Induction of tolerance to human arylsulfatase A in a mouse model of metachromatic leukodystrophy

Abstract

A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.

Figure 1

In vitro mutagenesis of the wildtype hASA cDNA and expression of hASA-C69S in cultured cells. (A) Exchange of three nucleotides (underlined) of the wildtype hASA cDNA resulted in the substitution of cysteine by serine at position 69 (bold letters), the loss of an ApaLI restriction site and the gain of a ScaI site. (B) The hASA-C69S cDNA was cloned under the control of the chicken β-actin promoter of the eukaryotic expression vector pTVC. (C) BHK cells were transiently transfected with pTVC harboring the hASA-C69S in 5′-3′ or, as a control, in 3′-5′ orientation. For additional controls, pBEH-HT14/CP8 bearing the wildtype hASA cDNA under the control of the SV40 promoter (15), the empty TVC plasmid (pTVC) or no DNA was used. ASA was quantified by ELISA (upper panel) or activity assays (lower panel) using cell homogenates (bars) and conditioned medium (closed circles). (D) Immunofluorescence of wildtype (wt) and mutant hASA (C69S) in BHK cells transiently transfected with pBEH-HT14/CP8 and pTVC hASA-C69S 5′-3′, respectively. The red perinuclear hASA staining of the original color images is visible as a bright signal after conversion into the black and white image. The DAPI staining of the nuclei appears gray.

Figure 2

Transgene expression and MLD-like phenotype of transgenic ASA−/− mice. Bars indicate means ± SD. (A) hASA-C69S concentrations in serum of 234 transgenic ASA−/−mice. hASA-C69S was undetectable in eight mice due to serum levels below 0.01 μg/mL. (B) Tissue distribution of hASA-C69S in mice with serum levels between 0.2 and 0.9 μg/mL. Tissue and serum levels were normalized on hASA-C69S levels of brain. n = 5. (C) Sulfatide concentration in tissues of six-month-old wildtype control mice (closed bars), non-transgenic ASA−/− mice (open bars) and transgenic ASA−/− mice (hatched bars; hASA-C69S serum level: 0.2–0.9 μg/mL). Sulfatide was quantified by TLC and normalized on the cholesterol concentration. n = 5. (D) Swimming velocity. n = 10. * P < 0.05 compared with wildtype controls. hASA-C69S serum levels of transgenic ASA−/− mice: 0.2–3.1 μg/mL.

Figure 3

Histology at age 11–12 months (A) hASA-C69S expression in the CNS of transgenic ASA−/− mice. Immunostaining of brain stem (A1) and cerebellum (A2) detects high hASA-C69S levels in neuronal perikarya of brain stem nuclei and the Purkinje cell layer (arrow). (B) Co-immunostaining of hASA-C69S (red) and F4/80 (green) reveals low transgene expression levels in liver (B1). Higher magnification (B2) detects expression in some F4/80-negative (open arrows) and -positive cells (closed arrows) with small nuclei, but not in hepatocytes (one nucleus is indicated by an arrowhead). (C) Cervical spinal cord of a conventional (C1–C3) and a transgenic ASA−/− mouse (C4–C6). Sections (100 μm) were incubated with alcian blue to visualize sulfatide storage. C1, C4 – overviews to outline the regions shown at higher magnification. gm – gray matter, wm – white matter, dh – dorsal horn, dt – dorsal tract, vh – ventral horn. (D) Alcian blue-incubated sections (100 μm) through kidney of a conventional (D1, D2) and a transgenic ASA−/− mouse (D3, D4). D1, D3 – overview; regions shown at higher magnification are boxed. Storage material is seen in the thick ascending limbs of Henle’s loop (TAL) of the inner (iS-oM) and outer stripe of the outer medulla (oS-oM), and to a lesser extent in the TALs of the medullary rays of the cortex (Co). D2, D4 – cortex. (E) Electron micrographs of hepatocytes. E1 – wildtype control mouse; E2, E3 – transgenic mouse expressing hASA-C69S on the wildtype background (serum level 0.91 μg/mL). Most lysosomes of the transgenic mouse show a normal morphology (asterices). However, some contain glycogen particles (open arrows in E3). Au-tophagic vacuoles do not contain glycogen (closed arrow). gl – cytosolic glycogen, bc –bile canaliculus, arrowheads – cell boundary.

Figure 4

General phenotype of transgenic ASA−/− mice (hatched bars and symbols), non-transgenic ASA−/− mice (open bars and symbols) and wildtype mice (closed bars and symbols). Bars and points indicate means ± SDs. (A) Body weight. n = 10. (B) Organ weights at age 6 mo. n = 10. (C) Littering. The litter frequency indicates the total number of litters produced by five to seven permanent breeding pairs mated at age five to nine weeks for six months. Litter sizes and lethality before age five weeks was evaluated for 33–64 litters. (D) Fraction of male pups among 83–363 newborns. (E) Activity of sulfatases in serum. n = 3. hASA-C69S serum levels of transgenic ASA−/− mice: 0.3–0.7 μg/mL. (F) Serum activities of β-hexosaminidase (β-hex), α-galactosidase (α-gal), β-galactosidase (β-gal) and lysosomal acid phosphatase (LAP). Same mice as in (E).

Figure 5

Immune responses of mice treated by weekly injection of hASA. (A) Lethality during treatment for up to ten weeks using 20 mg/kg (conventional ASA−/− mice [closed triangles, n = 20] and wildtype controls [open squares, n = 5]) or 15 mg/kg hASA (transgenic ASA−/− mice [closed circles, n = 20]). As a control, some conventional ASA−/− mice were treated with buffer (open triangles, n = 10). Asterisk- due to severe side effects treatment was terminated after four injections. (B) α-hASA antibody titers of mice treated for up to 17 weeks with 2.5 mg/kg hASA. Same legend as in (A). hASA-C69S serum concentrations of transgenic ASA−/− mice: < 0.01–1.70 μg/mL. Bars indicate means ± SDs, n = 4–5.

Figure 6

Sulfatide levels after weekly injection of 2.5 mg/kg hASA into transgenic ASA−/−mice for 17 weeks (hatched bars; hASA serum levels < 0.01–1.70 μg/mL). Age-matched transgenic ASA−/− mice mock-treated with buffer (open bars) and untreated wildtype mice (closed bars) were used as controls. Sulfatide was quantified by TLC and normalized on cholesterol. Bars represent means ± SDs, n = 5–6. P values are indicated (Student t-test).