Molecular architecture determines brain delivery of a transferrin-receptor targeted lysosomal enzyme

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

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miR-143 regulates lysosomal enzyme transport across blood-brain barrier and improves CNS treatment for Hurler syndrome

Molecular Genetics and Metabolism ◽

10.1016/j.ymgme.2019.11.328 ◽

2020 ◽

Vol 129 (2) ◽

pp. S125-S126

Author(s):

Dao Pan ◽

Yi Lin ◽

Xiaohong Wang ◽

Kevin Rose ◽

Mei Dai ◽

...

Keyword(s):

Blood Brain Barrier ◽

Lysosomal Enzyme ◽

Brain Barrier ◽

Hurler Syndrome ◽

Blood Brain ◽

Enzyme Transport

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High β-glucosidase (GBA) activity not attributable to GBA1 and GBA2 in live normal and enzyme-deficient fibroblasts may emphasise the role of additional GBAs

Biological Chemistry ◽

10.1515/hsz-2015-0144 ◽

2015 ◽

Vol 396 (11) ◽

pp. 1241-1246 ◽

Cited By ~ 3

Author(s):

Klaus Harzer ◽

Yildiz Yildiz

Keyword(s):

Lysosomal Enzyme ◽

Gaucher Disease ◽

Wild Type ◽

Activity Profile ◽

Glucosidase Activity ◽

Mouse Cells ◽

Enzyme Deficient

Abstract Beta-glucosidases (GBA) include GBA1, GBA2 and other β-glucosidases (non-GBA1-2). GBA1 is a lysosomal and GBA2 an extra-lysosomal enzyme. GBA1- and GBA2-deficient genetic conditions, with different phenotypes, are glucosylceramide (GC; the main GBA substrate) accumulating diseases. To study the activity profile of GBA, live fibroblasts were loaded with radioactive GC. The GC metabolism was measured in wild-type, GBA1-deficient (Gaucher disease) and GBA2-deficient (Gba2-/- mouse) cells. The differences found allowed the prediction of marked proportions of GBA1, GBA2, and particularly non-GBA1-2 (probably including GBA3, a cytosolic β-glucosidase) activity for wild-type cells. The high proportion of non-GBA1-2 suggests an important role of these enzymes.

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Function of the two mannose 6-phosphate receptors in lysosomal enzyme transport

Biochemical Society Transactions ◽

10.1042/bst0240133 ◽

1996 ◽

Vol 24 (1) ◽

pp. 133-136 ◽

Cited By ~ 40

Author(s):

H. Munier-Lehmann ◽

F. Mauxion ◽

B. Hoflack

Keyword(s):

Lysosomal Enzyme ◽

Enzyme Transport ◽

Mannose 6 Phosphate

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Alterations in EGF-Endocytosis, Lysosomal Enzyme Transport and Maturation of Cathepsins in Juvenile Neuronal Ceroid Lipofuscinosis Fibroblasts

International Clinical Neuroscience Journal ◽

10.15171/icnj.2020.02 ◽

2019 ◽

Vol 7 (1) ◽

pp. 6-15

Author(s):

Jaime Cárcel-Trullols

Keyword(s):

Cell Surface ◽

Lysosomal Enzyme ◽

Egf Receptor ◽

Cell Function ◽

Neuronal Ceroid Lipofuscinosis ◽

Cathepsin L ◽

Western Blots ◽

Juvenile Neuronal Ceroid Lipofuscinosis ◽

Ceroid Lipofuscinosis ◽

Enzyme Transport

Background: Juvenile neuronal ceroid lipofuscinosis (JNCL), one of the most frequent forms of the NCL storage diseases, is known to be caused by loss-of-function mutations in ceroid-lipofuscinosis neuronal protein 3 (CLN3), but its cell function has not been fully elucidated. We previously reported increased lysosomal pH in CLN3 deficient cells. In the present study, we analysed the consequences of this effect in the endo-lysosomal pathways in CLN3 cells. Methods: The present study investigated different endo-lysosomal pathways in control, CLN2, CLN3 human skin fibroblasts under high and low proteolysis conditions. Cell surface biotinylation after EGF (2 ng/mL) stimulation, EGF phosphorylation (Tyr-845), retromer and cation-independent mannose-6- phosphate receptor (CI-MPR) levels and stability, EGF degradation pathways and cathepsin L and D levels were analysed by western blots. Caveolae mediated endocytosis was analysed by flow cytometry. CIMPR subcellular localization was ascertained by immunocytochemistry, confocal microscopy and further image analysis. Results: Whereas caveolae-mediated endocytosis was not affected in CLN3 cells, clathrin-mediated epidermal growth factor (EGF) internalization was reduced, along with EGF receptor (EGFR) phosphorylation. In addition, cell surface EGFR levels and recycling to the cell membrane were increased. EGFR lysosomal degradation was impaired and our results suggest that the receptor was diverted to proteasomal degradation. We also analysed the machinery responsible for lysosomal hydrolase transport to the lysosome and found increased stability of CIMPR, a major receptor implicated in the transport of hydrolases. The subcellular distribution of the CI-MPR was also altered in CLN3 cells, since it accumulated within the Trans-Golgi network (TGN) and did not progress into the lysosomes. In addition, we found a reduced turnover of retromer subunits, a complex that retrieves the CI-MPR from endosomes to the TGN. Finally and as a possible consequence of these alterations in lysosomal enzyme transport, cathepsin L and D maturation were found suppressed in CLN3 cells. Conclusion: Altogether, these results point to increased lisosomal pH as a pivotal event causing various alterations in intracellular traffic associated to the development of JNCL disease.

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Bacterial morphine dehydrogenase further defines a distinct superfamily of oxidoreductases with diverse functional activities.

Biochemical Journal ◽

10.1042/bj2990805 ◽

1994 ◽

Vol 299 (3) ◽

pp. 805-811 ◽

Cited By ~ 32

Author(s):

N C Bruce ◽

D L Willey ◽

A F Coulson ◽

J Jeffery

Keyword(s):

Pseudomonas Putida ◽

Gluconic Acid ◽

Functional Activities ◽

Coenzyme Binding ◽

Substrate Reduction ◽

Eukaryotic Proteins

Pseudomonas putida morphine dehydrogenase is shown to be closely homologous to 18 proteins, defining a superfamily within which morphine dehydrogenase particularly resembles two bacterial, 2,5-dioxo-D-gluconic acid reductases, and two eukaryotic proteins of unknown functions. Relationships within the superfamily are extensive and complex. Residue identities between protein pairs range from 29-90%. Three subgroups are proposed. Nevertheless, on the basis of residue conservations/exchanges it is suggested that the nicotinamide coenzyme binding and substrate reduction occur in all the enzymes by broadly analogous mechanisms, among which some probable differences are identified.

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The I-Cell model: the molecular basis for abnormal lysosomal enzyme transport in mucolipidosis II and mucolipidosis III

Genetic and Metabolic Disease in Pediatrics ◽

10.1016/b978-0-407-02312-3.50010-7 ◽

1985 ◽

pp. 91-110 ◽

Cited By ~ 1

Author(s):

William S. Sly ◽

Vasantha Sundaram

Keyword(s):

Lysosomal Enzyme ◽

Molecular Basis ◽

Cell Model ◽

Enzyme Transport ◽

Mucolipidosis Ii

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Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice

Science Translational Medicine ◽

10.1126/scitranslmed.aay1163 ◽

2020 ◽

Vol 12 (545) ◽

pp. eaay1163 ◽

Cited By ~ 16

Author(s):

Julie C. Ullman ◽

Annie Arguello ◽

Jennifer A. Getz ◽

Akhil Bhalla ◽

Cathal S. Mahon ◽

...

Keyword(s):

Blood Brain Barrier ◽

Lysosomal Enzyme ◽

Brain Barrier ◽

Enzyme Treatment ◽

Brain Delivery ◽

In Vivo Models ◽

Blood Brain ◽

Brain Exposure ◽

Transport Vehicle ◽

The Brain

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

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