Dynamics of retrovirus-mediated gene transfer.

Abstract

Retroviruses are used as gene delivery vehicles in genetic treatment. Retroviral transduction is a complex process that is comprised of numerous interrelated steps, both in solution and after internalization of retroviruses into target cells. Mathematical modeling and quantitative experiments are necessary to understand and predict the response of highly interactive biological systems. Physico-chemical factors in solution, such as calf serum and polybrene concentrations exhibit coupled action and affect significantly the efficiency of gene transfer and the dynamical pattern of retroviral decay. Intracellular events are complicated by a kinetic interplay between events in the retroviral life cycle and the cell cycle of the target cells. A combination of mathematical modeling and experiments is used to study the dynamics and identify some of the rate limiting steps of retroviral transduction. The predictions of the model on the effect of cell culture parameters such as the target cell density and viral titers are in agreement with experimental observations in the literature. The model also identifies the intracellular stability of retroviral vectors as a potentially limiting step of the transduction process. Most importantly, predictions of the model are used for experimental design and interpretation. Based on the model predictions a method is developed to examine the cell cycle specificity of retroviral transduction. It overcomes the problems of cell synchronization methods and of overlapping time scales of retroviral decay and the doubling time of the target cells. Two novel methods are devised and mathematically analyzed to measure the post-penetration half-life of Moloney Murine Leukemia Virus (MMuLV)-derived vectors in NIH-3T3 cells. The results show that retroviral vectors lose their activity as a function of the time spent in the cell cytoplasm, with a half-life that ranges between 5.4-7.5 hr. Such a short half-life may limit the process of retrovirus-mediated gene transfer. This thesis demonstrates that a combination of mathematical analysis and experimental work can contribute to quantitative understanding of the life cycle of retroviral vectors. This is necessary for the successful implementation of gene therapy and a better understanding of disease pathogenesis.