High overexpression of the human α-galactosidase A gene driven by its promoter in transgenic mice:: Implications for the treatment of Fabry disease

Background: Human alpha-galactosidase A (alpha-Gal A) is the lysosomal enzyme that cleaves alpha-galactosyl residues from glycoconjugates and is the deficient enzyme in Fabry disease. To date, there have been no studies on the regulation of this "housekeeping" gene. Methods. Transgenic mice were established with either 1) a 13.3-kilobase (kb) human genomic fragment that contained 246 bp of 5'- and approximately 2.8 kb of 3'- untranslated sequences, or 2) an "intronless" construct derived from the genomic sequence with the 5' and 3' flanking regions intact. Tissues that expressed high levels of alpha-Gal A activity were examined by light and electron microscopy. Results: Transgenic mice were generated with 2 and 12 copies of the genomic sequence (Lines 1 and 2) or about 60 copies of the intronless construct (Lines 3 and 4). In mice hemizygous for the genomic transgene (Lines 1 and 2), tissue alpha-Gal A activities were 12 to 155 times higher than those in the respective wild-type tissue, depending on tissue and transgene copy number. Of note, the high overexpression did not alter the cellular or subcellular cytoarchitecture. In contrast, alpha-Gal A activities expressed by mice that carried the intronless construct were only two- to sixfold more than in wild-type tissues in which the genomic transgene was highly expressed. Conclusions: The remarkably high levels of alpha-Gal A expression in transgenic mice carrying the intact genomic sequence versus the intronless construct suggested that the genomic sequence contained a strong intronic enhancer element. Identification of this regulatory element or elements may be useful in efforts to overexpress human alpha-Gal A for gene therapy endeavors. In addition, overexpression of human alpha-Gal A did not affect cellular morphology, which indicates that its overexpression in gene therapy endeavors should be safe.