Reversal of peripheral and central neural storage and ataxia after recombinant enzyme replacement therapy in α-mannosidosis mice

Abstract

Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including alpha-mannosidosis. Here we show that the neuropathology of a mouse model for alpha-mannosidosis can be efficiently treated using recombinant human alpha-mannosidase (rhLAMAN). After intravenous administration of different doses (25-500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (<70%) in visceral tissues, higher doses were needed for a clear effect in central and peripheral nervous tissues. A distinct reduction (<50%) of brain storage required repeated high-dose injections (500 U/kg), whereas lower doses (250 U/kg) were sufficient for clearance of stored substrates in peripheral neurons of the trigeminal ganglion. Successful transfer across the blood-brain barrier was evident as the injected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storage vacuoles. Importantly, the decrease in neuronal storage in the brain correlated with an improvement of the neuromotor disabilities found in untreated alpha-mannosidosis mice. Uptake of rhLAMAN seems to be independent of mannose-6-phosphate receptors, which is consistent with the low phosphorylation profile of the enzyme. These data suggest that high-dose injections of low phosphorylated enzymes might be an interesting option to efficiently treat LSDs with CNS involvement.

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

“…Therefore, transgenic and nontransgenic knockout mice as well as wild‐type mice were analyzed for storage and secondary pathological changes 8, 16. TLC analyses revealed comparable accumulation of oligosaccharides linked to 2‐9 mannose residues (Man 2 ‐Man 9 ) between both genotypes in brain (Fig.…”

“…In order to investigate the chronic impact of this ERT regimen and whether chronic treatment can compensate for a lower dose, 4‐6 weeks old mice with an established blood‐brain barrier23, 24 were biweekly injected with 125 and 500 U/kg rhLAMAN (corresponding to 4.4 and 15.6 mg/kg per bw), respectively. ERT was completed at an age of 16‐18 weeks, a time point where alpha‐mannosidosis mice display a prominent storage phenotype 16, 17. Of note, for this and the following experiments prior to harvesting brain and other organs, mice were perfused with phosphate buffer to avoid contamination with plasma‐derived rhLAMAN.…”

“…We next investigated the clearance of rhLAMAN from circulation as well as the accessibility of the CNS to the injected enzyme by injecting one‐single high enzyme dose (1000 U/kg, corresponding to 31.2 mg/kg per bw) to obtain measurable enzyme activity in brain 16. At different time points, after administration (5 min, 1 h, 5 h, and 16 h), blood was taken and LAMAN activity determined.…”

“…Oligosaccharide extraction and thin layer chromatography (TLC) analysis of neutral oligosaccharides was performed essentially as described previously 16. A detailed description of oligosaccharide extraction, TLC and high‐performance liquid chromatography (HPLC) analyses can be found under Data S1.…”

“…So far, several LSDs including Morbus Gaucher (Type I), mucopolysaccharidoses (I, II, VI), Morbus Fabry, and Morbus Pompe have been shown to benefit from ERT 14. We have previously demonstrated the value of high‐dose short‐term ERT on clearance of primary brain substrate storage in alpha‐mannosidase‐deficient (knockout) mice,15 a valid mouse model for the human disease, using rhLAMAN of a laboratory scale batch 16, 17, 18. However, immunological responses to the injected enzyme associated with high mortality precluded long‐term ERT of this mouse strain.…”