Retrovirally expressed human arylsulfatase A corrects the metabolic defect of arylsulfatase A-deficient mouse cells

Bone marrow stem cell-based gene transfer in a mouse model for metachromatic leukodystrophy: effects on visceral and nervous system disease manifestations

“…14 The specific activity of MSCV-asa-encoded hASA is around 40 mU hASA/ g hASA in cultured mouse cells and in livers of treated animals (see above). 17 Thus, 20 ng ASA/mg tissue protein resemble roughly 1.0 mU/mg ASA or 100% of the normal activity. Therefore it can be concluded, that ASA levels at least in the range of the normal ASA activity are required to achieve improvement of the histopathology.…”

“…However, we have performed extensive control experiments to verify the structural and functional integrity of the MSCV-asa-encoded hASA polypeptide following expression in cultured mouse cells. 17 Thus, the spe- cific activity, secretion, intracellular sorting and half-life of the enzyme was normal and it corrected the metabolic defect of ASA-deficient mouse glial cells upon intracellular expression or endocytosis. Furthermore, hASA isolated from the brain of treated mice hydrolyzed sulfatide in an in vitro assay efficiently.…”

“…14,17 The activity assay is compromised by high background activities of unspecific sulfatases so that the hASA activity has to be calculated by subtraction of the mean sulfatase background activity (determined in livers of mocktreated, ASA-deficient mice) from the compound sulfatase activity measured (not shown). Division of the calculated data by corresponding ELISA data indicated a specific activity of hASA around 45 mU/ g on average (see below).…”

“…This value is close to the normal specific activity of gene-encoded hASA in human tissues and cell lines (40 mU/ g). 14,17 Since the ELISA is specific for hASA and thus superior to the activity assay in the precise evaluation of minor differences in hASA expression levels it was used for all other analyses.…”

Transport of Arylsulfatase A across the Blood-Brain Barrier in Vitro

“…ASA activity was measured with the artificial substrate p-nitrocatechol sulfate (13). ASA and protein concentrations were determined by an indirect sandwich ELISA specific for human ASA (14) and the Bio-Rad DC assay, respectively. For one experiment, rhASA was fractionated by Source 30Q anion exchange chromatography (GE Healthcare) using a linear gradient of 60 -500 mM NaCl in 10 mM sodium phosphate (pH 7.5) for elution (method available in patent application 20080003211 on the FreshPatents website).…”