Circulating alpha-galactosidase A derived from transduced bone marrow cells: relevance for corrective gene transfer for Fabry disease. Hum Gene Ther 1999 Aug 10;10(12):1931-9
Date
08/31/1999Pubmed ID
10466627DOI
10.1089/10430349950017293Scopus ID
2-s2.0-0033543108 (requires institutional sign-in at Scopus site) 45 CitationsAbstract
Fabry disease is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). We previously engineered a retrovirus encoding human alpha-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete alpha-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous alpha-gal A, we constructed a retroviral vector (pUMFG/alpha-gal A/FLAG) containing a fusion form of alpha-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type alpha-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome-enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/alpha-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed alpha-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.
Author List
Takenaka T, Qin G, Brady RO, Medin JAAuthor
Jeffrey A. Medin PhD Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBone Marrow Cells
Bone Marrow Transplantation
Cells, Cultured
DNA, Viral
Fabry Disease
Fibroblasts
Genetic Therapy
Genetic Vectors
Humans
Leukocytes, Mononuclear
Male
Mice
Polymerase Chain Reaction
Recombinant Fusion Proteins
Retroviridae
Skin
Transduction, Genetic
alpha-Galactosidase
