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Transduction efficiencies can also be improved by straightforward environmental or culture medium alterations that maximize the efficiency of extra cellular steps of infection. For example, by reducing the decay rate of recombinant retroviruses, which rapidly lose infectivity with time at 37oC, transduction efficiencies can be improved.

The rapid decay of infectivity (the half-life at 37oC is about 6 to 8 hours) of retroviruses reduces transduction efficiencies because retrovirus binding and infection occurs over a period of several hours, during which time most of the infectivity of the retroviruses is lost. Infection continues for several hours because the virus particles are large (100 nm) and diffuse slowly.

Retrovirus particles move about 300 mum in one half-life (7 hours), or about one-tenth the distance from the top of the culture medium fluid to the surface of the cells. With current cell culture configurations, most of the virus particles lose their infectivity long before they reach the surface of the target cells. The decay rate of retroviruses could, in principle, be reduced by genetic methods once the mechanism of decay is known.
For now, simpler strategies have been adopted, such as transduction at 32oC instead of 37oC, which reduced the decay rate and increased transduction efficiencies. A second method of minimizing the effects of retroviral decay is to increase the encounter frequency, by centrifugation or convection, between the target cells and the virus particles.

Centrifugation increased the transduction efficiency of adherent NIH-3T3 fibroblasts three to ten-fold and non-adherent CD34+ blood cells six-fold. Convection of virus particles past target cells immobilized onto a porous membrane increased transduction efficiencies up to ten-fold. Both methods increased the rate at which virus particles bound and infected the target cells, and therefore decreased the adverse impact of retroviral decay on transduction efficiency.

Given further refinement, these methods have the potential to improve substantially the efficiency of most ex vivo gene transfer protocols. Transduction efficiencies can also be increased by altering the composition of the culture medium. For instance, addition of cationic polymers (e.g., polybrene, protamine, DEAE-dextran) or cationic lipids (e.g., 2,3-dioleyloxy-N-{2(sperminecarboxamido)ethyl}-N,N-dimethyl-1-propaninium trifluoroacetate (DOSPA) and dioleylph osphatidyl ethanolamine (DOPE)) to the culture medium before or during infection increases virus binding and transduction efficiency 10-fold or more.

The mechanism of enhancement has not been completely elucidated, but it is thought that the polymers adsorb to either the virus particles and/or the surface of the cell, and reduce the electrostatic repulsion between the two negatively charged entities. A better understanding of the mechanisms that underlie the enhancement of infection by cationic polymers and lipids might offer strategies for the design of better ‘binding enhancers’ and thus increase transduction efficiency.

It may also be possible to improve the culture environment for infection by removing substances from the culture medium, either viral or non-viral, that inhibit infection. Inhibitors can block infection by binding to the virus particles, binding to the virus receptors, or by other mechanisms that interfere with the normal life cycle of the virus particles. One recent study found that medium conditioned by virus-producing cells inhibited infection, and the authors speculated that non-infectious virus particles, or VAPs not associated with virus particles, were blocking infection by binding to virus receptors.

A recent study in our laboratory demonstrated that high-molecular-weight proteoglycans, present in viral stocks, inhibited infection. These high-molecular-weight inhibitors will most likely be co-concentrated with the virus particles by conventional concentration methods, producing a high-titer virus stock with, nonetheless, low transduction efficiency. To produce virus stocks with higher titers and transduction efficiencies, methods are needed that remove or eliminate these large-molecular-weight inhibitors, rather than co-concentrate them with the virus particles.