title: Viral vector-mediated [corrected] expression of human collagen Q in cultured cells.
abstract: Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene (COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1alpha-ACHE-IRES-COLQ) or in two separate constructs (EF1alpha-ACHE and EF1alpha-COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES-EGFP downstream of the CMV promoter (pAAV-CMV-COLQ-IRES-EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.
authors: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-Ku, Nagoya, Japan.
authors: Mikako
authors: M
authors: Ito
authors: Akio
authors: A
authors: Masuda
authors: Shinsuke
authors: S
authors: Jinno
authors: Takeshi
authors: T
authors: Katagiri
authors: Eric
authors: E
authors: Krejci
authors: Kinji
authors: K
authors: Ohno
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics*; D000818:Animals; D003094:Collagen / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics*; D041681:NIH 3T3 Cells; D012190:Retroviridae / Q000235:genetics*
chemical_list: D009124:Muscle Proteins; D003094:Collagen; D000110:Acetylcholinesterase; C114027:COLQ protein, human