ABSTRACT
Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.
Subject(s)
Dependovirus , Gene Transfer Techniques , Liver , Mucopolysaccharidosis VI/therapy , Animals , Bone and Bones/metabolism , Bone and Bones/pathology , Cats , Dependovirus/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Genetic Therapy , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Glycosaminoglycans/metabolism , HEK293 Cells , Humans , Liver/metabolism , Motor Activity/drug effects , Motor Activity/genetics , Mucopolysaccharidosis VI/enzymology , Mucopolysaccharidosis VI/pathology , N-Acetylgalactosamine-4-Sulfatase/genetics , N-Acetylgalactosamine-4-Sulfatase/metabolism , Phenotype , Treatment OutcomeABSTRACT
One way that food processors in the United States have been controlling food-borne pathogens in a non-thermal manner is the application of electron beam (e-beam) radiation. The development of an increased resistance of Escherichia coli O157:H7 to various stressors such to pH, temperature, ionic strength, and antibiotics has been demonstrated. The objective of this study was to determine if the D(10)-value for E. coli O157:H7 (E. coli) in ground beef increases due to repetitive exposure to e-beam at sub-lethal levels. Ground beef samples were inoculated with E. coli and incubated to approximately 10(9) CFU/g followed by e-beam processing. Survivors were enumerated using a standard spread-plating technique. Colonies of E. coli survivors from the highest e-beam dose were isolated and grown for the next cycle of inoculation in ground beef and e-beam processing. Five such consecutive cycles of isolation and e-beam processing were performed. The D(10)-values for E. coli survivors following each cycle of e-beam processing were calculated from survivor curves. The D(10)-values increased (P<0.05) with subsequent cycles of e-beam processing, starting at 0.24+/-0.03 kGy for E. coli ATCC strain 35150 and reaching 0.63+/-0.02 kGy for E. coli isolate L3. Following four cycles of e-beam processing, the isolate L3 increased (P<0.05) its radio-resistance and survived an e-beam dose of 3.0 kGy. Therefore, our data demonstrates that e-beam can efficiently inactivate E. coli in food products; however, similar to other inactivation techniques, E. coli has a capability to develop increased resistance to e-beam if the same populations of E. coli in food products are repetitively subjected to e-beam processing. Although the exact mechanism for the development of increased radio-resistance of E. coli to e-beam is unclear at the moment, based on the available literature regarding increased resistance of E. coli to various stressors, it is likely that some genetic mechanism in involved. Therefore, we are currently investigating this hypothesis with micro-arrays.