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.

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.

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.

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.

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.

Newborn Screening for Mucopolysaccharidosis I: Moving Forward Learning from Experience.

There have been significant advances allowing for the integration of mucopolysaccharidosis I into newborn screening programs. Initial experiences using a single-tier approach for this disorder have highlighted shortcomings that require immediate remediation. The recent evaluation of a second-tier biomarker integrated into the MPS I newborn screening protocol has been demonstrated to greatly improve the precision and predictive value of newborn screening for this disorder. This commentary urges newborn screening programs to learn from these experiences and improve newborn screening for mucopolysaccharidosis I and future mucopolysaccharidoses newborn screening programs by implementation of a second-tier biomarker analyte.

Central nervous system pathology in preclinical MPS IIIB dogs reveals progressive changes in clinically relevant brain regions.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

Sub-region-Specific Optic Nerve Head Glial Activation in Glaucoma.

Glaucoma, a multifactorial neurodegenerative disease characterized by progressive loss of retinal ganglion cells and their axons in the optic nerve, is a leading cause of irreversible vision loss. Intraocular pressure (IOP) is a risk factor for axonal damage, which initially occurs at the optic nerve head (ONH). Complex cellular and molecular mechanisms involved in the pathogenesis of glaucomatous optic neuropathy remain unclear. Here we define early molecular events in the ONH in an inherited large animal glaucoma model in which ONH structure resembles that of humans. Gene expression profiling of ONH tissues from rigorously phenotyped feline subjects with early-stage glaucoma and precisely age-matched controls was performed by RNA-sequencing (RNA-seq) analysis and complementary bioinformatic approaches applied to identify molecular processes and pathways of interest. Immunolabeling supported RNA-seq findings while providing cell-, region-, and disease stage-specific context in the ONH in situ. Transcriptomic evidence for cell proliferation and immune/inflammatory responses is identifiable in early glaucoma, soon after IOP elevation and prior to morphologically detectable axon loss, in this large animal model. In particular, proliferation of microglia and oligodendrocyte precursor cells is a prominent feature of early-stage, but not chronic, glaucoma. ONH microgliosis is a consistent hallmark in both early and chronic stages of glaucoma. Molecular pathways and cell type-specific responses strongly implicate toll-like receptor and NF-κB signaling in early glaucoma pathophysiology. The current study provides critical insights into molecular pathways, highly dependent on cell type and sub-region in the ONH even prior to irreversible axon degeneration in glaucoma.

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.

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.

In-vivo cortical thickness estimation from high-resolution T1w MRI scans in healthy and mucopolysaccharidosis affected dogs.

Cortical thickness measurement estimated from high-resolution anatomical MRI scans may serve as a marker of cortical atrophy in clinical research applications. Most of the working algorithms and pipelines are optimized for human in-vivo data analyses that offer robust and reproducible measures. As animal-models are widely utilized in many preclinical phases of clinical trials the need for an optimized automated MRI data analysis to yield reliable data is warranted. We present a processing pipeline optimized for cortical thickness estimation of canine brains in native and template spaces. Preliminary results of 5 healthy and 5 mucopolysaccharidosis (MPS) dogs demonstrate single-canine mean/median cortical thickness in range of 2.69-3.58mm in native space and 3.26-4.15mm in template space. Our MRI generated values exceed previous histological measurements (observed mean about 2mm) in limited literature reports. Randomly selected manual measures corroborated the ranges defined by estimated cortical thickness probability density functions. Geometric transformations between native and template spaces change absolute mean/median cortical thickness values, but do not change the data nature and properties since the Pearson correlation coefficients between different space estimates were 0.84 for mean values and 0.89 for median values. No significant difference in total cortical thickness between MPS and age-and gender-matched dogs was observed.

Creating effective biocontainment facilities and maintenance protocols for raising specific pathogen-free, severe combined immunodeficient (SCID) pigs.

Severe combined immunodeficiency (SCID) is defined by the lack of an adaptive immune system. Mutations causing SCID are found naturally in humans, mice, horses, dogs, and recently in pigs, with the serendipitous discovery of the Iowa State University SCID pigs. As research models, SCID animals are naturally tolerant of xenotransplantation and offer valuable insight into research areas such as regenerative medicine, cancer therapy, as well as immune cell signaling mechanisms. Large-animal biomedical models, particularly pigs, are increasingly essential to advance the efficacy and safety of novel regenerative therapies on human disease. Thus, there is a need to create practical approaches to maintain hygienic severe immunocompromised porcine models for exploratory medical research. Such research often requires stable genetic lines for replication and survival of healthy SCID animals for months post-treatment. A further hurdle in the development of the ISU SCID pig as a biomedical model involved the establishment of facilities and protocols necessary to obtain clean SPF piglets from the conventional pig farm on which they were discovered. A colony of homozygous SCID boars and SPF carrier sows has been created and maintained through selective breeding, bone marrow transplants, innovative husbandry techniques, and the development of biocontainment facilities.

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.

Diffusion tensor imaging findings suggestive of white matter alterations in a canine model of mucopolysaccharidosis type I.

Purpose We investigated fractional anisotropy (FA) and radial diffusivity (RD) in a canine model of mucopolysaccharidosis (MPS). We hypothesized that canines affected with MPS would exhibit decreased FA and increased RD values when compared to unaffected canines, a trend that has been previously described in humans with white matter diseases. Methods Four unaffected canines and two canines with MPS were euthanized at 18 weeks of age. Their brains were imaged using high-resolution diffusion tensor imaging (DTI) on a 7T small-animal magnetic resonance imaging system. One hundred regions of interest (ROIs) were placed in each of four white matter regions: anterior and posterior regions of the internal capsule (AIC and PIC, respectively) and anterior and posterior regions of the centrum semiovale (ACS and PCS, respectively). For each specimen, average FA and RD values and associated 95% confidence intervals were calculated from 100 ROIs for each brain region. Results For each brain region, the FA values in MPS brains were consistently lower than in unaffected dogs, and the RD values in MPS dogs were consistently higher, supporting our hypothesis. The confidence intervals for affected and unaffected canines did not overlap in any brain region. Conclusion FA and RD values followed the predicted trend in canines affected with MPS, a trend that has been described in humans with lysosomal storage and dysmyelinating diseases. These findings suggest that the canine model parallels MPS in humans, and further indicates that quantitative DTI analysis of such animals may be suitable for future study of disease progression and therapeutic response in MPS.

Novel region of interest interrogation technique for diffusion tensor imaging analysis in the canine brain.

Purpose We describe a novel technique for measuring diffusion tensor imaging metrics in the canine brain. We hypothesized that a standard method for region of interest placement could be developed that is highly reproducible, with less than 10% difference in measurements between raters. Methods Two sets of canine brains (three seven-week-old full-brains and two 17-week-old single hemispheres) were scanned ex-vivo on a 7T small-animal magnetic resonance imaging system. Strict region of interest placement criteria were developed and then used by two raters to independently measure diffusion tensor imaging metrics within four different white-matter regions within each specimen. Average values of fractional anisotropy, radial diffusivity, and the three eigenvalues (λ1, λ2, and λ3) within each region in each specimen overall and within each individual image slice were compared between raters by calculating the percentage difference between raters for each metric. Results The mean percentage difference between raters for all diffusion tensor imaging metrics when pooled by each region and specimen was 1.44% (range: 0.01-5.17%). The mean percentage difference between raters for all diffusion tensor imaging metrics when compared by individual image slice was 2.23% (range: 0.75-4.58%) per hemisphere. Conclusion Our results indicate that the technique described is highly reproducible, even when applied to canine specimens of differing age, morphology, and image resolution. We propose this technique for future studies of diffusion tensor imaging analysis in canine brains and for cross-sectional and longitudinal studies of canine brain models of human central nervous system disease.

Diffusion tensor imaging tensor shape analysis for assessment of regional white matter differences.

Purpose The purpose of this study was to investigate a novel tensor shape plot analysis technique of diffusion tensor imaging data as a means to assess microstructural differences in brain tissue. We hypothesized that this technique could distinguish white matter regions with different microstructural compositions. Methods Three normal canines were euthanized at seven weeks old. Their brains were imaged using identical diffusion tensor imaging protocols on a 7T small-animal magnetic resonance imaging system. We examined two white matter regions, the internal capsule and the centrum semiovale, each subdivided into an anterior and posterior region. We placed 100 regions of interest in each of the four brain regions. Eigenvalues for each region of interest triangulated onto tensor shape plots as the weighted average of three shape metrics at the plot's vertices: CS, CL, and CP. Results The distribution of data on the plots for the internal capsule differed markedly from the centrum semiovale data, thus confirming our hypothesis. Furthermore, data for the internal capsule were distributed in a relatively tight cluster, possibly reflecting the compact and parallel nature of its fibers, while data for the centrum semiovale were more widely distributed, consistent with the less compact and often crossing pattern of its fibers. This indicates that the tensor shape plot technique can depict data in similar regions as being alike. Conclusion Tensor shape plots successfully depicted differences in tissue microstructure and reflected the microstructure of individual brain regions. This proof of principle study suggests that if our findings are reproduced in larger samples, including abnormal white matter states, the technique may be useful in assessment of white matter diseases.

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease.

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and ß-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl3) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 µg/mL). It is concluded that GdCl3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

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.

Diffusion tensor imaging analysis of the brain in the canine model of Krabbe disease.

PURPOSE: The goal of this study was to compare the diffusion tensor imaging (DTI) metrics from an end-stage canine Krabbe brain evaluated by MR imaging ex vivo to those of a normal dog brain. We hypothesized that the white matter of the canine Krabbe brain would show decreased fractional anisotropy (FA) values and increased apparent diffusion coefficient (ADC) and radial diffusivity (RD) values. METHODS: An 11-week-old Krabbe dog was euthanized after disease progression. The brain was removed and was placed in a solution of 10% formalin. MR imaging was performed and compared to the brain images of a normal dog that was similarly fixed post-mortem. Both brains were scanned using similar protocols on a 7 T small-animal MRI system. For each brain, maps of ADC, FA, and RD were calculated for 11 white-matter regions and five control gray-matter regions. RESULTS: Large decreases in FA values, increases in ADC values, and increases in RD (consistent with demyelination) values, were seen in white matter of the Krabbe brain but not gray matter. ADC values in gray matter of the Krabbe brain were decreased by approximately 29% but increased by approximately 3.6% in white matter of the Krabbe brain. FA values in gray matter were decreased by approximately 3.3% but decreased by approximately 29% in white matter. RD values were decreased by approximately 27.2% in gray matter but increased by approximately 20% in white matter. CONCLUSION: We found substantial abnormalities of FA, ADC, and RD values in an ex vivo canine Krabbe brain.