Effects of flocculation on retrovirus processing, delivery and transduction

by Landázuri, Natalia

Abstract (Summary)
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.
Bibliographical Information:

Advisor:Niren Murthy; Andrés J. García; Joseph M. Le Doux; Mark R. Prausnitz; H. Trent Spencer

School:Georgia Institute of Technology

School Location:USA - Georgia

Source Type:Master's Thesis

Keywords:biomedical engineering


Date of Publication:04/13/2005

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