Recombinant retroviruses are used extensively for gene therapy purposes because they permit stable gene transfer and expression. However, due to the low diffusivities (~2 x 10^-4 cm²/h) and short half-lives (4-9 h) of retroviral vectors, their ability to transfer, in the active form, from a bulk suspension to the target cell surface is very limited. In addition, the repulsive forces generated due to the net negative charge on both the retroviral envelope and the target cell membrane result in low binding efficiencies. As a consequence of these effects, only a small fraction of target cells is typically transduced. Furthermore, the transduction process becomes even less effective when the titers of the retrovirus-containing medium harvested from the packaging cell line are very low. Various improved transduction methods have been developed which use different physico-chemical approaches to overcome these limitations. However, the increase in the transduction efficiency using high concentrations of vectors and improved methods also results in a proportional increase in the integrated vector copy number of the transduced cells. Retroviral genome integration can cause oncogene activation leading to leukemia induction, a process referred to as insertional mutagenesis, the risk of which increases as the number of integrated copies per transduced cell goes up. Hence, retroviral transduction methods need to be optimized not only to yield high transduction efficiencies but also to deliver a reduced range of vector copy numbers per cell. A quantitative understanding of the process by which cells are transduced using retroviral vectors provides a means to accelerate the optimization of this process.

A model was developed for the standard geometrical arrangement where a homogeneous layer of cells at the bottom of a culture dish is contacted with an overlying suspension of viral vectors treated as uniformly-sized spheres. As well as undergoing Brownian diffusion, the viral particles are allowed to settle, either under normal gravity (conventional static protocol) or under augmented forces (centrifugation-based protocol). Thus, the model accounts for the following extracellular processes: viral mass transfer by diffusion and convection, extracellular viral decay, and irreversible binding to the cell surface; as well as all of the essential intracellular steps: intracellular decay, reverse transcription, transport of the pre-integration complex to the vicinity of the nucleus, its import into the nucleus and, finally, its integration into the cell's genome. The internalization of the surface-bound virus into the cell cytoplasm and the integration of viral DNA with cell chromosome are assumed to take place sufficiently rapidly that their rates are non-limiting. The model also incorporates the kinetics of multiple vector insertions and, hence, can be used to calculate not only the transduction efficiency but also the integrated copy number.

The retroviral vectors packaged from PG13 cells with Gibbon Ape Leukemia Envelope and K-562 target cells were used as the experimental system. The binding of retroviral vectors to the target cells was found to be a rate-limiting step in the transduction process. This retroviral vector was found to remain as a stable intermediate inside the cytoplasm. The initial active retrovirus concentration in the extracellular medium, which cannot be easily quantified by experimental measurements, was estimated by fitting the experimental data obtained under static conditions with the model predictions. The influence of the viral vector numbers per target cell on the transduction efficiency of K562 cells under static and centrifugation conditions was investigated and compared with the model predictions. The model-predicted centrifugal transduction efficiencies were in close agreement with the experimental results. The model-predicted relationship of transduction efficiency with integrated copy number was also in good agreement with the experimental data reported by others for the same virus-cell system. The model was used to develop an experimental strategy which uses a multi-exposure centrifugation protocol to increase the transduction efficiency while minimizing the integrated copy number.

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