Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo.
The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 10(7) transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 10(10) TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.
Coleman
Jason E
JE
Department of Neuroscience, University of Florida McKnight Brain Institute and College of Medicine, Gainesville, FL 32610-0255, USA.
Huentelman
Matthew J
MJ
Kasparov
Sergey
S
Metcalfe
Beverly L
BL
Paton
Julian F R
JF
Katovich
Michael J
MJ
Semple-Rowland
Susan L
SL
Raizada
Mohan K
MK
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D010286:Paraventricular Hypothalamic Nucleus / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000196:embryology / Q000378:metabolism; D017552:Solitary Nucleus / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D019065:Virus Assembly / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase