Biochemical characterization of fluorescent‐labeled recombinant human alpha‐l‐iduronidase in vitro

Abstract

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein’s characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-L-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled protein. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to non-labeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan (GAG) storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

Discovery of allosteric regulators with clinical potential to stabilize alpha-L-iduronidase in mucopolysaccharidosis type I

“…As previously reported, rhIDUA administration increased IDUA activity in cell models [ 28 ]. Importantly, co-administration of rhIDUA with GT-01803 to patient-derived fibroblasts showed a marked dose-dependent increase in IDUA cell activity compared with the administration of rhIDUA alone (p ≤ 0.0001) ( Fig 6A ) (mean rhIDUA 1.25 nM: 53.37 nmol/h·mg, mean rhIDUA 1.25 nM + compound GT-01803 50 μM, mean difference: 33.75±7.255 nmol/h·mg).…”

A Humoral Immune Response Alters the Distribution of Enzyme Replacement Therapy in Murine Mucopolysaccharidosis Type I

“…The uptake of rhIDU into murine macrophages was only partially inhibited by mannose 6-phosphate, whereas uptake of rhIDU into human fibroblasts was nearly abolished with the addition of mannose 6-phosphate, consistent with prior results. 8 Murine Fc receptor blocking agent did not substantially affect the uptake of rhIDU into either murine macrophages or human fibroblasts. However, the blocking agents do not bind to all Fc receptors, limiting our ability to draw conclusions about the mechanism of antibody-bound rhIDU uptake into macrophages.…”

“…At 16 weeks of age, MPS I mice previously sensitized to rhIDU (n = 10) and naive to rhIDU (n = 6) were administrated one dose of 1.57 mg/kg body weight of Alexa Fluor 680 labeled rhIDU (AF680-rhIDU; degree of labeling 1.3 mol label/mol protein), which was previously showed to retain its biochemical properties (including enzymatic activity, cellular uptake, and lysosomal delivery). 8 …”

Comparative study of idursulfase beta and idursulfase in vitro and in vivo

“…Cultured fibroblasts were labeled with 35 SO 4 as described elsewhere. 21 , 22 Briefly, cultured fibroblasts were starved with serum- and sulfate-free DMEM (Welgene, Gyeonsan-si, Korea) for one day and incorporated with 35 S-radioisotope-labeled Na 2 35 SO 4 (20 μCi, Perkin Elmer) in sulfate-free DMEM containing 8% dialyzed FBS for 2 days. After that, radioisotope-labeled fibroblasts were treated with idursulfase and idursulfase beta for 24 h in DMEM containing 5% FBS.…”