Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.     

Multispecies Epidemiologic Surveillance Study after an Outbreak of Yersiniosis at an African Green Monkey Research Facility

After an outbreak of Yersinia enterocolitica at a NHP research facility, we performed a multispecies investigation of the prevalence of Yersinia spp. in various mammals that resided or foraged on the grounds of the facility, to better understand the epizootiology of yersiniosis. Blood samples and fecal and rectal swabs were obtained from 105 captive African green monkeys (AGM), 12 feral cats, 2 dogs, 20 mice, 12 rats, and 3 mongooses. Total DNA extracted from swab suspensions served as template for the detection of Y. enterocolitica DNA by real-time PCR. Neither Y. enterocolitica organisms nor their DNA were detected from any of these samples. However, Western blotting revealed the presence of Yersinia antibodies in plasma. The AGM samples revealed a seroprevalence of 91% for Yersinia spp. and of 61% for Y. enterocolitica specifically. The AGM that were housed in cages where at least one fatality occurred during the outbreak (clinical group) had similar seroprevalence to that of AGM housed in unaffected cages (nonclinical group). However, the nonclinical group was older than the clinical group. In addition, 25%, 100%, 33%, 10%, and 10% of the sampled local cats, dogs, mongooses, rats, and mice, respectively, were seropositive. The high seroprevalence after this outbreak suggests that Y. enterocolitica was transmitted effectively through the captive AGM population and that age was an important risk factor for disease. Knowledge regarding local environmental sources of Y. enterocolitica and the possible role of wildlife in the maintenance of yersiniosis is necessary to prevent and manage this disease.Abbreviations: AGM, African green monkeyYersinia enterocolitica is a zoonotic, gram-negative member of the family Enterobacteriaceae and the causative agent of mesenteric lymphadenitis, terminal ileitis, acute gastroenteritis, and septicemia in domestic animals, wildlife, and primates. The bacterium has a very broad host range and has been detected in more than 110 species of animals worldwide, including mammals, birds, and reptiles.^3,4,19 People of all ages can become infected with pathogenic strains of Y. enterocolitica. Clinical illness is more frequent in children and young adults, with asymptomatic infection being more common in adults.²³ Latent infection by Y. enterocolitica occurs in free-living wild rodents, which excrete the organism in their feces.^6,14 Contaminated food and water are common vehicles for the transmission of this pathogen.^5,10Y. enterocolita presents high antigenic variability. There are approximately 34 O-antigen and 20 H-antigen serogroups.¹² In primates, serotypes O3, O5/27, and O9 have relatively low pathogenicity, mainly causing diarrhea, but serotype O8 is highly pathogenic and can cause septicemia.^11,24 NHP appear to be quite susceptible to infection with Y. enterocolitica, and many fatal cases of yersiniosis have been reported worldwide.^3,4,16,24,26In 2012, Y. enterocolitica was identified as the causative agent of an outbreak in captive African green monkeys (AGM; Chlorocebus sabaeus) on the island of St Kitts, West Indies, where approximately 4% of AGM in a local research facility died. Affected AGM presented with mucohemorrhagic diarrhea, marked dehydration, lethargy, and depression, often followed by death. Samples of the spleen, liver and lungs of affected monkeys yielded 15 bacterial isolates, all of which were identified as Y. enterocolitica by biochemical analysis and sequence comparison of the 16S rRNA gene. Phenotypic and genotypic analysis of the recovered isolates revealed homogeneity among the recovered bacteria, and all isolates gave a random amplified polymorphic DNA pattern resembling that of genotype D in serotypes O:7,8.²⁷The objectives of the current study were to: 1) describe the prevalence of yersiniosis in AGM, dogs (Canis lupus familaris), cats (Felis catus), and peridomestic wildlife including small Asian mongooses (Herpestes javanicus), mice (Mus spp.) and rats (Rattus norvegicus) that resided or foraged within the perimeter of the NHP research institute where the outbreak of yersiniosis occurred; 2) identify potential reservoirs of infection; and 3) better understand the epizootiology of this pathogen in the Caribbean. This information may be helpful for developing reliable and sensitive diagnostic methods and can serve as a baseline for developing effective biosecurity protocols and prophylactic measures, including vaccination and the use of probiotics.     

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid alpha-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.