title: Viral gene delivery: optimized protocol for production of high titer lentiviral vectors.
abstract: HIV-derived lentiviral vectors (LVV) are among the most commonly used gene delivery vehicles. Their production in high quantities, which enables concentration of viral particles to high titers, is important for their successful application in both biomedical research and gene therapy. LVV are produced by co-transfection of three or more plasmids into a packaging cell line followed by several purification and concentration steps. Protocols currently in circulation differ from each other but the direct comparison of their efficacy based on the published information is extremely difficult because more than one variable may be changed and essential information may be omitted. We systematically evaluated three protocols and found that one single modification described here, using FuGene(®) 6 in the co-transfection step, increase LVV output almost 20 times as compared to the most commonly used calcium phosphate (CaPO4) transfection technique. Unexpectedly FuGene(®) 6 was also much more efficient than another widely used reagent, Superfect. Dependent on requirements, this permits a dramatic downscaling of the packaging stage of viral production, and/or super-concentration of LVV to achieve stronger expression. For example we were able to prepare ∼25 μL of high titer LVV suitable for injections into rodent brain using a single T75 cm(2) cell culture flask of packaging cells. The same output would require up to 20 times more packaging cells and reagents following conventional protocols. We illustrate the potential of our approach using transfection of primary neuronal cultures with LVV expressing an optogenetic actuator channelrhodopsin-2. Our observations should help to achieve reproducible production of high titer LVV for experimental and potential therapeutic applications.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK.
authors.forename: James
authors.initials: J
authors.lastname: Hewinson
authors.forename: Julian F R
authors.initials: JF
authors.lastname: Paton
authors.forename: Sergey
authors.initials: S
authors.lastname: Kasparov
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D002130:Calcium Phosphates / Q000737:chemistry; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D014162:Transfection / Q000379:methods*; D019562:Viral Load
chemical_list: D002130:Calcium Phosphates; D007202:Indicators and Reagents; C020243:calcium phosphate; D000469:Alkaline Phosphatase