Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice.

Preclinical studies of enzyme-replacement therapy for Fabry disease (deficient alpha-galactosidase A [alpha-Gal A] activity) were performed in alpha-Gal A-deficient mice. The pharmacokinetics and biodistributions were determined for four recombinant human alpha-Gal A glycoforms, which differed in sialic acid and mannose-6-phosphate content. The plasma half-lives of the glycoforms were approximately 2-5 min, with the more sialylated glycoforms circulating longer. After intravenous doses of 1 or 10 mg/kg body weight were administered, each glycoform was primarily recovered in the liver, with detectable activity in other tissues but not in the brain. Normal or greater activity levels were reconstituted in various tissues after repeated doses (10 mg/kg every other day for eight doses) of the highly sialylated AGA-1 glycoform; 4 d later, enzyme activity was retained in the liver and spleen at levels that were, respectively, 30% and 10% of that recovered 1 h postinjection. Importantly, the globotriaosylceramide (GL-3) substrate was depleted in various tissues and plasma in a dose-dependent manner. A single or repeated doses (every 48 h for eight doses) of AGA-1 at 0.3-10.0 mg/kg cleared hepatic GL-3, whereas higher doses were required for depletion of GL-3 in other tissues. After a single dose of 3 mg/kg, hepatic GL-3 was cleared for > or =4 wk, whereas cardiac and splenic GL-3 reaccumulated at 3 wk to approximately 30% and approximately 10% of pretreatment levels, respectively. Ultrastructural studies demonstrated reduced GL-3 storage posttreatment. These preclinical animal studies demonstrate the dose-dependent clearance of tissue and plasma GL-3 by administered alpha-Gal A, thereby providing the in vivo rationale-and the critical pharmacokinetic and pharmacodynamic data-for the design of enzyme-replacement trials in patients with Fabry disease.

Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Quantitative determination of globotriaosylceramide by immunodetection of glycolipid-bound recombinant verotoxin B subunit.

An assay for the neutral glycosphingolipid, globotriaosylceramide (Galalpha1-4Galbeta1-4Glcbeta1-1Cer; GL-3), was developed based on the B subunit of Escherichia coli verotoxin (VTB). The VTB gene was isolated, overexpressed in E. coli, and purified by a single immunoaffinity chromatographic step using a monoclonal anti-VTB IgG-agarose column. Purified recombinant VTB was used to develop an enzyme-linked immunosorbent assay (ELISA) to determine the GL-3 concentrations in plasma and tissue extracts from normal individuals and patients and mice with alpha-galactosidase A deficiency (human Fabry disease). The mean (+/-1 SD) plasma GL-3 concentrations in affected male and female heterozygotes with Fabry disease were 12.6 +/- 3.7 and 1.1 +/- 0.7 microg/ml, respectively, whereas normal individuals had 0.9 +/- 0.4 microg/ml. In 5- to 6-month-old mice with alpha-galactosidase A deficiency, the average GL-3 concentrations in spleen, kidney, liver, heart, and plasma were 2790 +/- 400, 1100 +/- 93, 378 +/- 67, and 196 +/- 28 ng/mg wet wt and 5. 1 +/- 2.0 microg/ml, respectively, whereas tissues from wild-type mice contained very low or undetectable GL-3 levels. This ELISA assay should prove useful for determining the GL-3 levels, as well as for monitoring the effectiveness of therapeutic endeavors in patients with Fabry disease.

Murine alpha-N-acetylgalactosaminidase: isolation and expression of a full-length cDNA and genomic organization: further evidence of an alpha-galactosidase gene family.

Recent characterization of the human sequences encoding two lysosomal hydrolases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-GalNAc) revealed that these two enzymes with distinct enzymatic activities shared about 50% overall amino acid identity and that their genomic sequences had a conserved common gene structure. These findings suggested that these genes, which are located on different chromosomes, arose by duplication and divergence from a common ancestral gene. To further compare this alpha-galactosidase gene family, the murine alpha-GalNAc cDNA and genomic sequences were isolated and characterized. The full-length cDNA contained an open-reading frame of 1245 bp encoding a 415 amino acid polypeptide and had 5' and 3' untranslated regions of 94 and 333 bp, respectively. The coding region had 81% nucleotide and 81.9% amino acid identities with those of the corresponding human alpha-GalNAc sequence. Northern analysis revealed a single transcript of approximately 1.9 kb. The functional integrity of the cDNA was demonstrated by transient expression in COS-1 cells. The murine alpha-GalNAc genomic sequence spanned approximately 9 kb and was identical in structure with the human alpha-GalNAc gene with eight introns interrupting the coding sequence at identical positions. In addition, the deduced amino acid sequence of the murine alpha-GalNAc gene was highly homologous with alpha-GalNAc and alpha-Gal A genes from other species providing further support for a common evolutionary ancestor of the alpha-galactosidase gene family. The availability of the murine gene will permit additional evolutionary comparisons, structure/function analyses, and the generation of mice with alpha-GalNAc deficiency by gene targeting.

Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

The entire genomic sequence and cDNA expression of mouse alpha-galactosidase A.

The full-length cDNA and genomic sequences encoding mouse alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22), a lysosomal galactohydrolase, were isolated and characterized. The cDNA's open reading frame encoded 419 amino acids and had 82% nucleotide (nt) and 78% amino acid identity with the human sequence, although the carboxy terminus of the mouse alpha-Gal A polypeptide was 10 amino acids shorter. The functional integrity of the mouse cDNA was demonstrated by transient expression in COS-1 cells. Northern analysis revealed two mRNA species of about 1.6 and 3.4 kb due to alternative polyadenylation signals. The entire 14.4-kb mouse genomic sequence was determined; each of its seven exons was interrupted by intronic sequence at the identical positions as the exons in the human gene. The mouse 5' flanking region (250 nt) had one Sp1, site, five CAAT boxes, and no TATA box and had 67% identity with the human promoter region. The gene contained 18 complete or partial Alu-repetitive elements (13 type 1 and 5 type 2 repeats), and three putative functional AATAAA consensus polyadenylation signals were identified 72, 1668, and 1682 nt after the TAA termination codon. Use of the 72-nt site and the 1866 and/or 1682 sites were consistent with the shorter and longer transcripts. The availability of the full-length cDNA and genomic sequence encoding mouse alpha-Gal A should facilitate structure/function studies of this lysosomal glycosidase and the construction of alpha-Gal A-deficient mice by targeted gene disruption.