentage of WT values. For statistical analysis, see Table 1. (D) Representative TLC gel from Rh-sulfatide test on liver and brain extracts from the indicated mice groups. (E) Assessment of central motor conduction in untreated and mock-treated Arsa^–/– mice, 12-month-old GT mice (pool), and age-matched WT controls (n = 15 mice per group). The GT group showed significantly lower CCT as compared with 6-month-old and age-matched Arsa^–/– mice; comparison with WT mice showed normalization of CCT (*P < 0.05, **P < 0.01). (F) Behavioral evaluations of GT mice. Mean latencies on rotarod ± SEM for each day are indicated. The GT group was indistinguishable from age-matched WT controls (left panel). Twelve-month-old GT mice in group B had a significantly improved performance compared with 6-month-old Arsa^–/– mice, demonstrating correction of the neurological deficit present at the time of treatment (right panel). For statistical analysis, see Table 3 (n = 15–30 mice per group.). GalCer, galactosylceramide.

Figure 2. Correction of sulfatide storage and neuronal damage in the CNS of GT-treated mice.

Transverse semithin sections of the hippocampal CA2–3 regions (A and B) and fimbria (C), the cerebellar Purkinje cell layer (D and E), and white matter (F) of untreated and mock-treated Arsa^–/– mice (6 and 12 months old, as indicated), and 12-month-old GT mice. Cells with pathological features were already detectable in the pyramidal cell layer of the hippocampus and in the Purkinje cell layer of cerebellum at 6 months (left panels). Lipid storage (arrowheads) was detected throughout the white matter, particularly in the fimbria and cerebellum. The neuronal damage became more severe and the number and size of metachromatic deposits increased significantly in 12-month-old mice (central panels). A marked reduction of sulfatide-containing metachromatic granuli in the white matter and of neuronal damage in CA2–3 and in the Purkinje cell layer was observed in GT mice (right panels). Scale bar: 120 μm (A and D); 80 μm (B, C, and F); and 50 μm (E). (G) Morphometric analysis of neuronal damage, shown as percentage of total counted neurons. Neurons in the CA2–3 and in the Purkinje cell layer were protected from age-related degeneration. In 12-month-old group B treated mice, reduction of degenerating neurons as compared with that of 6-month-old untreated mice was observed, indicating neuronal rescue (*P < 0.05).

Figure 3. Correction of PNS pathology in GT-treated mice.

Semithin sections and EM images from sciatic nerve and DRG are shown. (A) Metachromatic deposits (arrows) were detected in the SC cytoplasm of 6-month-old untreated Arsa^–/– mice. (B) By 12 months of age, the number and size of metachromatic deposits in SCs increased, and demyelination of nerve fibers became more apparent (arrowhead), as also shown at higher magnification in C and in detail by EM in E. (D) In GT mice, neither sulfatide storage nor demyelination was observed. (F) Metachromatic deposits and intracytoplasmatic vacuolation were present in sensory neurons as well as in satellite cells and SCs in DRG of 12-month-old Arsa^–/– mice. (G) In GT mice, these alterations were almost completely absent. Scale bar: 20 μm (A, B, D, F, and G); 5 μm (C); and 1.5 μm (E).

Figure 4. Enzyme biodistribution within different cell types in the nervous system of GT-treated mice.

Immunofluorescence and confocal analysis of brain sections of representative mice. Single confocal planes and orthoprojections acquired in Z-stack are shown from individual and merged fluorescent signals. (A) HA signal was detected within microglia cells stained for the F4/80 marker. (B) ARSA-HA was correctly localized within the Lamp1⁺ lysosomal compartment of microglia, here stained for the isolectin B4 (IsoB4) marker. (C and E) HA signal was also detected within NeuN⁺ neurons and calbindin⁺ Purkinje cells (Calb) in close contact with IsoB4⁺ microglia, indicating the occurrence of enzyme transfer from ARSA-producing microglia to neurons. (D and F) The tagged enzyme was correctly sorted to the lysosomal compartment in NeuN⁺ neurons and calbindin⁺ Purkinje cells. (G–I) ARSA-HA was also detected in GFAP-positive astrocytes (G, magnification shown in far right panel) and in a few MBP-positive (H) and CNPase-positive (I) oligodendrocytes, as shown by arrows in magnified panels on the right. Scale bar: 90 μm (E, G, and H); 80 μm (A and I); 60 μm (C and magnified panels in G and I); 50 μm (D); 40 μm (magnified panel in H); 30 μm (F); and 20 μm (B).

Figure 5. Enzyme biodistribution in neurogenic areas of the CNS and in the PNS of GT-treated mice.

Immunofluorescence and confocal analysis of brain (A–C), sciatic nerve (D), and DRG (E) sections of representative mice. Single confocal planes and orthoprojections acquired in Z-stack are shown from individual and merged fluorescent signals. (A–C) The ARSA-HA was found in adult neural stem cell areas, such as in the hippocampus (A and B), within different cell types, including NeuN, GFAP- and nestin-positive cells, and in the subventricular zone (C), within GFAP-positive and nestin-positive cells. (D and E) In the PNS, ARSA-HA was detected in S100-positive SCs in the sciatic nerve (D) as well as in sensory neurons in DRG (E). Scale bar: 60 μm (A); 50 μm (E); 40 μm (B and C); and 15 μm (D). TPIII, Topro III.

Figure 6. Enzyme biodistribution in ARSA-HA/FAH^–/– chimeras.

(A) Immunohistochemistry of a liver section from a representative chimeric mouse showing FAH⁺ hepatocytes from ARSA-HA–transgenic donors repopulating the FAH^–/– recipient. Scale bar: 20 μm. (B) Western blot analysis of liver samples from chimeric (nos. 17, 19, 23, and 26), control FAH^+/– (Co), GT-treated (GT: 2 different animals), and transgenic mice. Blot hybridization with anti-HA antibody (upper panel) showed ARSA-HA expression in chimeric mice and detectable protein in the GT mouse. GAPDH hybridization was used for normalization (lower panel). The engraftment of ARSA-HA, FAH⁺ hepatocytes was quantified by the percentage of FAH⁺ cells on liver sections (C) and by ARSA-HA activity of liver samples, expressed as fold increase compared with FAH^+/– levels (D). LV content in the liver is expressed in CpC (right y axes in C and D). (E) ARSA-HA activity was detected in the serum of GT and chimeric mice, indicating that the tagged enzyme was secreted into the bloodstream. (F–J) Confocal immunofluorescence and Western blot analysis of kidney (F and G), DRG (H), and brain (I and J) from representative mice. For Western blots, anti-HA hybridization is shown in the upper panels, anti-GAPDH in the lower panels, and MW markers on the right (in kDa). ARSA-HA signal was detected in the kidney and DRG of transgenic, GT, and chimeric mice. In contrast, the signal was detected only in the brain of transgenic and GT mice, not of chimeric mice, indicating that ARSA-HA could not reach the brain from the bloodstream. Scale bar: 20 μm.