Clinical and pathological characterization of ophthalmic disease in a canine model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a rare lysosomal storage disease caused by α-L-iduronidase enzyme deficiency, resulting in glycosaminoglycan (GAG) accumulation in various cell types, including ocular tissues. Ocular manifestations in humans are common with significant pathological changes including corneal opacification, retinopathy, optic nerve swelling and atrophy, and glaucoma. Available treatments for MPS I are suboptimal and there is limited to no effect in treating the ocular disease. The goal of this study was to characterize the clinical and pathological features of ocular disease in a line of MPS I affected dogs, including changes not previously reported. A total of 22 dogs were studied; 12 MPS I were affected and 10 were unaffected. A subset of each underwent complete ophthalmic examination including slit lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry, and ultrasonic pachymetry. Globes were evaluated microscopically for morphological changes and GAG accumulation. Clinical corneal abnormalities in affected dogs included edema, neovascularization, fibrosis, and marked stromal thickening. Intraocular pressures were within reference interval for affected and unaffected dogs. Microscopically, vacuolated cells containing alcian blue positive inclusions were detected within the corneal stroma, iris, ciliary body, sclera, and optic nerve meninges of affected dogs. Ganglioside accumulation was identified by luxol fast blue staining in rare retinal ganglion cells. Increased lysosomal integral membrane protein-2 expression was demonstrated within the retina of affected animals when compared to unaffected controls. Results of this study further characterize ocular pathology in the canine model of MPS I and provide foundational data for future therapeutic efficacy studies.

Newborn screening and the recommended uniform screening panel: Optimal submissions and suggested improvements based on an advocacy organization's decade-long experience.

The National MPS Society, Inc., founded in 1974, is a rare disease advocacy non-profit with a tripartite mission addressing the needs of the mucopolysaccharidosis and mucolipidosis communities through advocacy, research, and family and patient support. The Recommended Uniform Screening Panel (RUSP) of conditions for newborn screening (NBS), legislatively mandated in 2008, was implemented in 2010 by the Secretary of Health and Human Services (HSS), through the adoption of 29 core conditions. Since its inception the RUSP has grown to 35 core conditions. Each addition followed a defined nomination process that has itself undergone further definition over time. Since the adoption of the RUSP, the Society has nominated two conditions that have been approved by the Advisory Committee on Heritable Disorders in Children and Newborns (ACHDNC) and forwarded to the Secretary of HSS for inclusion on the RUSP. This history places the Society in a position to reflect on the process of successfully nominating conditions. Additionally, the Society is well placed by this experience to provide observations on the RUSP process. We will highlight best practices for pending and future nominations and reflect on potential improvements to the process and infrastructure of NBS, the RUSP, and the ACHDNC.

Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Temporospatial Development of Neuropathologic Findings in a Canine Model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidosis (MPS) IIIB is a neuropathic lysosomal storage disease characterized by the deficient activity of a lysosomal enzyme obligate for the degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). The pathogenesis of neurodegeneration in MPS IIIB is incompletely understood. Large animal models are attractive for pathogenesis and therapeutic studies due to their larger size, outbred genetics, longer lifespan, and naturally occurring MPS IIIB disease. However, the temporospatial development of neuropathologic changes has not been reported for canine MPS IIIB. Here we describe lesions in 8 brain regions, cervical spinal cord, and dorsal root ganglion (DRG) in a canine model of MPS IIIB that includes dogs aged from 2 to 26 months of age. Pathological changes in the brain included early microscopic vacuolation of glial cells initially observed at 2 months, and vacuolation of neurons initially observed at 10 months. Inclusions within affected cells variably stained positively with PAS and LFB stains. Quantitative immunohistochemistry demonstrated increased glial expression of GFAP and Iba1 in dogs with MPS IIIB compared to age-matched controls at all time points, suggesting neuroinflammation occurs early in disease. Loss of Purkinje cells was initially observed at 10 months and was pronounced in 18- and 26-month-old dogs with MPS IIIB. Our results support the dog as a replicative model of MPS IIIB neurologic lesions and detail the pathologic and neuroinflammatory changes in the spinal cord and DRG of MPS IIIB-affected dogs.

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

The post-natal development of intraocular pressure in normal domestic cats (Felis catus) and in feline congenital glaucoma.

Intraocular pressure (IOP) is the most consistent risk factor for progressive vision loss in glaucoma. Cats with recessively inherited feline congenital glaucoma (FCG) exhibit elevated IOP with gradual, painless progression of glaucoma similar to humans and are studied as a model of glaucoma in humans and animals. Here, post-natal development of IOP was characterized in normal domestic cats and in cats with FCG caused by a homozygous LTBP2 mutation. Rebound tonometry (TonoVet®, ICare Oy, Finland) was used to measure IOP non-invasively, 2-3 times weekly in 63 FCG and 33 normal kittens, of both sexes, from eyelid opening until 3-6 months of age. IOPs in the left and right eyes of both FCG and normal kittens were compared by paired t-test and linear regression. One-way ANOVA and Tukey-Kramer post-tests were used to compare IOP of cats grouped by age and disease status. A p-value <0.05 was considered significant. In the second week of life, mean IOP was 7.16 mmHg (SD = 1.3) in normal kittens and 8.72 mmHg (SD = 1.4) in kittens with FCG. Mean IOP at age 10 weeks was significantly higher in FCG (19.8 mmHg; 95% CI = 17.7, 21.9  mmHg) than in normal kittens (13.2 mmHg; 95% CI = 11.9, 14.5  mmHg). At 3 months of age, IOP in normal cats reached adult values while IOP in FCG cats continued to increase through at least six months of age. These results provide ranges for normal IOP values in young kittens and confirm that IOP is significantly higher than normal by 10wks of age in this spontaneous feline glaucoma model.

Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs.

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T(-)B(-)NK(+) SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T(-)B(-)NK(+) SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

Schirmer tear test I in dogs: results comparing placement in the ventral vs. dorsal conjunctival fornix.

OBJECTIVE: To compare Schirmer tear test I (STT I) values obtained from placement of tear strips in the ventral and dorsal conjunctival fornices in dogs. PROCEDURE: Schirmer tear test I was performed on each eye of 16 clinically normal dogs (32 eyes) in a crossover study. Initial tear strip placement site was randomized for each eye. Alternative placement site measurements were obtained after 1 week. RESULTS: The mean (± standard deviation, SD) STT I for dorsal and ventral conjunctival fornices was 20.44 (±4.46) mm/min and 23.56 (±3.98) mm/min, respectively. STT I values obtained from the ventral conjunctival fornix were significantly greater than those obtained from the dorsal conjunctival fornix (P = 0.004). CONCLUSIONS: Schirmer tear test I values were significantly greater with tear strips placed in the ventral conjunctival fornix.

Effect of topical latanoprost 0.005% on intraocular pressure and pupil diameter in normal and glaucomatous cats.

OBJECTIVE: To determine the effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in cats with inherited primary congenital glaucoma (PCG) and normal cats. ANIMALS STUDIED AND PROCEDURES: IOP and PD were measured in both eyes (OU) of 12 adult cats (six normal, six PCG), three times per week for 3 weeks prior to, for 3 weeks during, and for 2 weeks following twice-daily treatment with 0.005% latanoprost to the right eye (OD) and vehicle to the left (control) eye (OS). IOP and PD were measured hourly, for 8 h, 1 day prior to, and on the first and last days of treatment. Aqueous humor flow rate (AHF) was determined at baseline and at the end of the treatment phase in six normal cats. RESULTS: Mean IOP was significantly lower in treated vs. control eyes of PCG cats, for up to 8 h following a single latanoprost treatment, and a maximal IOP reduction of 63% occurred in treated eyes at 3 h. Latanoprost acutely lowered IOP in cats with PCG, but this effect appeared to diminish over 3 weeks of treatment. AHF was modestly increased in the treated eyes of normal cats after 3 weeks of latanoprost treatment, although IOP was not significantly affected. Latanoprost caused miosis, with rebound mydriasis at 24 h posttreatment, in the treated eyes of all cats. CONCLUSIONS: Further research is needed to determine the suitability and efficacy of latanoprost treatment for long-term IOP-lowering in cats with PCG or other forms of glaucoma.

Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I.

BACKGROUND: Treatment with intravenous enzyme replacement therapy and hematopoietic stem cell transplantation for mucopolysaccharidosis (MPS) type I does not address joint disease, resulting in persistent orthopedic complications and impaired quality of life. A proof-of-concept study was conducted to determine the safety, tolerability, and efficacy of intra-articular recombinant human iduronidase (IA-rhIDUA) enzyme replacement therapy in the canine MPS I model. METHODS: Four MPS I dogs underwent monthly rhIDUA injections (0.58 mg/joint) into the right elbow and knee for 6 months. Contralateral elbows and knees concurrently received normal saline. No intravenous rhIDUA therapy was administered. Monthly blood counts, chemistries, anti-rhIDUA antibody titers, and synovial fluid cell counts were measured. Lysosomal storage of synoviocytes and chondrocytes, synovial macrophages and plasma cells were scored at baseline and 1 month following the final injection. RESULTS: All injections were well-tolerated without adverse reactions. One animal required prednisone for spinal cord compression. There were no clinically significant abnormalities in blood counts or chemistries. Circulating anti-rhIDUA antibody titers gradually increased in all dogs except the prednisone-treated dog; plasma cells, which were absent in all baseline synovial specimens, were predominantly found in synovium of rhIDUA-treated joints at study-end. Lysosomal storage in synoviocytes and chondrocytes following 6 months of IA-rhIDUA demonstrated significant reduction compared to tissues at baseline, and saline-treated tissues at study-end. Mean joint synovial GAG levels in IA-rhIDUA joints were 8.62 ± 5.86 µg/mg dry weight and 21.6 ± 10.4 µg/mg dry weight in control joints (60% reduction). Cartilage heparan sulfate was also reduced in the IA-rhIDUA joints (113 ± 39.5 ng/g wet weight) compared to saline-treated joints (142 ± 56.4 ng/g wet weight). Synovial macrophage infiltration, which was present in all joints at baseline, was abolished in rhIDUA-treated joints only. CONCLUSIONS: Intra-articular rhIDUA is well-tolerated and safe in the canine MPS I animal model. Qualitative and quantitative assessments indicate that IA-rhIDUA successfully reduces tissue and cellular GAG storage in synovium and articular cartilage, including cartilage deep to the articular surface, and eliminates inflammatory macrophages from synovial tissue. CLINICAL RELEVANCE: The MPS I canine IA-rhIDUA results suggest that clinical studies should be performed to determine if IA-rhIDUA is a viable approach to ameliorating refractory orthopedic disease in human MPS I.

Neonatal gene therapy with a gamma retroviral vector in mucopolysaccharidosis VI cats.

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

Accelerated clinical disease and pathology in mucopolysaccharidosis type IIIB and GalNAc transferase double knockout mice.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a neuropathic lysosomal storage disorder (LSD) resulting from an inherited deficiency of N-acetyl-α-D-glucosaminidase (Naglu) activity, an enzyme required to degrade the glycosaminoglycan heparan sulfate (HS). A deficiency in Naglu activity leads to lysosomal accumulation of HS as a primary storage substrate, and the gangliosides GM2 and GM3 as secondary accumulation products. To test the effect on neuropathogenesis of ganglioside accumulation, we bred mice deficient in both Naglu and GalNaAcT activities. The latter is the enzyme required for synthesis of GM2 and other complex gangliosides. Contrary to our expectation and to double knockout (DKO) studies where GalNAcT was knocked out in combination with other LSDs, our DKO mice showed a drastically shortened lifespan (24.5±1.4 weeks, versus 50.5±0.9 weeks (MPS IIIB), and 38.6±1.2 weeks (GalNAcT)). To confirm that HS storage was the primary element resulting in the accelerated disease in our DKO mice, and not a locus tightly linked to the Naglu gene, we replicated our study with MPS IIIA mice, and found a virtually identical result (27.5±1.8 weeks, versus 53.8±1.6 weeks). All DKO mice showed motor signs of hind limb ataxia and hyper-extension, which were not seen in single KO or normal mice. At approximately 5 months of age, the MPS IIIB-DKO showed a unique pattern of vacuolization and nerve fiber degeneration in the corpus callosum, seen only in the DKO mice, as well as the relatively early intracytoplasmic vacuolation of many neurons and glia characteristic of the MPS IIIB mice. We analyzed motor performance on a rocking Rota-Rod beginning at 3 months of age. The MPS IIIA-DKO and MPS IIIB-DKO mice showed impaired performance and were statistically different from all parental lines. In particular, the MPS IIIB-DKO mice were significantly different from the parent MPS IIIB strains at 3, 5, and 6 months (p≤0.0245). In conclusion we identified an accelerated phenotype associated with MPS IIIB within a DKO model system which showed white matter changes, with attendant performance deficits and a drastically shortened lifespan. This was in stark contrast to our expectations of a salutary response to the elimination of GM2. Despite this, the accelerated pathology and clinical signs represent a potentially improved system to study MPS IIIB neuropathogenesis as well as the role of complex gangliosides in normal CNS function.

Specific antibody titer alters the effectiveness of intrathecal enzyme replacement therapy in canine mucopolysaccharidosis I.

Intrathecal enzyme replacement therapy is an experimental option to treat central nervous system disease due to lysosomal storage. Previous work shows that MPS I dogs receiving enzyme replacement with recombinant human alpha-l-iduronidase into the cisterna magna showed normal brain glycosaminoglycan (GAG) storage after three or four doses. We analyzed MPS I dogs that received intrathecal enzyme in a previous study using an assay that detects only pathologic GAG (pGAG). To quantify pGAG in MPS I, the assay measures only those GAG which display terminal iduronic acid residues on their non-reducing ends. Mean cortical brain pGAG in six untreated MPS I dogs was 60.9±5.93 pmol/mg wet weight, and was 3.83±2.64 in eight normal or unaffected carrier animals (p<0.001). Intrathecal enzyme replacement significantly reduced pGAG storage in all treated animals. Dogs with low anti-iduronidase antibody titers showed normalization or near-normalization of pGAG in the brain (mean 8.17±6.17, n=7), while in dogs with higher titers, pGAG was reduced but not normal (mean 21.9±6.02, n=4). Intrathecal enzyme therapy also led to a mean 69% reduction in cerebrospinal fluid pGAG (from 83.8±26.3 to 27.2±12.3 pmol/ml CSF). The effect was measurable one month after each dose and did not differ with antibody titer. Prevention of the immune response to enzyme may improve the efficacy of intrathecal enzyme replacement therapy for brain disease due to MPS I.

Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes.

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

Differential Transcriptomic Profiles Following Stimulation with Lipopolysaccharide in Intestinal Organoids from Dogs with Inflammatory Bowel Disease and Intestinal Mast Cell Tumor.

Lipopolysaccharide (LPS) is associated with chronic intestinal inflammation and promotes intestinal cancer progression in the gut. While the interplay between LPS and intestinal immune cells has been well-characterized, little is known about LPS and the intestinal epithelium interactions. In this study, we explored the differential effects of LPS on proliferation and the transcriptome in 3D enteroids/colonoids obtained from dogs with naturally occurring gastrointestinal (GI) diseases including inflammatory bowel disease (IBD) and intestinal mast cell tumor. The study objective was to analyze the LPS-induced modulation of signaling pathways involving the intestinal epithelia and contributing to colorectal cancer development in the context of an inflammatory (IBD) or a tumor microenvironment. While LPS incubation resulted in a pro-cancer gene expression pattern and stimulated proliferation of IBD enteroids and colonoids, downregulation of several cancer-associated genes such as Gpatch4, SLC7A1, ATP13A2, and TEX45 was also observed in tumor enteroids. Genes participating in porphyrin metabolism (CP), nucleocytoplasmic transport (EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) exhibited a similar pattern of altered expression between IBD enteroids and IBD colonoids following LPS stimulation. In contrast, genes involved in anion transport, transcription and translation, apoptotic processes, and regulation of adaptive immune responses showed the opposite expression patterns between IBD enteroids and colonoids following LPS treatment. In brief, the crosstalk between LPS/TLR4 signal transduction pathway and several metabolic pathways such as primary bile acid biosynthesis and secretion, peroxisome, renin-angiotensin system, glutathione metabolism, and arachidonic acid pathways may be important in driving chronic intestinal inflammation and intestinal carcinogenesis.

Timing is everything: Clinical courses of Hunter syndrome associated with age at initiation of therapy in a sibling pair.

Hunter syndrome, or mucopolysaccharidosis (MPS) II, is a rare lysosomal disorder characterized by progressive, multi-system disease. As most symptoms cannot be reversed once established, early detection and treatment prior to the onset of clinical symptoms are critical. However, it is difficult to identify affected individuals early in disease, and therefore the long-term outcomes of initiating treatment during this optimal time period are incompletely described. We report long-term clinical outcomes of treatment when initiated prior to obvious clinical signs by comparing the courses of two siblings with neuronopathic Hunter syndrome (c.1504 T > G[p.W502G]), one who was diagnosed due to clinical disease (Sibling-O, age 3.7 years) and the other who was diagnosed before disease was evident (Sibling-Y, age 12 months), due to his older sibling's findings. The brothers began enzyme replacement therapy within a month of diagnosis. Around the age of 5 years, Sibling-O had a cognitive measurement score in the impaired range of <55 (average range 85-115), whereas Sibling-Y at this age received a score of 91. Sibling-O has never achieved toilet training and needs direct assistance with toileting, dressing, and washing, while Sibling-Y is fully toilet-trained and requires less assistance with daily activities. Both siblings have demonstrated sensory-seeking behaviors, hyperactivity, impulsivity, and sleep difficulties; however, Sibling-O demonstrates physical behaviors that his brother does not, namely biting, pushing, and frequent elopement. Since the time of diagnosis, Sibling-O has had significant joint contractures and a steady deterioration in mobility leading to the need for an adaptive stroller at age 11, while Sibling-Y at age 10.5 could hike more than 6 miles without assistance. After nearly a decade of therapy, there were more severe and life-limiting disease manifestations for Sibling-O; data from caregiver interview indicated substantial differences in Quality of Life for the child and the family, dependent on timing of ERT. The findings from this sibling pair provide evidence of superior somatic and neurocognitive outcomes associated with presymptomatic treatment of Hunter syndrome, aligned with current considerations for newborn screening.

Arterial pathology in canine mucopolysaccharidosis-I and response to therapy.

Mucopolysaccharidosis-I (MPS-I) is an inherited deficiency of α-L-iduronidase (IdU) that causes lysosomal accumulation of glycosaminoglycans (GAG) in a variety of parenchymal cell types and connective tissues. The fundamental link between genetic mutation and tissue GAG accumulation is clear, but relatively little attention has been given to the morphology or pathogenesis of associated lesions, particularly those affecting the vascular system. The terminal parietal branches of the abdominal aorta were examined from a colony of dogs homozygous (MPS-I affected) or heterozygous (unaffected carrier) for an IdU mutation that eliminated all enzyme activity, and in affected animals treated with human recombinant IdU. High-resolution computed tomography showed that vascular wall thickenings occurred in affected animals near branch points, and associated with low endothelial shear stress. Histologically these asymmetric 'plaques' entailed extensive intimal thickening with disruption of the internal elastic lamina, occluding more than 50% of the vascular lumen in some cases. Immunohistochemistry was used to show that areas of sclerosis contained foamy (GAG laden) macrophages, fibroblasts and smooth muscle cells, with loss of overlying endothelial basement membrane and claudin-5 expression. Lesions contained scattered cells expressing nuclear factor-κß (p65), increased fibronectin and transforming growth factor ß-1 signaling (with nuclear Smad3 accumulation) in comparison to unaffected vessels. Intimal lesion development and morphology was improved by intravenous recombinant enzyme treatment, particularly with immune tolerance to this exogenous protein. The progressive sclerotic vasculopathy of MPS-I shares some morphological and molecular similarities to atherosclerosis, including formation in areas of low shear stress near branch points, and can be reduced or inhibited by intravenous administration of recombinant IdU.

Biodistribution and pharmacodynamics of recombinant human alpha-L-iduronidase (rhIDU) in mucopolysaccharidosis type I-affected cats following multiple intrathecal administrations.

The storage disorder mucopolysaccharidosis type I (MPS I) is caused by a deficiency in lysosomal α-L-iduronidase activity. The inability to degrade glycosaminoglycans (GAG) results in lysosomal accumulation and widespread tissue lesions. Many symptoms of MPS I are amenable to treatment with recombinant human α-L-iduronidase (rhIDU), however, peripherally administered rhIDU does not cross the blood-brain barrier and has no beneficial effects in the central nervous system (CNS). A feline model of MPS I was used to evaluate the CNS effects of rhIDU following repeated intrathecal (IT) administration. Twelve animals were randomized into four groups based on the time of euthanasia and tissue evaluation following three repeat IT administrations of 0.1 mg/kg rhIDU or placebo on Study Days 1, 4 or 5, and 9. Two days after the final IT injection, the mean tissue α-L-iduronidase (IDU) activity in the brains of the two treated animals were approximately 3-times higher (50.1 and 54.9 U/mg protein) than the activity found in normal cat brains (mean of 18.3 U/mg), and remained higher than untreated MPSI brain at 1 month (2.4 and 4.1 U/mg protein) before returning to near-baseline levels after 2 months. This activity corresponded with decreased brain GAG concentrations after 2 days (1.4 and 2.0 µg/mg) and 1 month (0.9 and 1.1 µg/mg) which approached levels observed in normal animals (0.7 µg/mg). Attenuation of GAG, gangliosides GM2 and GM3, and cholesterol reaccumulation was identified at both two days and one month following final IT injection. No adverse effects attributable to IT rhIDU administration were observed. IT rhIDU may be an effective means for providing enzyme replacement therapy for the central manifestations of MPS I.

Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I.

Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.

An exonic insertion in the NAGLU gene causing Mucopolysaccharidosis IIIB in Schipperke dogs.

Mucopolysaccharidosis (MPS) IIIB (Sanfilippo syndrome B; OMIM 252920), is a lysosomal storage disease with progressive neurological signs caused by deficient activity of alpha-N-acetylglucosaminidase (NAGLU, EC 3.2.1.50). Herein we report the causative variant in the NAGLU gene in Schipperke dogs and a genotyping survey in the breed. All six exons and adjacent regions of the NAGLU gene were sequenced from six healthy appearing and three affected Schipperkes. DNA fragment length and TaqMan assays were used to genotype privately owned Schipperkes. A single variant was found in exon 6 of MPS IIIB affected Schipperkes: an insertion consisting of a 40-70 bp poly-A and an 11 bp duplication of the exonic region preceding the poly-A (XM_548088.6:c.2110_2111ins[A(40_70);2100_2110]) is predicted to insert a stretch of 13 or more lysines followed by either an in-frame insertion of a repeat of the four amino acids preceding the lysines, or a frameshift. The clinically affected Schipperkes were homozygous for this insertion, and the sequenced healthy dogs were either heterozygous or homozygous for the wild-type allele. From 2003-2019, 3219 Schipperkes were genotyped. Of these, 1.5% were homozygous for this insertion and found to be clinically affected, and 23.6% were heterozygous for the insertion and were clinically healthy, the remaining 74.9% were homozygous for the wild-type and were also clinically healthy. The number of dogs homozygous and heterozygous for the insertion declined rapidly after the initial years of genotyping, documenting the benefit of a DNA screening program in a breed with a small gene pool. In conclusion, a causative NAGLU variant in Schipperke dogs with MPS IIIB was identified and was found at high frequency in the breed. Through genotyping and informed breeding practices, the prevalence of canine MPS IIIB has been drastically reduced in the Schipperke population worldwide.