Correction of brain oligodendrocytes by AAVrh.10 intracerebral gene therapy in metachromatic leukodystrophy mice

Abstract

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in glial cells and neurons, the result of an inherited deficiency of arylsulfatase A (ARSA; EC 3.1.6.8) and myelin degeneration in the central and peripheral nervous systems. No effective treatment is currently available for the most frequent late infantile (LI) form of MLD, which results in rapid neurological degradation and early death after the onset of clinical manifestations. To potentially arrest or reverse disease progression, ARSA enzyme must be rapidly delivered to brain oligodendrocytes of patients with LI MLD. We previously showed that brain gene therapy with adeno-associated virus serotype 5 (AAV5) driving the expression of human ARSA cDNA under the control of the murine phosphoglycerate kinase (PGK) promoter alleviated most long-term disease manifestations in MLD mice. Herein, we evaluated the short-term effects of AAVrh.10 driving the expression of human ARSA cDNA under the control of the cytomegalovirus/β-actin hybrid (CAG/cu) promoter in 8-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 2 months, and in contrast to results with the AAV5-PGK-ARSA vector, a single intrastriatal injection of AAVrh.10cuARSA resulted in correction of brain sulfatide storage, accumulation of specific sulfatide species in oligodendrocytes, and associated brain pathology in the injected hemisphere. Better potency of the AAVrh.10cuARSA vector was mediated by higher neuronal and oligodendrocyte transduction, axonal transport of the AAVrh.10 vector and ARSA enzyme, as well as higher CAG/cu promoter driven expression of ARSA enzyme. These results strongly support the use of AAVrh.10cuARSA vector for intracerebral gene therapy in rapidly progressing early-onset forms of MLD.

FIG. 1.

AAVrh.10 (solid columns) and AAV5 (shaded columns) vector genome copy number per 2n genome (VGC) in whole brain (A) and in serial coronal brain sections of the injected hemisphere (B) in 10-month-old MLD mice, 2 months after injection of AAVrh.10cuARSA or AAV5-PGK-ARSA vector into the right striatum. S1 to S8 refer to 1-mm coronal brain sections. The gray asterisk (B, inset) indicates the injection site in the striatum. All results are expressed as mean±SEM of five mice. *p<0.05; **p<0.01.

FIG. 2.

(A–C) Arylsulfatase A (ARSA) expression (ng/mg protein) assessed by ELISA in the injected hemisphere (A), serial 1 mm-coronal brain sections of the injected hemisphere (B), and in the noninjected hemisphere (C) of 10-month-old MLD mice, 2 months after injection of AAVrh.10cuARSA or AAV5-PGK-ARSA vector into the right striatum. (D) ARSA activity in the whole injected hemisphere of the same AAVrh.10-or AAV5-treated mice, assessed by the degradation of an artificial substrate of the enzyme (pNCS, p-nitrocatechol sulfate). (E) ARSA mRNA levels driven by the CAG/cu (CAG) or PGK promoter. Results are expressed as means±SEM of three or four samples (arbitrary unit). 4NC, 4-nitrocatechol; Bs, brainstem, Cb, cerebellum; CeSC, cervical spinal cord; S2, S4, S6, and S8 refer to 1-mm coronal brain sections; UT, untreated; WT, wild type. Results are expressed as means±SEM of four or five mice. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

FIG. 3.

(A and B) Cartography of ARSA expression in the brain of 10-month-old MLD mice, 2 months after a single injection of AAVrh.10cuARSA (A) or AAV5-PGK-ARSA (B) vector into the right striatum. (C and D) Representative immunostaining of human ARSA enzyme in a sagittal brain section of AAVrh.10-treated (C) and AAV5-treated mice (D). (E–J) After a single injection of AAVrh.10cuARSA vector into the right striatum, ARSA-positive cells were observed in several brain regions including the striatum (E), cortex (F), hippocampus (G), thalamus (H), ventral tegmental area (I), and interposed nucleus (J). cc, corpus callosum; ctx, cortex; hpf, hippocampus formation; hy, hypothalamus; ip, interposed nucleus; mgp, medial globus pallidus; pcg, pontine central nucleus; pg, pontine nucleus; sc, superior colliculus; sn, substantia nigra; STR, striatum; th, thalamus; vta, ventral tegmental area. Scale bars: 50 μm.

FIG. 4.

Immunostaining with anti-ARSA (A) and anti-GFP (B) antibodies in the right internal capsule after a single injection of AAVrh.10cuARSA or AAVrh.10cuGFP vector into the right striatum. Cells having the morphology of oligodendrocytes and expressing ARSA (arrows in A) were 2-fold more abundant than cells having the morphology of oligodendrocytes and expressing GFP (arrows in B), suggesting a cross-correction of oligodendrocytes after injection of the AAVrh.10cuARSA vector. Nuclei are stained blue. Scale bars: 25 μm. Insets: Double immunostaining with CNPase (a marker for oligodendrocytes) and (A) ARSA or (B) GFP.

FIG. 5.

Schematic representation of GFP and ARSA expression in the ipsilateral hemisphere after a single injection of AAVrh.10cuGFP vector (A and B) or AAVrh.10cuARSA vector (C and D) into the right striatum (A and C) or the right VTA (B and D) of MLD mice. Solid lines represent reciprocal projections, dashed lines anterograde (efferent) projections, and dotted lines retrograde (afferent) projections. Insets for (A–D) show representative GFP or ARSA expression in selected brain areas. cc, corpus callosum; ctx, cortex; hpf, hippocampal formation; hy, hypothalamus; int, internal capsules; ip, interpositus nucleus; my, medulla; ml, medial lemniscus; soc, superior olivary complex; ob, olfactory bulbs; ot, olfactory tubercle; pag, periaqueductal gray; pcg, pontine central nucleus; pg, pontine nucleus; purkinje, Purkinje cells; sc, superior colliculus; STR/str, striatum; st term, stria terminalis; th, thalamus; vta, ventral tegmental area. Scale bars: 50 μm.

FIG. 6.

(A) Correction of sulfatide storage as assessed by Alcian blue staining in the corpus callosum of wild-type (WT), untreated (UT), and AAVrh.10- and AAV5-treated MLD mice at 10 months of age, 2 months after treatment. In UT MLD mice, storage of sulfatides is particularly evident in macrophages and oligodendrocytes. Sulfatide accumulation is decreased but still present in AAV5-treated mice, whereas it is no longer detectable in AAVrh.10-treated mice. Insets: Higher magnification showing sulfatide storage in oligodendrocytes of UT mice that is corrected in AAVrh.10-treated mice, but not in AAV5-treated mice. (B) Immunostaining of astrocytes (glial fibrillary acidic protein [GFAP], red) in the anterior part of the corpus callosum, showing marked astrogliosis in UT mice that is prevented in AAVrh.10-treated mice, but to a much lesser extent in AAV5-treated mice at 10 months of age (2 months after treatment). (C) Immunostaining of microglia (Iba1, red) in the frontal cortex. Ten-month-old UT MLD mice display increased numbers of amoeboid microglial cells (yellow arrows), which correspond to activated microglia. At 10 months of age (2 months after treatment) these cells are not present in AAVrh.10-treated mice, which display only ramified microglia (blue arrows) corresponding to resting nonactivated microglia, as in WT mice, whereas they are still present in AAV5-treated mice. Scale bars: 100 μm.

FIG. 7.

Ratio of sulfatide content to galactosylceramide content (Sulf/GalCer ratio) in the brain of 10-month-old (A) and 18-month old (B) MLD mice, 2 months after a single intrastriatal injection of AAVrh.10cuARSA or AAV5-PGK-ARSA vector. (C) Sulfatide species identified by their fatty acid moiety in the brains of normal (WT), untreated (UT), and AAV5- or AAVrh.10-treated MLD mice at 10 months of age (2 months after treatment). Results are expressed as means±SEM (three mice per group). *p<0.05, **p<0.01, ***p<0.001.