Nature Biotechnology pp 289 - 295 and pp 264 - 265

A new approach promises to cure hemophilia from within. Until now, individuals with this debilitating blood disease have had to resort to injections of clotting factors to prevent blood loss and hemorrhages. These injections not only must be carried out frequently because of the instability of the exogenous factors in the blood, but also may become ineffective when an individual develops antibodies against the injected factors. Researchers have now found a potential way to circumvent these problems using an implanted device that converts inactive clotting proteins in a hemophiliac’s blood into active clotting factors. The approach may one day represent a possible alternative treatment for hemophilia patients that develop antibodies against injected clotting factors.

Conventionally, hemophiliacs, who lack the clotting factors Factor VIII or IX, are treated by direct transfusion of the missing factor. But such treatment is short-lived and expensive, and comes with inherent risks of disease transmission, as was tragically demonstrated in the 1980s when many hemophiliacs contracted AIDS and hepatitis. More recently, recombinant DNA technology provided synthetic clotting factors, lowering the disease risk of treatments, but raising the cost. Even more troubling, 15%-25% of patients treated in this manner develop an immune response to the foreign clotting factors themselves, severely limiting the effectiveness of treatments.

Now Harvey Pollard and colleagues have designed an implanted semi-permeable chamber that contains beads coated with clotting Factors Xa or XIIa. These factors convert inactive clotting Factor VII---already present in a hemophiliac’s blood---to the active form, VIIa, which directly activates Factor X and enables normal blood coagulation, thus bypassing the need for Factors VIII or IX that are missing in hemophilia patients. In a trial using rhesus monkeys whose blood had been depleted of Factor VIII and cannot coagulate, the authors show that these implants can successfully generate requisite clotting factors for at least one month. As the chamber uses proteins already present in the hemophiliac’s blood, the approach has the added advantage of avoiding deleterious immunological responses that can reduce the effectiveness of current treatments.

Nature Biotechnology pp 321 - 326

Imagine being able to follow the expression of any gene in a living organism no matter where in the organism that gene is located. Scientists have taken the first steps in achieving that goal using a technology termed magnetic resonance imaging (MRI). Their approach can image expression of a marker gene as it occurs deep within the interior of a living animal---a feat previously impossible using other technologies. The authors suggest that the technique could be used to visualize changes in gene expression in a living mouse embryo in utero, providing new insights into the role of particular genes in developmental processes.

Until now, scientists have only been able to visualize gene expression in tissues that can be penetrated by light. Many current confocal microscopy methods also use wavelengths of light that damage cells over time, making the visualization of gene expression in a living embryo impractical over prolonged periods. In contrast, the MRI method can image the expression of genes active deep within an organism and is more suitable for studying live specimens because it is less harmful to cell integrity.

With this in mind, Thomas Meade and colleagues have designed an MRI contrast agent that is activated solely in the presence of a gene of interest. This contrast agent-1-(2-) beta-galactopyranosyloxy)propyl)-1,4,7,
10-tetraazacyclododecane)gadolinium(III) or EgadMe for short-yields a robust MRI signal only when it is exposed to the product of the gene encoding beta-galactosidase. By specifically cleaving a sugar from the EgadMe molecule, beta-galactosidase, liberates atoms within EgadME that interact with water molecules and produce a detectable magnetic resonance signal.

To test the system, the authors injected EgadMe into two-celled Xenopus embryos and then introduced nucleic acid encoding beta-galactosidase into only one of the two cells. When the fully developed animal was then viewed using an MRI instrument, the tissues derived from the beta-galactosidase injected cells produced a high-intensity signal that could be viewed in real time. The authors confirmed that these tissues were actually beta-galactosidase expressing cells using a conventional detection system.

Nature Biotechnology pp 333 - 338

Scientists have succeeded in turning choloroplasts---the tiny granules in plant cells that generate energy from sunlight---into miniature factories for manufacturing therapeutic proteins. By inserting the gene encoding human somatotropin (hST) into the DNA of chloroplasts in tobacco plants, they have expressed soluble, active therapeutic protein at levels 300-fold higher than those previously attained using conventional transgenic plants containing genetically modified nuclear DNA. As chloroplast DNA is not transferred to pollen, the new type of transgenic plants may represent a more effective approach for containing foreign genes in the environment.

Until now, most genetic engineering has focused on introducing foreign genes into nuclear DNA, not the small amount of DNA that is located outside the nucleus in chloroplasts. In the present work, Jeffrey Staub and his colleagues show for the first time that hST---a fully active human secretory protein---can be expressed at high levels in tobacco when chloroplast DNA is engineered to carry the human gene. Using an antibiotic marker to track the foreign gene, they also show that crosses between these genetically modified plants and wild-type tobacco result in offspring that contain no inserted genes within their nuclear or chloroplast DNA. What’s more, the authors could detect no foreign genes within the pollen of transgenic plants.

The ability to produce high levels of hST in tobacco plants with engineered chloroplasts has the potential to significantly reduce the cost of manufacturing the therapeutic, reduces the risk of foreign gene transfer to weedy relatives in the environment, and avoids the risk of viral contamination that can occur when recombinant protein is produced in mammalian cell culture.

Nature Biotechnology pp 339 - 342 and pp 266 - 267

Crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are becoming increasingly popular with farmers as a way of increasing crop yield and reducing insecticide use. However, concerns have been raised that widespread cultivation of these transgenic crops may accelerate the rate at which insects acquire resistance to the toxin, rendering obsolete both Bt crops and natural Bt insecticides used to control insects infesting organic crops. To combat this, farmers have adopted strategies in which non-Bt crops are planted as isolated "refuges" within Bt crop fields, but the effectiveness of this strategy has remained unclear. Now, a group of researchers has published the first field trial data testing the effects of the size and type of refuge on insect survival. Their results indicate that Bt crop fields containing isolated refuges of non-Bt plants are far more effective at suppressing insect resistance than fields in which non-Bt crops are mixed among Bt plants. They also indicate that spraying a refuge with a different insecticide---a practice preferred by farmers to reduce pest damage to non-Bt crops in refuges---may actually increase the likelihood of the emergence of resistance.

Insect resistance to the Bt toxin is generally a recessive trait that is only manifest when both copies (homozygous) of the resistance gene are inherited. Thus, insects that contain one copy of the resistance gene are still susceptible to killing by the Bt toxin. Refuge strategies attempt to increase the likelihood that resistant homozygous insects mate with susceptible insects, as the resultant progeny are likely to be susceptible to the Bt toxin.

Over a period of two years, Tony Shelton and his colleagues carried out two trials in which Bt-resistant diamondback moths were released onto plots of Bt broccoli. By carrying out the trials in upstate New York, the authors ensured that resistant diamondback moths released onto the plots could not overwinter and transfer their resistance to the wild moth population. In the first trial, Shelton’s team planted Bt broccoli in plots comprising all Bt plants, Bt plants containing an isolated refuge (20% of whole plot), Bt plants mixed with non-Bt plants (20% of total plants), or all non-Bt plants. Results indicated that the plot containing the isolated refuge was most effective at suppressing the emergence of resistance, presumably because more Bt-susceptible insects survived to breed with resistant insects. In a second trial, the researcher’s investigated the effect of spraying a different insecticide onto the non-Bt refuge. The finding that resistance was more likely to occur in treated than in untreated refuges suggests that the practice of spraying non-Bt crops in refuges may be detrimental to resistance control strategies.