The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

SummaryNitric oxide (NO) is emerging as an important signalling molecule with diverse physiological functions in plants. In the current study, changes in gene expression in response to 0.1 m M and 1.0 m M sodium nitroprusside (SNP), a donor of NO, were studied in Arabidopsis using the whole genome ATH1 microarray, representing over 24 000 genes. We observed 342 up-regulated and 80 down-regulated genes in response to NO treatments. These included 126 novel genes with unknown functions. Most of these changes were specific to NO treatment, as we observed a reverse trend when the plants were treated with NO scavenger,

Duchenne muscular dystrophy (DMD) is a dystrophin-deficient lethal muscle disease. To date, the catastrophic muscle wasting phenotype has only been seen in dystrophin-deficient humans and dogs. While Duchenne-like symptoms have been observed in more than a dozen dog breeds, the mutation is often not known and research colonies are rarely established. Here we report an independent canine DMD model originally derived from the Pembroke Welsh corgi breed. The affected dogs presented clinical signs of muscular dystrophy. Immunostaining revealed the absence of dystrophin and up-regulation of utrophin. Histopathologic examination showed variable fiber size, central nucleation, calcification, fibrosis, neutrophil and macrophage infiltration and cardiac focal vacuolar degeneration. Carrier dogs also displayed mild myopathy. The mutation was identified as a long interspersed repetitive element-1 (LINE-1) insertion in intron 13 which introduced a new exon containing an in-frame stop codon. Similar mutations have been seen in human patients. A colony was generated by crossing carrier females with normal males. Affected puppies had a normal birth weight but they experienced a striking growth delay in the first 5 days. In summary, the new corgi DMD model offers an excellent opportunity to study DMD pathogenesis and to develop novel therapies.

Oncolytic adenoviruses, which selectively replicate in and subsequently kill cancer cells, have emerged as a promising approach for treatment of tumors resistant to other modalities. Although preclinical results have been exciting, single-agent clinical efficacy has been less impressive heretofore. The immunogenicity of adenoviruses, and consequent premature abrogation of replication, may have been a partial reason. Improving the oncolytic potency of agents has been hampered by the inability to study host-vector interactions in immune-competent systems, since human serotype adenoviruses do not productively replicate in animal tissues. Therefore, approaches such as immunomodulation, which could result in sustained replication and subsequently increased oncolysis, have not been studied. Utilizing the osteocalcin promoter for restricting the replication of a canine adenovirus to dog osteosarcoma cells, we generated and tested the first nonhuman oncolytic adenovirus. This virus effectively killed canine osteosarcoma cells in vitro and yielded a therapeutic benefit in vivo. Canine osteosarcoma is the most frequent malignant disease in large dogs, with over 8000 cases in the United States annually, and there is no curative treatment. Therefore, immunomodulation for increased oncolytic potency could be studied with clinical trials in this population. This could eventually translate into human trials.

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Highly abbreviated micro-dystrophin genes have been intensively studied for Duchenne muscular dystrophy (DMD) gene therapy. Following adeno-associated virus (AAV) gene transfer, robust microgene expression is achieved in murine DMD models in the absence of immune suppression. Interestingly, a recent study suggests that AAV gene transfer in dystrophic dogs may require up to 18 weeks' immune suppression using a combination of three different immune-suppressive drugs (cyclosporine, mycophenolate mofetil, and anti-dog thymocyte globulin). Continued immune suppression is not only costly but also may cause untoward reactions. Further, some of the drugs (such as anti-dog thymocyte globulin) are not readily available. To overcome these limitations, we developed a novel 5-week immune suppression scheme using only cyclosporine and mycophenolate mofetil. AAV vectors (either AV.RSV.AP that expresses the heat-resistant human alkaline phosphatase gene, or AV.CMV.lDys that expresses the canine R16-17/H3/DC microgene) at 2.85 · 10 12 vg particles were injected into adult dystrophic dog limb muscles under the new immune suppression protocol. Sustained transduction was observed for nearly half year (the end of the study). The simplified immune suppression strategy described here may facilitate preclinical studies in the dog model.

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G M1 gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal β-D-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G M1 ganglioside. To characterize the genetic mutation responsible for feline G M1 gangliosidosis, the normal sequence of feline β-galactosidase cDNA first was defined. The feline β-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the β-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human β-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal β-galactosidase was sequenced, the mutation responsible for feline G M1 gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G M1 gangliosidosis in humans. Feline β-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline β-galactosidase cDNA restored activity in transfected G M1 fibroblasts to 18-times normal. β-Galactosidase protein levels in G M1 tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant β-galactosidase protein did not reach the lysosome. Additionally, G M1 cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G M1 gangliosidosis. CIHR Author Manuscript CIHR Author Manuscript CIHR Author ManuscriptThe lysosomal enzyme β-D-galactosidase (βgal, EC 3.2.1.23) cleaves terminal galactose residues from a variety of molecules, including gangliosides G A1 and G M1 . Deficiency of βgal is known to cause two lysosomal storage diseases: G M1 gangliosidosis (neuronopathic) and Morquio B Disease (mucopolysaccharidosis IVB, non-neuronopathic [3,4]. Although the in vivo biochemical effect of the Arg482His mutation often is difficult to discern because it occurs most frequently in compound heterozygotes, patients homozygous for the G1445A substitution present with the infantile (most severe) form of G M1 gangliosidosis [3,7,8]. A similar mutation, Arg482Cys, also produced no residual βgal activity after expression in G M1 gangliosidosis fibroblasts [4].Feline G M1 gangliosidosis, first described in a Siamese cat in 1971 [9], models the juvenile form of the human disease. Onset of clinical neurological disease in affected cats occurs at approximately 3.5 months of age with a fine head or limb tremor. G M1 mutant cats have progressive dysmetria and ambulatory difficulties, with blindness and epileptiform seizures in the terminal disease stage at 9-10 months of age. In the current st...