Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction.

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.

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines.

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.

Prophylactic Prednisolone Promotes AAV5 Hepatocyte Transduction Through the Novel Mechanism of AAV5 Coreceptor Platelet-Derived Growth Factor Receptor Alpha Upregulation and Innate Immune Suppression.

Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day -1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.

Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a ß-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.

Mutant mice derived by ICSI of evaporatively dried spermatozoa exhibit expected phenotype.

Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at -80 °C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze-drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.

Translational studies of intravenous and intracerebroventricular routes of administration for CNS cellular biodistribution for BMN 250, an enzyme replacement therapy for the treatment of Sanfilippo type B.

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.

Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type B Mice.

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.