Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept

Abstract

Galactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and β-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and β-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms. Here, we describe a proof-of-concept, preclinical study toward the development of enzyme replacement therapy for galactosialidosis, using a recombinant human PPCA. We show that the recombinant enzyme, taken up by patient-derived fibroblasts, restored cathepsin A, neuraminidase-1, and β-galactosidase activities. Long-term, bi-weekly injection of the recombinant enzyme in a cohort of mice with null mutation at the PPCA (CTSA) locus (PPCA ^-/- ), a faithful model of the disease, demonstrated a dose-dependent, systemic internalization of the enzyme by cells of various organs, including the brain. This resulted in restoration/normalization of the three enzyme activities, resolution of histopathology, and reduction of sialyloligosacchariduria. These positive results underscore the benefits of a PPCA-mediated enzyme replacement therapy for the treatment of galactosialidosis.

Keywords: ERT; galactosialidosis; lysosomal NEU1; lysosomal glycoproteinosis; lysosomal storage disease; lysosomes; recombinant Protective protein/cathepsin A; β-GAL.

Graphical abstract

Figure 1

SDS-PAGE analysis of rhPPCA under reducing conditions (A and B) Representative images showing the purity of rhPPCA on a Coomassie brilliant blue (CBB)-stained polyacrylamide gel (A) and on an immunoblot probed with anti-hPPCA antibody (B).

Figure 2

rhPPCA is delivered to the lysosome in patient-derived GS fibroblasts (A) K_uptake determination of rhPPCA by Hanes-Woolf regression analysis following 24 h uptake in GS fibroblasts. rhPPCA shows a high-affinity K_uptake of 1−3 nM, n = 12. (B) Dose-dependent and receptor-mediated uptake of rhPPCA in the absence or presence of the competitive inhibitor, M6P, n = 11. (C) Immunoblot of lysates derived from GS fibroblasts treated with increasing amount of rhPPCA and probed with an anti-hPPCA antibody. Dose-dependent detection of ∼30 kDa of the mature form of rhPPCA confirms the lysosomal delivery and conversion of the precursor into a mature and active form. (D–F) Cathepsin A (D), NEU1 (E), and β-GAL (F) activities performed in cell lysates following treatment with rhPPCA. Normal human fibroblasts were used as positive control, n = 3.

Figure 3

rhPPCA restores cathepsin A activity and corrects NEU1 and β-GAL activities in PPCA^–/– mice (A−D) Cathepsin A (CA) activity measured in liver, spleen, heart, and kidney lysates from PPCA^–/– mice treated with rhPPCA (doses: 0.2, 0.6, 2.0, 6.0, and 20 mg/kg, corresponding to groups 4, 5, 6, 7, and 8, respectively) for 8 weeks from untreated wild-type (WT) FVB/NJ mice (group 1) and from untreated PPCA^–/– (KO) mice (group 3). Group 9, designated 20.0 + R, represents the recovery group sacrificed 1 week after the final injection, n ≥ 4. (E–P) NEU1 (E–H), β-GAL (I–L), and β-HEX (M–P) activities measured in the same set of tissues as above, n ≥ 3. KO∗, PPCA^–/– + CPH. All of the graphs are presented as mean ± SD. Statistical analyses were performed using the Brown-Forsythe and Welch ANOVA tests. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

rhPPCA is taken up by cells of PPCA^–/– visceral organs and reduces lysosomal vacuolation (A) Representative immunohistochemistry images of liver, kidney, and spleen from mice treated with 20 mg/kg rhPPCA (group 8) showing widespread distribution of rhPPCA. Scale bar, 50 μm. (B) Representative histopathology images of the liver, kidney, heart, and spleen of untreated WT and PPCA^–/– (KO) mice and of PPCA^–/– (KO) mice treated with 2 mg/kg rhPPCA (groups 1, 3, and 6, respectively). Scale bars, 50 μm, 100 μm, 50 μm, and 50 μm, respectively.

Figure 5

Quantification of lysosomal vacuolation in PPCA^–/– mice after treatment with rhPPCA (A–H) Histopathological analyses of kidney (A–C), liver (D–F), spleen (G), and heart (H). Correction of tissue morphology after rhPPCA treatment of PPCA^–/– mice was quantified based on the extent of lysosomal vacuolation in cells of these organs. Mean path score of individual mice was graded: 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. Scores obtained from mice treated with rhPPCA (doses: 0.2, 0.6, 2.0, 6.0, and 20 mg/kg, corresponding to groups 4, 5, 6, 7, and 8, respectively) for 8 weeks were plotted with scores from WT FVB/NJ mice (group 1) and from untreated PPCA^–/– (KO) mice (group 3). Group 9, designated 20.0 + R, represents the recovery group sacrificed 1 week after the final injection. Each dot represents an individual mouse in the study, n ≥ 9.

Figure 6

rhPPCA reduces urinary sialylated oligosaccharides in PPCA^–/– mice in a dose-dependent manner (A) Urinary sialic acid after 4 (mid-study), 8 (end of study), and 8 + 1 (group 9, 20.0 + recovery) weeks of treatment with rhPPCA was compared to the levels measured at week 0, used as baseline. (B and C) Sialic acid levels were compared among doses (0.2, 0.6, 2.0, 6.0, and 20 mg/kg) at 4 (B) and 8 and 8 + 1 (recovery group) (C) weeks, respectively. KO∗, group 3, PPCA^–/– (KO) + CPH. Urinary sialic acid levels were normalized to a creatinine concentration; data are presented as mean ± SD. The dots in the graphical bars represent values obtained from each single mouse. Statistical analyses were performed using the Brown-Forsythe and Welch ANOVA tests, n ≥ 4. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001. The asterisk (∗) is used to label groups compared to KO∗ = group 3; #, used to label comparisons among groups treated with different doses of rhPPCA.