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There has been only limited success in using recombinant retroviruses to transfer genes for the purposes of human gene therapy, in part because the average number of genes delivered to the target cells (transduction efficiency) is often too low to achieve the desired therapeutic effect [Miller, AD. 1990. Blood 76:271-278; Mulligan RC. 1993. Science 260:926-932; Orkin SH, Motulsky AG. 1995. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Bethesda, MD: National Institutes of Health.]. One strategy to improve transduction efficiency is to focus on understanding and improving the processes used to produce recombinant retroviruses. In this report, we characterized the dynamics of retrovirus production and decay in batch cultures of virus producer cells using a simple mathematical model, a recombinant retrovirus encoding the Escherichia coli lacZ gene, and quantitative assays for virus activity and number. We found that the rate at which recombinant retroviruses spontaneously lose their activity (decay) is a strong function of temperature, decreasing roughly 2-fold for every 5 degrees C reduction in temperature, whereas the rate at which retroviruses are produced is only weakly affected by temperature, decreasing about 10% for every 5 degrees C reduction in temperature. In addition, we developed a simple mathematical model of virus production and decay that predicted that the virus titer in batch cultures of virus producer cells would reach a maximum steady-state at a rate that is inversely proportional to the virus decay rate and to a level that is proportional to the ratio of the virus production rate to the virus decay rate. Consistent with the model, we observed that the steady-state levels of virus titer increased more than 3-fold when the cell culture temperature was reduced from 37 to 28 degrees C. Despite their higher titers, virus stocks produced at 28 degrees C, when used in undiluted form so as to mimic human gene transfer protocols, did not transduce substantially more cells than virus stocks produced at 37 degrees C. The implications of our findings on the production of retroviruses for use in human gene therapy protocols are discussed. |
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