The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection MOI for predicting gene transfer eventsReport as inadecuate




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Genetic Vaccines and Therapy

, 2:6

First Online: 04 August 2004Received: 24 October 2003Accepted: 04 August 2004

Abstract

BackgroundAlthough lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection MOI as a parameter for the prediction of gene transfer events.

MethodsLentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector – target cell contact and adsorption periods were studied. MOI between 0–32 was assessed on commonly used cell lines as well as a new cell line.

ResultsWe demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0–32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained.

ConclusionSeveral variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.

List of AbbreviationsCAEVcaprine arthritis-encephalitis virus

DMEMDulbecco-s modified Eagle-s medium

EGFPenhanced green fluorescent protein

EIAVequine infectious anaemia virus

FCSFetal Calf Serum

FITCfluorescein isothiocyanate

FIVthe feline immunodeficiency virus

HIVHuman Immunodeficiency Virus

JDVJembrana disease virus

MOIMultiplicity of Infection

MoMLVMoloney murine leukaemia virus

PCphosphatidylcholine

PSphosphatidylserine

VPCvector-producing cell

VSV-GVesicular Stomatitis Virus G glycoprotein

Electronic supplementary materialThe online version of this article doi:10.1186-1479-0556-2-6 contains supplementary material, which is available to authorized users.

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Author: Bing Zhang - Pat Metharom - Howard Jullie - Kay AO Ellem - Geoff Cleghorn - Malcolm J West - Ming Q Wei

Source: https://link.springer.com/



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