title: Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector.
abstract: Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.
authors.affiliation: Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: R M
authors.initials: RM
authors.lastname: Wazen
authors.forename: P
authors.initials: P
authors.lastname: Moffatt
authors.forename: S F
authors.initials: SF
authors.lastname: Zalzal
authors.forename: N G
authors.initials: NG
authors.lastname: Daniel
authors.forename: K A
authors.initials: KA
authors.lastname: Westerman
authors.forename: A
authors.initials: A
authors.lastname: Nanci
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002114:Calcinosis / Q000378:metabolism* / Q000821:virology; D002460:Cell Line; D003746:Dental Enamel Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D008334:Mandible / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis; D051381:Rats; D017208:Rats, Wistar; D013977:Tibia / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
chemical_list: C098519:Ambn protein, rat; D003746:Dental Enamel Proteins; D049452:Green Fluorescent Proteins