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High Overexpression of the Human α-Galactosidase A Gene Driven by Its Promoter in Transgenic Mice: Implications for the Treatment of Fabry Disease
  1. Grace A. Ashley,
  2. Robert J. Desnick,
  3. Ronald E. Gordon,
  4. Jon W. Gordon
  1. From the Departments of Human Genetics (G.A.A., R.J.D., J.W.G.), Mount Sinai School of Medicine of New York University, New York, NY
  2. Pathology (R.E.G.), Mount Sinai School of Medicine of New York University, New York, NY
  3. Obstetrics and Gynecology (J.W.G.), Mount Sinai School of Medicine of New York University, New York, NY.
  1. Address correspondence to: Jon W. Gordon, MD, PhD, Department of Obstetrics and Gynecology, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, Box 1175, New York, NY 10029. Email: jgordon{at}smtplink.mssm.edu
  2. This work was supported in part by grants to R.J.D. from the National Institutes of Health, which include a Merit Award (5 R37 DK34045), a grant (5 M01 RR00071) for the Mount Sinai General Clinical Research Center, and a grant (5 P30 HD28822) for the Mount Sinai Child Health Research Center. J.W.G. is Mathers Professor.

Abstract

Background Human α-galactosidase A (α-Gal A) is the lysosomal enzyme that cleaves α-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 α-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 α-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, α-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 α-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 α-Gal A for gene therapy endeavors. In addition, overexpression of human α-Gal A did not affect cellular morphology, which indicates that its overexpression in gene therapy endeavors should be safe.

Key Words
  • α-galactosidase A
  • Fabry disease
  • transgenic mice
  • gene regulation
  • promoter sequences

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