Effects of flocculation on retrovirus processing, delivery and transduction

dc.contributor.advisor Le Doux, Joseph M.
dc.contributor.author Landazuri, Natalia en_US
dc.contributor.committeeMember García, Andrés J.
dc.contributor.committeeMember H. Trent Spencer
dc.contributor.committeeMember Prausnitz, Mark R.
dc.contributor.committeeMember Murthy, Niren
dc.contributor.department Biomedical Engineering en_US
dc.date.accessioned 2005-07-28T17:56:44Z
dc.date.available 2005-07-28T17:56:44Z
dc.date.issued 2005-04-13 en_US
dc.description.abstract The efficiency of retrovirus-mediated gene transfer can be dramatically enhanced by inducing flocculation of viruses. Addition of oppositely charged polymers to virus stocks resulted in the formation of virus-polymer complexes. The complexes specifically incorporated virus particles and only few other proteins, were not cytotoxic, did not reduce the stability of the viruses, and were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion. Increases in the rate of transport of viruses correlated with increases in the rate of transduction, as the polymers did not affect the efficiency of post-binding steps of transduction. The formation of virus-polymer complexes also permitted concentrating viruses and purifying the stocks from inhibitors of transduction. Pelleting of the complexes followed by resuspension of the pellet in a reduced volume of fresh cell culture medium resulted in substantial enhancement of transduction. Purified virus stocks could be used in smaller quantities than unprocessed stocks to achieve a given level of gene transfer and reduced uncertainties about the relationship between the amount of virus used and the number of genes transferred. When using high concentrations of purified viruses, the efficiency of gene transfer was dependent on the number of envelope proteins displayed on the surface of each virus particle. Viruses with a low number of envelope proteins transduced cells more efficiently than did viruses with a high number of envelope proteins, and allowed more integrations of the transgene per target cell. In contrast, when the number of envelope proteins per virus particle was high, transduction appeared to be limited by a reduction in availability of functional receptors for viruses pseudotyped with the same envelope. Taken together, this novel method for processing retrovirus stocks and a better understanding of major limitations of transduction should simplify efforts to predict the outcome of retrovirus transduction protocols and should help to increase the likelihood that human gene therapy protocols will succeed. en_US
dc.description.degree Ph.D. en_US
dc.format.extent 3207155 bytes
dc.format.mimetype application/pdf
dc.identifier.uri http://hdl.handle.net/1853/6884
dc.language.iso en_US
dc.publisher Georgia Institute of Technology en_US
dc.subject Flocculation en_US
dc.subject Polymers
dc.subject Lentivirus
dc.subject Retrovirus
dc.subject Gene therapy
dc.subject Gene transfer
dc.title Effects of flocculation on retrovirus processing, delivery and transduction en_US
dc.type Text
dc.type.genre Dissertation
dspace.entity.type Publication
local.contributor.advisor Le Doux, Joseph M.
local.contributor.corporatename Wallace H. Coulter Department of Biomedical Engineering
local.contributor.corporatename College of Engineering
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