title: Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy.
abstract: The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells. Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies.
authors.affiliation: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, PO Box 54, Dunedin 9054, New Zealand; Brain Health Research Centre, University of Otago, PO Box 54, Dunedin 9054, New Zealand. Electronic address: stephanie.hughes@otago.ac.nz.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Hughes
authors.affiliation: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, PO Box 54, Dunedin 9054, New Zealand. Electronic address: hopka758@student.otago.ac.nz.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Hope
authors.affiliation: Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand. Electronic address: Janet.Xu@lincolnuni.ac.nz.
authors.forename: Janet Boyu
authors.initials: JB
authors.lastname: Xu
authors.affiliation: Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand. Electronic address: Nadia.Mitchell@lincoln.ac.nz.
authors.forename: Nadia L
authors.initials: NL
authors.lastname: Mitchell
authors.affiliation: Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand. Electronic address: David.Palmer@lincoln.ac.nz.
authors.forename: David N
authors.initials: DN
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015316:Genetic Therapy; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009472:Neuronal Ceroid-Lipofuscinoses / Q000196:embryology / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D012756:Sheep
chemical_list: C113332:CLN5 protein, human; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins
keywords: 4′,6-diamidino-2-phenylindole; AAV; Batten disease; CIDR; CLN5; DAPI; Endo H; Endoglycosidase H; FBS; Fluorescent storage bodies; GFAP; GFP; Gene therapy; LVMNDU3; Lentiviral vector; M6P; M6PR; MAP; MOI; NCL; Neural cell culture; Neuronal ceroid lipofuscinoses; PAGE; PBS; PDI; PNGase F; PVDF; SDS; Sheep; TU; Transduction; adeno-associated virus; controlled internal drug (progesterone) release device; days in vitro; div; foetal bovine serum; glial fibrillary acidic protein; green fluorescent protein; lentiviral derived vector with a myeloid sarcoma virus U3 element; mannose-6-phosphate; mannose-6-phosphate receptor; microtubule associated protein; multiplicity of infection; neuronal ceroid lipofuscinosis; peptide-N-glycosidase F; phosphate buffered saline, pH7.2; polyacrylamide gel electrophoresis; polyvinylidene fluoride; protein disulphide isomerase; sodium dodecyl sulphate; transducing units