Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Summary Objective: The purpose of this study was to define the cardiovascular abnormalities present in young and adult cats affected with the lysosomal storage diseases mucopolysaccharidosis (MPS) I and MPS VI. Method: Eighteen cats affected with MPS I and 10 cats affected with MPS VI were evaluated by physical examination, electrocardiography and echocardiography. Electrocardiography (ECG) was performed on all MPS I and 9 of the MPS VI cats. Twelve unaffected cats underwent complete examinations for comparison purposes. Results: No cardiovascular abnormalities were noted on physical examination. Measured ECG intervals were normal in affected cats; however, sinus arrhythmia was noted more frequently than in the unaffected cats. Significant echocardiographic abnormalities included aortic valve thickening, regurgitation and aortic root dilation. Significant mitral valve thickening was also noted. The severity of changes increased in older affected cats. Conclusion: As affected animals increased in age, more cardiac abnormalities were found with increasing severity. Significant lesions included the mitral and aortic valves and ascending aorta, but myocardial changes were not recognized. MPS I and MPS VI cats have similar cardiovascular findings to those seen in children and constitute important models for testing new MPS therapies.

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We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (γc) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34+ bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting ∼75% of Cf2Th cells. An average of 11% of highly enriched canine CD34+ cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine γc cDNA produced a γc protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34+ BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Mucopolysaccharidosis III (MPS III) is characterized by lysosomal accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). In humans, the disease manifests in early childhood, and is characterized by a progressive central neuropathy leading to death in the second decade. This disease has also been described in mice (MPS IIIA and IIIB), dogs (MPS IIIA), emus (MPS IIIB) and goats (MPS IIID). We now report on dogs with naturally occurring MPS IIIB, detailing the clinical signs, diagnosis, histopathology, tissue enzymology and substrate levels. Two 3-year-old Schipperke dogs were evaluated for tremors and episodes of stumbling. Examination of the animals found signs consistent with cerebellar disease including dysmetria, hind limb ataxia and a wide-based stance with truncal swaying. There were mildly dystrophic corneas and small peripheral foci of retinal degeneration. Magnetic resonance imaging of the brain and skeletal radiographs were normal. Intracytoplasmic granules were found in the white cells of peripheral blood and cerebral spinal fluid, and in myeloid lineages in bone marrow. Electrophoresis of urinary GAGs indicated the presence of HS, while assays of cultured fibroblasts found N -acetyl-α-D-glucosaminidase (Naglu) activity of between 4.3% and 9.2% of normal. Owing to neurological deterioration, both dogs were euthanized, and post-mortem examinations were performed. Biochemical studies of liver and kidney from both animals demonstrated profound deficiency of Naglu activity and abnormally high GAG levels. Pathology of the brain included severe cerebellar atrophy, Purkinje cell loss, and cytoplasmic vacuolation in neurons and perithelial cells throughout the central nervous system. Pedigree analyses and Naglu levels of family members supported an autosomal recessive mode of inheritance. Using an obligate heterozygote, a breeding colony has been established to aid in understanding the pathogenesis of MPS IIIB and testing of potential therapies.

Mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease, is one of the more prevalent inherited diseases in cats and is commonly found in cats with Siamese ancestry. The prevalence of 2 known MPS VI mutations in cats was investigated in 101 clinically normal Siamese cats, in 2 cats with clinical signs of MPS VI, and in 202 cats from 4 research colonies. The mutation L476P which causes a severe clinical phenotype, was present on both alleles in the known MPS VI cats from Italy and North America and was present in all research colonies that originated from North America. However, LA76P was not detected in the Siamese population screened. In contrast, the mutation D520N, which causes a mild clinical phenotype, was identified in 23 of 202 (11.4%) alleles tested in Siamese cats from 3 continents, 2 of which were homozygous for D520N. Thus, the D520N mutation was widespread, and it is likely that cats inheriting both mutations (LA76P/D520N compound heterozygotes) would be in the general Siamese population, particularly in North America. Practitioners should note the high incidence of degenerative joint disease in these animals.

Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is an autosomal recessive lysosomal storage disorder resulting from the deficiency in the activity of the enzyme beta-glucuronidase (GUSB). To characterize the biochemical and molecular defect in GUSB-deficient MPS VII dogs, we have measured lysosomal enzyme activities, analyzed distribution of glycosaminoglycans (GAGs), and estimated the size and abundance of the GUSB gene product at the mRNA and protein level in normal, homozygous affected, and heterozygous carrier retinal pigment epithelium (RPE) samples. Compared to normal, only 2-5% and 40-60% of GUSB activity was detected in the affected and the carrier samples, respectively. The decrease in GUSB activity resulted in storage of GAGs predominantly heparan sulfate and chondroitin sulfate. A slight increase in storage of GAGs was also observed in the carrier sample. Northern blot analysis of affected and carrier RPE samples detected a 2.4 kb GUSB transcript similar in size and abundance to that of normal controls. In western blot analysis using anti-human GUSB antibody, three bands of size 78, 56, and 38 kDa were detected in normal samples, which were present at lower intensity in the carrier RPE samples and absent in the MPS VII-affected RPE samples. These results suggest that the mutant GUSB gene causes a posttranscriptional defect and produces an unstable protein.

Autologous transplantation of retrovirally transduced bone marrow (BM) or neonatal blood cells was carried out on eight cats (ranging in age from 2 weeks to 12 months) with mucopolysaccharidosis type VI (MPS VI). The transducing vector contained the full-length cDNA encoding human arylsulfatase B (hASB), the enzymatic activity deficient in this lysosomal storage disorder. Following transplantation, the persistence of transduced cells and enzymatic expression were monitored for more than 2 years. Five of the cats received no myeloablative preconditioning, two cats received 370–390 cGy of total body irradiation (TBI), and one cat received 190 cGy TBI. Evidence of transduced cells, as judged by enzymatic activity and PCR detection of the provirus, was demonstrated in granulocytes, lymphocytes, or BM cells of the treated animals up to 31 months after transplantation. Radiation preconditioning was not required to achieve these results, nor were they dependent on the recipient’s age. However, despite the long-term persistence of transduced cells, the levels of ASB activity in the transplanted animals was low, and no clinical improvements were detected. These data provide evidence for the long-term persistence of retrovirally transduced feline hematopoietic cells, and further documentation that engraftment of transduced cells can be achieved in the absence of myeloablation. Consistent with previous bone marrow transplantation studies, these results also suggest that to achieve clinical improvement of MPS VI, particularly in the skeletal system, high-level expression of ASB must be achieved in the treated animals and improved techniques for targeting the expressed enzyme to specific sites of pathology (eg chondrocytes) must be developed.

OBJECTIVE To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus. SAMPLE POPULATION Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPSVII. PROCEDURE The canine beta-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPSVII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles. RESULTS Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme. CONCLUSIONS The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS VII-carrier dogs from homozygous normal dogs.

Morphological changes in neurons with inborn defects of the lysosomal hydrolase, α-l-iduronidase, and with concomitant storage of glycosaminoglycans, were evaluated by Golgi staining in two animal models and compared to a similar study of a child with the same disease. Cortical pyramidal neurons in feline mucopolysaccharidosis type I often displayed axon hillock enlargements (meganeurites) and/or ectopic, secondary neuritic processes sprouting from this same region of the cell. The latter structures were prominent and often appeared longer than similar neurites reported in other neuronal storage discases. Although most meganeurites were aspiny, a few were observed which possessed spine-like processes or neurites. Other than these morphological changes in cortical pyramidal neurons, few other cell types displayed abnormalities demonstrable by Golgi impregnation. In the canine model of this disorder, abnormal Golgi-impregnated cortical neurons resembled more closely those seen in human mucopolysaccharidosis. That is, they possessed meganeurites which typically were aspiny in appearance. Ectopic neurite growth was not observed on any Golgi-impregnated neurons in the cases of canine or human mucopolysaccharidosis used in this study. The latter finding given the advanced ages of these cases, is consistent with the view that ectopic neuritogenesis seen in neuronal storage diseases may be subject to a developmental window, albeit one open well beyond the period of early postnatal maturation.

Alpha-mannosidosis, an inherited enzyme-deficiency disease, caused growth retardation, hepatomegaly, ocular abnormalities, and neurologic dysfunction in a Persian cat. Because of the clinical progression of this autosomal recessive, lysosomal storage disorder, the cat was euthanatized at 5 months of age.

Transplantation reussie dans 2 cas: donneur chat mâle, receveur chatte sœur, irradiee, dans les 2 cas les cultures mixtes de lymphocytes etaient negatives. Le chimerisme est verifie par le karyotype XY et par le polymorphisme d'arylsulfatase B

Nine XX true hermaphrodites and two XX males were discovered in a family of American cocker spaniels. The true hermaphrodites were partially-masculinized females with ovotestes; the XX males had malformed male external genitalia and cryptorchid aspermatogenic testes. Wolffian and Mullerian duct derivatives were present in both true hermaphrodites and XX males. All four sires of sex-reversed dogs were normal XY males; five of the dams were anatomically normal females and one was an XX true hermaphrodite. A second true hermaphrodite reproduced as a female, producing anatomically normal offspring.