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Quantitative analysis of retrovirus-mediated gene transfer into mammalian cells Tayi, Venkata Siva Ganesh

Abstract

Recombinant retroviruses (gammaretro-, lenti- and foamy-viral vectors) are used for gene therapy as well as for scientific research because of their ability to provide relatively stable gene transfer and expression. The process of retrovirus-mediated gene transfer into mammalian cells involves a series of transduction events that take place sequentially. A mathematical model for the retroviral transduction process was developed that incorporates the important extracellular and intracellular rate-limiting steps. The mathematical model was validated with experimental data obtained using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. The model predictions of transduction efficiency and integrated virus copy number were generally in good agreement with measured results acquired for both static and centrifugation-based gene transfer protocols. However, a deviation between the model calculations and the experimental transduction efficiency data was observed for centrifugation at high vector-to-cell ratios. To address this limitation, believed to be caused by the saturation of binding sites on the cell surface, a detailed experimental investigation of the binding and entry kinetics of retroviruses was performed. With the help of a mathematical representation for retroviral binding to, dissociation from and entry into mammalian cells, the kinetic rate constants for these three steps were experimentally quantified. The model was modified to incorporate these more complex kinetic steps and was shown to provide even better predictions of the experimental transduction results for the full range of vector-to-cell ratios investigated. The modified model was then used to optimize various retroviral transduction process parameters. Studies of the extracellular transport of viral vectors provided the optimal range of centrifugal forces needed to obtain the maximum transduction efficiency. Based on a simulated performance comparison of the three different types of recombinant retroviruses, lentiviral vectors were found to be very efficient for targeting hematopoietic stem cells. The mathematical model was able to provide a set of conditions where retroviral transduction protocols should yield high transduction efficiencies while maintaining the viral copy number in a lower, more desirable range.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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