Finally, transduction efficiencies can also be improved by increasing the efficiencies of the steps of infection that occur on or inside the cell. For example, virus binding (and transduction efficiency) can be improved by increasing the concentration of cell surface receptors. The recent cloning of the amphotropic retrovirus receptor, a sodium-dependent phosphate symport, has enabled researchers to measure, and alter, the tissue-specific expression of the receptor.
By culturing CD-4 enriched human peripheral blood lymphocytes in phosphate-free medium for 12 hours, transduction efficiency was increased more than ten-fold, presumably because expression of the amphotropic receptor was unregulated. Transduction efficiencies can also be increased by reducing the time required to complete intracellular steps of infection, thereby increasing the probability of completing transduction before the virus spontaneously loses infectivity.
One recent study found that reverse transcription does not occur exclusively in the cytoplasm as previously thought, but can occur outside the cell, inside extra cellular virus particles, when the virus particles are incubated in high concentrations of deoxyribo nucleoside triphosphates (dNTPs). Virus stocks that have been incubated with dNTPs contain significant amounts of viral DNA (reverse transcribed from the viral RNA) and are about 100-fold more efficient at infecting cells, possibly because the virus particles take less time to integrate into the chromosomal DNA once they enter the cytoplasm of the target cell.
It is also possible that virus particles that enter non-S phase cells, which generally have lower concentrations of dNTPs, might significantly benefit from having undergone at least some reverse transcription before cell entry. These studies demonstrated that reverse transcription can limit the efficiency of retroviral-mediated gene transfer, and that inefficiencies in reverse transcription can be partially overcome by incubation of viral stocks in dNTPs before transduction of the target cells.
Rapid entry into the nucleus is also crucial for maximizing transduction efficiency. Retroviral infection, with the exception of infection by human immunodeficiency virus, requires that the target cells pass through mitosis, most likely because the retroviral DNA complex cannot enter the nucleus until the nuclear envelope breaks down. In one experiment, quiescent cells, stimulated to divide only 6 hours after exposure to retroviruses, were not successfully infected, suggesting that intracellular virus particles are rapidly degraded.
If the intracellular half-life is shorter than the cell cycle rate, then the probability of infection will be strongly influenced by the cell cycle position and cycling rate of the host cell. Construction of recombinant retroviruses that have longer intracellular half-lives should significantly increase transduction efficiencies, especially in slowly dividing cells. Alternatively, transduction efficiencies might be increased by infecting cells at a stage of the cell cycle that maximizes the probability of successful transduction.
Another option is to design recombinant retroviruses that can infect cells that do not pass through mitosis. One approach involves the use of lent viruses (e.g., HIV, simian immunodeficiency virus (SIV)), which can infect non-dividing cells. Another strategy is to construct Mo-MuLV-based retroviruses that have sequences from HIV that allow them to pass through the nuclear envelope.
Both strategies are being actively pursued but vectors based on non-human viruses (e.g., SIV or Mo-MuLV) are preferred for obvious safety considerations. The development of recombinant retroviruses that could infect quiescent cells would not only enhance transduction efficiencies but would also greatly expand the number of target tissues that could be treated by retroviral-mediated gene transfer.