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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 affected and 10 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.

Supplemental materials for the manuscript "Differential transcriptomic profiles following stimulation with lipopolysaccharide in intestinal organoids from dogs with inflammatory bowel disease and intestinal mast cell tumor" were submitted to Cancers.

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.

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.

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.

Recombinant human alpha-N-acetylglucosaminidase-insulin-like growth factor-2 (rhNAGLU-IGF2) is an investigational enzyme replacement therapy for Sanfilippo B, a lysosomal storage disease. Because recombinant human NAGLU (rhNAGLU) is poorly mannose 6-phosphorylated, we generated a fusion protein of NAGLU with IGF2 to permit its binding to the cation-independent mannose 6-phosphate receptor. We previously administered rhNAGLU-IGF2 intracerebroventricularly to Sanfilippo B mice, and demonstrated therapeutic restoration of NAGLU, normalization of lysosomal storage, and improvement in markers of neurodegeneration and inflammation. Here, we studied repeated intracerebroventricular rhNAGLU-IGF2 delivery in both murine and canine Sanfilippo B to determine potential effects on their behavioral phenotypes and survival. Treated mice showed improvement in disease markers such as heparan sulfate glycosaminoglycans, beta-hexosaminidase, microglial activation, and lysosomal-associated membrane protein-1. Sanfilippo B mice treated with rhNAGLU-IGF2 displayed partial normalization of their stretch attend postures, a defined fear pose in mice (p

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.

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.

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.

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.

Supplemental Material, Combined_supplemental_materials-Harm_et_al for Temporospatial Development of Neuropathologic Findings in a Canine Model of Mucopolysaccharidosis IIIB by Tyler A. Harm, Shannon J. Hostetter, Ariel S. Nenninger, Bethann N. Valentine, N. Matthew Ellinwood and Jodi D. Smith in Veterinary Pathology

Purpose We compared fractional anisotropy and radial diffusivity measurements between pediatric canines affected with mucopolysaccharidosis I and pediatric control canines. We hypothesized that lower fractional anisotropy and higher radial diffusivity values, consistent with dysmyelination, would be present in the mucopolysaccharidosis I cohort. Methods Six canine brains, three affected with mucopolysaccharidosis I and three unaffected, were euthanized at 7 weeks and imaged using a 7T small-animal magnetic resonance imaging system. Average fractional anisotropy and radial diffusivity values were calculated for four white-matter regions based on 100 regions of interest per region per specimen. A 95% confidence interval was calculated for each mean value. Results No difference was seen in fractional anisotropy or radial diffusivity values between mucopolysaccharidosis affected and unaffected brains in any region. In particular, the 95% confidence intervals for mucopolysaccharidosis affected and unaffected canines frequently overlapped for both fractional anisotropy and radial diffusivity measurements. In addition, in some brain regions a large range of fractional anisotropy and radial diffusivity values were seen within the same cohort. Conclusion The fractional anisotropy and radial diffusivity values of white matter did not differ between pediatric mucopolysaccharidosis affected canines and pediatric control canines. Possible explanations include: (a) a lack of white matter tissue differences between mucopolysaccharidosis affected and unaffected brains at early disease stages; (b) diffusion tensor imaging does not detect any existing differences; (c) inflammatory processes such as astrogliosis produce changes that offset the decreased fractional anisotropy values and increased radial diffusivity values that are expected in dysmyelination; and (d) our sample size was insufficient to detect differences. Further studies correlating diffusion tensor imaging findings to histology are warranted.

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.

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.

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.

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.

BackgroundLarge animal models, such as the dog, are increasingly being used over rodent models for studying naturally occurring diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between currently used animal models (e.g. mouse) 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.ResultsOrganoids were propagated from isolation of adult intestinal stem cells (ISC) from whole jejunal tissue as well as endoscopically obtained duodenal, ileal and colonic biopsy samples of healthy dogs and GI cases, including inflammatory bowel disease (IBD) and intestinal carcinomas. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization and transmission electron microscopy, and organoids mimicked the in vivo tissue environment. 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.ConclusionsIn summary, our findings establish the canine GI organoid systems as a novel model to study naturally occurring intestinal diseases in dogs and humans. Furthermore, canine organoid systems will help to elucidate host-pathogen interactions contributing to GI disease pathogenesis.

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.

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.

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

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 β-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 trans...

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management.

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.

The glaucomas are a group of diseases characterized by optic nerve damage that together represent a leading cause of blindness in the human population and in domestic animals. Here we report a mutation in LTBP2 that causes primary congenital glaucoma (PCG) in domestic cats. We identified a spontaneous form of PCG in cats and established a breeding colony segregating for PCG consistent with fully penetrant, autosomal recessive inheritance of the trait. Elevated intraocular pressure, globe enlargement and elongated ciliary processes were consistently observed in all affected cats by 8 weeks of age. Varying degrees of optic nerve damage resulted by 6 months of age. Although subtle lens zonular instability was a common feature in this cohort, pronounced ectopia lentis was identified in less than 10% of cats examined. Thus, glaucoma in this pedigree is attributed to histologically confirmed arrest in the early post-natal development of the aqueous humor outflow pathways in the anterior segment of the eyes of affected animals. Using a candidate gene approach, significant linkage was established on cat chromosome B3 (LOD 18.38, θ = 0.00) using tightly linked short tandem repeat (STR) loci to the candidate gene, LTBP2. A 4 base-pair insertion was identified in exon 8 of LTBP2 in affected individuals that generates a frame shift that completely alters the downstream open reading frame and eliminates functional domains. Thus, we describe the first spontaneous and highly penetrant non-rodent model of PCG identifying a valuable animal model for primary glaucoma that closely resembles the human disease, providing valuable insights into mechanisms underlying the disease and a valuable animal model for testing therapies.

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±1.4weeks, versus 50.5±0.9weeks (MPS IIIB), and 38.6±1.2weeks (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±1.8weeks, versus 53.8±1.6weeks). 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 5months 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 3months 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 6months (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.

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 per mg wet weight, and was 3.83 ± 2.64 in eight normal or unaffected carrier animals (p

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 MPS1 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 mcg/mg) and 1 month (0.9 and 1.1 mcg/mg) which approached levels observed in normal animals (0.7 mcg/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 or rhIDU antibody response 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.

Intrathecal (IT) recombinant human α-L-iduronidase (rhIDU) has been shown to reduce mean brain glycosaminoglycans (GAGs) to normal levels in MPS I dogs. In this study, we examined storage in neuroanatomical regions of the MPS I dog brain, including frontal lobe, cerebellum, basal ganglia, thalamus, hippocampal formation, and brainstem, to determine the response of these functional regions to treatment with IT rhIDU. GAG storage in untreated MPS I dogs was significantly different from normal dogs in all examined sections. GAG levels in normal dogs varied by region: frontal lobe (mean 2.36 ± 0.54 µg/mg protein), cerebellum (2.67 ± 0.33), basal ganglia and thalamus (3.51 ± 0.60), hippocampus (3.30 ± 0.40), and brainstem (3.73 ± 1.10). Following intrathecal treatment, there was a reduction in GAG storage in each region in all treatment groups, except for the brainstem. Percent reduction in GAG levels from untreated to treated MPS I dogs in the deeper regions of the brain was 30% for basal ganglia and thalamus and 30% for hippocampus, and storage reduction was greater in superficial regions, with 61% reduction in the frontal lobe and 54% in the cerebellum compared to untreated MPS I dogs. Secondary lipid storage in neurons was also reduced in frontal lobe, but not in the other brain regions examined. Response to therapy appeared to be greater in more superficial regions of the brain, particularly in the frontal lobe cortex.

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 demonstrate 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 NF-κβ (p65), increased fibronectin and transforming growth factor β-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 morphologic 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.

The editors of this issue have assembled an exemplary group of expert reviews that provide an effective overview of the current use of large animal models in genetic disease research and describe the importance of these models in bringing new, safe, and effi cacious treatments to clinical practice. In this essay we focus on both practical and technical considerations for the conduct of this type of research from the standpoint of those who prepare and review institutional animal care and use committee (IACUC) protocols. We discuss the history of these types of research subjects and practical considerations for the use of these models. As IACUC membership often varies in expertise, we spend some time on issues that may seem obvious to the expert but that may not be readily apparent to nonscientist IACUC members. Many of our observations are based on our own and our colleagues’ experiences in the preparation and review of protocols, refl ecting common questions or themes raised during the review process. We do not address issues relevant to individual species but rather the general considerations, including the advantages and constraints, of maintaining and using these large animal models for the study of genetic disease.

Severe mucopolysaccharidosis type I (MPS I) is a fatal neuropathic lysosomal storage disorder with significant skeletal involvement. Treatment involves bone marrow transplantation (BMT), and although effective, is suboptimal, due to treatment sequelae and residual disease. Improved approaches will need to be tested in animal models and compared to BMT. Herein we report on bone marrow transplantation to treat feline mucopolysaccharidosis I (MPS I). Five MPS I stably engrafted kittens, transplanted with unfractionated bone marrow (6.3x10(7)-1.1x10(9) nucleated bone marrow cells per kilogram) were monitored for 13-37 months post-engraftment. The tissue total glycosaminoglycan (GAG) content was reduced to normal levels in liver, spleen, kidney, heart muscle, lung, and thyroid. Aorta GAG content was between normal and affected levels. Treated cats had a significant decrease in the brain GAG levels relative to untreated MPS I cats and a paradoxical decrease relative to normal cats. The alpha-l-iduronidase (IDUA) activity in the livers and spleens of transplanted MPS I cats approached heterozygote levels. In kidney cortex, aorta, heart muscle, and cerebrum, there were decreases in GAG without significant increases in detectable IDUA activity. Treated animals had improved mobility and decreased radiographic signs of disease. However, significant pathology remained, especially in the cervical spine. Corneal clouding appeared improved in some animals. Immunohistochemical and biochemical analysis documented decreased central nervous system ganglioside storage. This large animal MPS I study will serve as a benchmark of future therapies designed to improve on BMT.

OBJECTIVE To determine whether 2- or 3-times-daily application of topical ophthalmic 0.005% latanoprost solution is more effective at lowering intraocular pressure (IOP) in clinically normal dogs. ANIMALS 9 clinically normal dogs. PROCEDURES For each dog, I drop of latanoprost 0.005% solution was applied to 1 eye every 8 or 12 hours each day for 5 days; the contralateral eye received topical ophthalmic treatment with 1 drop of saline (0.9% NaCl) solution at the times of latanoprost application. Ocular examinations of both eyes were performed every 6 hours starting 48 hours prior to and ending 42 hours after the treatment period. Following a 5-week washout interval, the procedures were repeated but the previously latanoprost-treated eye of each dog received latanoprost application at the alternate frequency. RESULTS Mean ± SD IOP reduction in the latanoprost-treated eyes was 31 ± 6.9% with 2-times-daily application and 33 ± 8.2% with 3-times-daily application. A 2-way repeated-measures ANOVA revealed significant differences in IOP with contributions by treatment (2 or 3 times daily), time of day (diurnal variation), and individual dog. The maximum mean daily IOP reduction in latanoprost-treated eyes was detected on day 3 of latanoprost treatment in each group. Eyes treated 3 times daily had significantly smaller pupil diameter and greater conjunctival hyperemia than eyes treated 2 times daily. CONCLUSIONS AND CLINICAL RELEVANCE The clinical importance of the ocular hypotensive effects of 3-times-daily topical ophthalmic application of 0.005% latanoprost solution in dogs with glaucoma warrants investigation.

There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by beta-glucuronidase (GUSB) deficiency. Intravenous injection of a retroviral vector expressing canine GUSB into neonatal MPS VII mice resulted in transduction of 6 to 35% of hepatocytes, which secreted GUSB into blood. Serum GUSB activity was stable for 6 months at 600 (low expression) to 10,000 (high expression) U/ml, and enzyme was modified appropriately with mannose 6-phosphate. The average serum GUSB activity (3531 U/ml) is the highest long-term expression reported for MPS VII mice after gene therapy. Secreted enzyme was taken up by other tissues, as the average enzyme activity was13% of normal in somatic organs and 2% of normal in brain. Low expression markedly reduced histopathological evidence of lysosomal storage in liver, spleen, kidney, small intestine, neurons, and glial cells. High expression appeared to be more effective than low expression at reducing lysosomal storage in aorta, heart valves, thymus, bronchial epithelium, cornea, and retinal pigmented epithelium. Future experiments will determine if greater pathological improvements will consistently be observed in retrovirus-treated MPS VII mice with higher serum GUSB activity relative to animals with lower activity and if these result in clinical benefits.