Transduction rate constant as more reliable index quantifying efficiency of retroviral gene delivery

Abstract

Although the efficiency of retrovirus-mediated gene delivery can be enhanced by several physicochemical approaches reported (e.g., addition of polycations and spinoculation), systematic analysis of retroviral transduction combined with experimental data remains to be challenged. With the aid of a reasonable mathematical description of an experimental system, we can therefore predict and optimize the retroviral gene delivery on a quantitative basis of understanding. In this study, we formulated a mathematical model involved with diffusion, decay and uptake of retroviral vectors onto the target cells resided on a solid culture surface. The model was solved analytically by the Laplace transform method. The analytical solutions were then fitted with experimental data to compute two unknown parameters: concentration of infectious retrovirus and transduction rate constant. Our results showed that the concentration of infectious retrovirus determined by the titration method was approximately hundred-fold lower than the one calculated by fitting experimental data with the mathematical solutions. More importantly, effects of polycation (i.e., Polybrene) on ex vivo retroviral transduction were illustrated in a quantitative way by estimating the transduction rate constant, which represents a more reliable parameter to determine the degree of transduction of a retroviral vector to a given target cell.

Determination of Infectious Retrovirus Concentration from Colony-Forming Assay with Quantitative Analysis

“…VSV-G can even convert noninfectious viral particles into infectious viral particles and increase transfection of plasmid DNA (1,24). We have also shown that the concentration of Polybrene increases k and titer (15). NIH 3T3 cells were transduced by VSV-G-pseudotyped MoMuLV with a higher k value and titer than human embryonic kidney epithelial 293 cells (data not shown).…”

“…The mathematical model used to quantitate the processes of retroviral diffusion, deactivation, and uptake was recently developed (see Appendix A) (15). The concentration profiles of infectious retrovirus in the supernatant (C) can be depicted mathematically as…”

“…This is due to different densities of receptors on the cellular surface (14) and different rates of mitotic activity, which affects retroviral transduction (11,21). However, it should be emphasized that the value of C 0 was conserved for the same batch of retroviral supernatant with different target cells and Polybrene concentration, while k was dependent on the target cell type and Polybrene concentration (15). Therefore, k is the parameter that represents the diversity of the experimental systems consisting of various retroviruses (e.g., ecotropic and VSV-Gpseudotyped MoMuLVs) and target cells (e.g., NIH 3T3 and 293 cells) at different conditions (e.g., Polybrene concentration).…”

“…Andreadis et al also estimated that only 1 to 8% of bulk infectious retrovirus was counted by determining the viral titer (2). Recently, we have shown that more accurate infectious retrovirus concentrations can be determined with an extended mathematical model and experimentally determined infectivity (e.g., colony count) (15,16). With this new method, a consistent value of concentration was obtained under different transduction conditions, e.g., different concentrations of Polybrene (15).…”

“…Recently, we have shown that more accurate infectious retrovirus concentrations can be determined with an extended mathematical model and experimentally determined infectivity (e.g., colony count) (15,16). With this new method, a consistent value of concentration was obtained under different transduction conditions, e.g., different concentrations of Polybrene (15). We have also shown that the efficiencies of retroviral transduction with different tropisms can be compared quantitatively (16).…”