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1.
J Biol Chem ; 298(12): 102625, 2022 12.
Article in English | MEDLINE | ID: mdl-36306823

ABSTRACT

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 µg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV.


Subject(s)
Brain Diseases , Mucopolysaccharidosis III , Cricetinae , Animals , Humans , Mice , Mucopolysaccharidosis III/drug therapy , Mucopolysaccharidosis III/metabolism , CHO Cells , Pilot Projects , Cricetulus , Hydrolases/metabolism , Brain/metabolism , Heparitin Sulfate/metabolism , Brain Diseases/metabolism , Lysosomes/metabolism , Disease Models, Animal
2.
Drug Deliv Transl Res ; 10(2): 425-439, 2020 04.
Article in English | MEDLINE | ID: mdl-31942701

ABSTRACT

BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing. We first assess the ability of enzyme to cross a potentially compromised blood-brain barrier in the Naglu-/- mouse model and then assess the potential for CI-MPR to be employed for receptor-mediated transport across the blood-brain barrier. In wild-type and Naglu-/- mice, CI-MPR expression in brain vasculature is high during the neonatal period but virtually absent by adolescence. In contrast, CI-MPR remains expressed through adolescence in non-affected non-human primate and human brain vasculature. Combined results from IV administration of BMN 250 in Naglu-/- mice and IV and ICV administration in healthy juvenile non-human primates suggest a limitation to therapeutic benefit from IV administration because enzyme distribution is restricted to brain vascular endothelial cells: enzyme does not reach target neuronal cells following IV administration, and pharmacological response following IV administration is likely restricted to clearance of substrate in endothelial cells. In contrast, ICV administration enables central nervous system enzyme replacement with biodistribution to target cells.


Subject(s)
Acetylglucosaminidase/administration & dosage , Acetylglucosaminidase/genetics , Blood-Brain Barrier/chemistry , Insulin-Like Growth Factor II/administration & dosage , Mucopolysaccharidosis III/drug therapy , Receptor, IGF Type 2/metabolism , Recombinant Fusion Proteins/administration & dosage , Acetylglucosaminidase/therapeutic use , Administration, Intravenous , Animals , Disease Models, Animal , Enzyme Replacement Therapy , Female , Infusions, Intraventricular , Insulin-Like Growth Factor II/therapeutic use , Male , Mice , Mice, Transgenic , Mucopolysaccharidosis III/genetics , Primates , Recombinant Fusion Proteins/therapeutic use , Translational Research, Biomedical
3.
Mol Ther Methods Clin Dev ; 6: 43-53, 2017 Sep 15.
Article in English | MEDLINE | ID: mdl-28664165

ABSTRACT

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), caused by inherited deficiency of α-N-acetylglucosaminidase (NAGLU), required for lysosomal degradation of heparan sulfate (HS), is a pediatric neurodegenerative disorder with no approved treatment. Intracerebroventricular (ICV) delivery of a modified recombinant NAGLU, consisting of human NAGLU fused with insulin-like growth factor 2 (IGF2) for enhanced lysosomal targeting, was previously shown to result in marked enzyme uptake and clearance of HS storage in the Naglu-/- mouse brain. To further evaluate regional, cell type-specific, and dose-dependent biodistribution of NAGLU-IGF2 (BMN 250) and its effects on biochemical and histological pathology, Naglu-/- mice were treated with 1-100 µg ICV doses (four times over 2 weeks). 1 day after the last dose, BMN 250 (100 µg doses) resulted in above-normal NAGLU activity levels, broad biodistribution, and uptake in all cell types, with NAGLU predominantly localized to neurons in the Naglu-/- mouse brain. This led to complete clearance of disease-specific HS and reduction of secondary lysosomal defects and neuropathology across various brain regions lasting for at least 28 days after the last dose. The substantial brain uptake of NAGLU attainable by this highest ICV dosage was required for nearly complete attenuation of disease-driven storage accumulations and neuropathology throughout the Naglu-/- mouse brain.

4.
Proc Natl Acad Sci U S A ; 111(41): 14870-5, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25267636

ABSTRACT

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood-brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [ß-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, ß-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and ß-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.


Subject(s)
Acetylglucosaminidase/therapeutic use , Brain/metabolism , Drug Delivery Systems , Insulin-Like Growth Factor II/therapeutic use , Mucopolysaccharidosis III/drug therapy , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/therapeutic use , Animals , Biomarkers/metabolism , Brain/pathology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Endocytosis , Fibroblasts/metabolism , Fibroblasts/pathology , Heparitin Sulfate/metabolism , Humans , Injections, Intraventricular , Liver/metabolism , Lysosomal Membrane Proteins/metabolism , Mice , Mucopolysaccharidosis III/pathology , Neurons/metabolism , Neurons/pathology , Protein Binding , beta-N-Acetylhexosaminidases/metabolism
5.
JIMD Rep ; 8: 63-72, 2013.
Article in English | MEDLINE | ID: mdl-23430522

ABSTRACT

The immune response to exogenous protein has been shown to reduce therapeutic efficacy in animal models of enzyme replacement therapy. A previously published study demonstrated an immunosuppressive regimen which successfully induced immune tolerance to α-L-iduronidase in canines with mucopolysaccharidosis I. The two key requirements for success were high-affinity receptor-mediated enzyme uptake, conferred by mannose 6-phosphate conjugation, and immunosuppression with low-dose antigen exposure. In this study, we attempted to induce immune tolerance to phenylalanine ammonia-lyase by producing a recombinant mannose 6-phosphate conjugated form and administering it to normal dogs according to the previously published tolerance induction regimen. We found that the recombinant conjugated enzyme was stable, could bind to the mannose 6-phosphate receptor with high affinity, and its uptake into fibroblast cells was mediated by this receptor. However, at the end of a tolerance induction period, all dogs demonstrated an antigen-specific immune response when challenged with increasing doses of unconjugated phenylalanine ammonia-lyase. The average time to seroconvert was not significantly different among three separate groups of test animals (n = 3 per group) and was not significantly different from one group of control animals (n = 3). None of the nine test group animals developed immune tolerance to the enzyme using this method. This suggests that high-affinity cellular uptake mediated by the mannose 6-phosphate receptor combined with a previously studied tolerizing regimen is not sufficient to induce immune tolerance to an exogenous protein and that other factors affecting antigen distribution, uptake, and presentation are likely to be important.

6.
Mol Genet Metab ; 104(3): 325-37, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21784683

ABSTRACT

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in the gene encoding tripeptidyl-peptidase 1 (TPP1). LINCL patients accumulate lysosomal storage materials in the CNS accompanied by neurodegeneration, blindness, and functional decline. Dachshunds homozygous for a null mutation in the TPP1 gene recapitulate many symptoms of the human disease. The objectives of this study were to determine whether intrathecal (IT) TPP1 treatment attenuates storage accumulation and functional decline in TPP1-/- Dachshunds and to characterize the CNS distribution of TPP1 activity. TPP1 was administered to one TPP1-/- and one homozygous wild-type (WT) dog. An additional TPP1-/- and WT dog received vehicle. Four IT administrations of 32 mg TPP1 formulated in 2.3 mL of artificial cerebrospinal fluid (aCSF) or vehicle were administered monthly via the cerebellomedullary cistern from four to seven months of age. Functional decline was assessed by physical and neurological examinations, electrophysiology, and T-maze performance. Neural tissues were collected 48 h after the fourth administration and analyzed for TPP1 activity and autofluorescent storage material. TPP1 was distributed at greater than WT levels in many areas of the CNS of the TPP1-/- dog administered TPP1. The amount of autofluorescent storage was decreased in this dog relative to the vehicle-treated affected control. No improvement in overall function was observed in this dog compared to the vehicle-treated TPP1-/- littermate control. These results demonstrate for the first time in a large animal model of LINCL widespread delivery of biochemically active TPP1 to the brain after IT administration along with a decrease in lysosomal storage material. Further studies with this model will be necessary to optimize the dosing route and regimen to attenuate functional decline.


Subject(s)
Aminopeptidases/pharmacology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/pharmacology , Lysosomes/metabolism , Neuronal Ceroid-Lipofuscinoses/drug therapy , Neuronal Ceroid-Lipofuscinoses/metabolism , Serine Proteases/pharmacology , Aminopeptidases/administration & dosage , Aminopeptidases/blood , Aminopeptidases/genetics , Aminopeptidases/therapeutic use , Animals , CHO Cells , Central Nervous System/metabolism , Chromatography, Gel , Chromatography, Ion Exchange , Cricetinae , Cricetulus , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/administration & dosage , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/blood , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/therapeutic use , Dogs , Electrophysiology , Fluorescence , Gene Knockout Techniques , Humans , Immunoassay , Immunoglobulin E/blood , Injections, Spinal , Magnetic Resonance Imaging , Maze Learning/drug effects , Recombinant Proteins/pharmacology , Serine Proteases/administration & dosage , Serine Proteases/blood , Serine Proteases/genetics , Serine Proteases/therapeutic use , Tripeptidyl-Peptidase 1
7.
Chem Commun (Camb) ; (14): 1642-3, 2003 Jul 21.
Article in English | MEDLINE | ID: mdl-12877481

ABSTRACT

Synthetic H-bonded molecular zippers contain no sequence information that can be used to engineer the selective binding interactions characteristic of biopolymers; reversing the sense of the amide bonds in the two binding partners generates a new orthogonal recognition motif and the mutually complementary binding partners form complexes an order of magnitude more stable than the corresponding mismatch complexes.


Subject(s)
Amino Acid Motifs , Biopolymers/chemistry , Protein Engineering/methods , Amino Acid Sequence , Binding Sites , Hydrogen Bonding , Macromolecular Substances , Molecular Structure
8.
Chemistry ; 8(23): 5435-46, 2002 Dec 02.
Article in English | MEDLINE | ID: mdl-12561316

ABSTRACT

Chemical double-mutant cycles have been used to quantify intermolecular functional-group interactions in H-bonded zipper complexes in chloroform. If the same interaction is measured in zippers of different overall stability, the double-mutant cycles can be combined to produce a triple-mutant box. This construct quantifies cooperativity between the functional group interaction of interest and the other interactions that are used to change the overall stability of the complexes. The sum of two edge-to-face aromatic interactions (-2.9 +/- 0.5 kJ mol-1) is shown to be insensitive to changes of up to 13.7 +/- 0.2 kJ mol-1 in the overall stability of the complex. In principle, enthalpic cooperative effects caused by entropy-enthalpy compensation could perturb the measurement of intermolecular interactions when using the double-mutant cycle approach, but these experiments show that, for this system, the magnitude of the effect lies within the error of the measurements.


Subject(s)
Models, Chemical , Hydrogen Bonding , Kinetics , Magnetic Resonance Spectroscopy , Molecular Conformation , Thermodynamics
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