Nature Biotechnology pp 163 - 167, pp 168 - 171 and pp 145 - 146

Is life possible without water? Work by scientists to resuscitate mammalian cells subjected to freezing or drying suggests it may be. In one remarkable experiment, cells were revived after desiccation for 5 days following treatment with trehalose, a simple sugar molecule that allows certain forms of life to exist entirely without water. The work suggests that trehalose will be an extremely useful additive for protecting mammalian cells during drying or cryopreservation, with implications for cell and tissue storage and transportation in basic research and biomedicine.

Some organisms astound us with their abilities to survive, even thrive, in the most extreme and unlikely conditions. Trehalose was originally identified as a life-preserving molecule in organisms such as baker’s yeast and tardigrades--bizarre microscopic bugs that can lie dormant in a mummified state for decades without any water. These organisms naturally synthesize trehalose, which preserves the structural integrity of cells, allowing them to revive and fully function when conditions become more hospitable.

Previous work had suggested that trehalose is of use for revitalizing frozen cells. For researchers aiming to use trehalose to protect mammalian cells, the big problem is how to get the molecule to penetrate cell membranes. In this issue, two new papers describe different approaches for incorporating trehalose into human cells. In the first approach, Fred Levine and colleagues have infected human cells with an adenovirus containing two genes encoding the enzymes for synthesizing trehalose. After thorough dehydration--to the point at which no water was detectable--the modified cells were consistently able to be resurrected after three days, and in some cases up to five days following desiccation. In the same study, trehalose was shown to have negligible toxic effects on mammalian cells. In a different approach, Mehmet Toner and colleagues have achieved similar results when reviving human cells following freezing. They used an engineered bacterial protein to ferry trehalose through the cell membrane and into cells. When these cells were frozen 72% survived, compared with only 0.4% survival for cells that didn’t receive trehalose.

Nature Biotechnology pp 194 - 198

For a potentially life-saving cancer drug, smaller may be better. Scientists have shown that a miniature version of the commercial monoclonal antibody Herceptin is not only as effective as the original molecule in killing tumors, but could also potentially avoid drawbacks such as antibody immunogenicity, poor tissue penetration, and problems with manufacture. The results suggest that antibody mimics may provide a promising new avenue in the design of antibody-based therapies.

Herceptin is an anticancer treatment that targets breast tumor cells expressing the HER2 gene. The drug is a member of a class of compounds called monoclonal antibodies that inhibit the growth of tumors by binding to their receptors and blocking the binding of growth factors, which otherwise would cause the cells to divide and multiply.

In the present study, Mark Green and Ramachandran Murali set out to assess what portions of an antibody are needed to retain tumor cell binding capacity. By modeling small protein molecules (peptides) so that they resemble only the part of the antibody known to bind to tumor receptors, they created several miniature antibody mimics. When these peptides were tested for biological activity, they found that one of the peptides, which had two different structural forms, showed moderate activity, with one form performing better than the other. In cell culture, the latter peptide was found to bind to receptors encoded by genes related to HER2, allowing them to inhibit the growth of tumor cells. Experiments in mice also showed that the peptide went to the tumors that contained the HER2 gene and inhibited growth.

Nature Biotechnology pp 208 - 212

Good news is on the way for anyone who craves the health benefits of soybeans, but hasn’t acquired a taste for tofu. Scientists have identified an enzyme for producing genistein--the substance that confers soybeans with their health-promoting benefits--and have transferred it into another plant. By demonstrating that this enzyme works efficiently in a well-studied laboratory plant, their work suggests food crops more suited to a Western palate might one day be engineered with the health-boosting nutrient.

The abundance of soya in the traditional Japanese diet is thought to contribute to a lower rate of heart disease, osteoporosis, and certain kinds of cancer in the population. Soybeans’ health benefits are attributable to high levels of isoflavone molecules, such as genistein, that resemble the sex hormone estrogen. In plants, isoflavones are almost exclusively found in soybeans and other legumes, where they help ward off diseases and play a role in nitrogen absorption. In humans, their biochemical action is still a bit of a mystery, but scientists believe they block certain harmful effects of estrogen by binding to estrogen receptors that relay hormone signals to certain tissues of the body.

Unfortunately, in most Western diets, the intake of soy-based foods is very low and the isoflavone content of soy is variable. Now a team of scientists headed by Brian McGonigle has identified isoflavone synthase--the enzyme responsible for isoflavone production in soy. Using the knowledge that isoflavone synthase has characteristics of an important and well-studied class of enzymes called cytochrome P450s, the scientists searched their collection of soybean DNA sequences for candidate genes and introduced them into a yeast strain that had been engineered to produce a plant version of another enzyme known to be involved in isoflavone synthesis. To see whether the yeast contained the enzyme they were looking for, they then isolated the yeast’s cellular membranes (which contained the accessory protein and the candidate protein from soybean) and chemical precursors for isoflavone synthesis. When they found a strain that produced genistein, they knew they had identified the isoflavone synthase gene. As a final step, they transferred the gene into Arabidopsis, a laboratory plant that normally doesn’t produce genistein, but does contain some of its precursors. Sure enough, in the transgenic plants, the newly introduced isoflavone synthase turned isoflavone precursors into genistein. Future work could investigate whether the enzyme can be introduced into crops destined for human consumption.

Nature Biotechnology pp 176 - 180 and pp 150 - 151

By combining the properties of two entirely different classes of virus, scientists may have stumbled upon a new way to deliver therapeutic genes to cells that significantly extends the length of time the gene is expressed. Bruce Baum and his colleagues have engineered an adenovirus vector with retroviral DNA sequences that enable therapeutic genes to be integrated in host chromosomes, allowing genes to be expressed far longer than with conventional adenoviruses. While the mechanism by which the viruses integrate is unclear, the approach may be useful in designing adenoviral gene therapies for inherited genetic disorders, which required prolonged expression of the therapeutic gene.

Derived from a causative agent of the common cold, adenoviruses are often used as gene therapy vectors in clinical trials. They are relatively easy to produce, capable of infecting quiescent or adult cells, and infect cells with high efficiency. A major disadvantage, however, is their inability to integrate into host cell DNA, which means that expression of the therapeutic gene can often be unstable and short lived. Retroviruses, on the other hand, are adept at integrating their DNA into the chromosome of host cells, which can be an advantage if long-term expression is required, such as for the treatment of inherited genetic disorders. To get around this problem, Bruce Baum and his colleagues have incorporated long terminal repeats (LTRs) from a mouse retrovirus into a standard adenoviral vector. Normally, these LTRs mediate retroviral integration in a reaction performed by the retroviral structural proteins, Env, Gag and Pol. Surprisingly, Baum and his colleagues found that the modified adenoviral could integrate a test gene into the host cell genome and drive its expression for far longer than previously shown, even in the absence of these retroviral structural proteins. Genetic analysis revealed that the vector was integrating into many different locations in the host cell chromosomes, but the mechanism of viral integration remains somewhat of a mystery.

Although the hybrid virus is far from application in the clinic, it seems likely that the authors may have found a new strategy for vector integration that could find application in other viral and even nonviral vector systems.