From The Lab: Biotechnology

Knocking Out Malaria?
Genetically engineered vaccine shows promise

Context: The world’s first vaccines were made from weak forms of disease-causing microbes. A more modern approach is to instead use one or a few of a microbe’s signature molecules. But the malaria para­site – Plasmodium – foils this strategy because at different developmental stages it has different surface proteins. However, the older approach also poses problems: insufficiently weakened parasites might bring on the disease, but overly weakened ones are ineffective. Researchers led by Kai Matuschewski at the Heidelberg University School of Medicine in Germany and Stefan Kappe at the Seattle Biomedical Research Institute have announced a promising mixture of new and old. They have, for the first time, made a malaria vaccine by weakening the parasite through genetic engineering.

Methods and Results: Plasmodium constantly changes both appearance and address, moving from mosquitoes’ guts to their salivary glands to mammals’ liver cells to their blood. With the newly sequenced Plasmodium genome as a guide, the researchers looked for genes the parasite needs to move from liver to blood. They found such a gene and completely deleted it from the version of Plasmodium that infects rodents. Then, they injected rats and mice with the genetically modified parasites. None got malaria. Two months after vaccination, the rodents were injected with 50,000 nonmodified parasites (the equivalent of hundreds of bites from infected mosquitoes). Encouragingly, none got sick.

Why it Matters: Malaria kills millions annually; the sickness stifles development in afflicted countries by keeping people from work and school and by draining health resources. The vaccine developed by Matuschewski and colleagues must still show that it is able to prevent disease in humans and for the long term. In mice, the vaccine must be administered two or three times, delivering thousands of parasites that can be grown only in mosquitoes’ salivary glands. To save lives, future whole-parasite vaccines must be more potent and easier to produce. Or perhaps knowledge gleaned from genetically modified vaccines will inspire more successful versions of other vaccines. If either happens, this devastating disease could be eradicated within a generation.

Source: Mueller, A. K., et al. 2005. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 433:164–7.

Seeing Missing Letters
Genetic test promises more accurate diagnoses

Context: There are many ways that a gene can go wrong. A single DNA “letter,” or nucleotide, in a sequence of letters may be replaced with another. Or a long string of letters may get deleted, reversed, or duplicated. With certain diseases, patients are likely to have mutated versions of the same gene but unlikely to have the same mutation. Current genetic tests overlook certain kinds of mutations; they are a bit like a word-processing program that can find misspelled words but not missing words or sentences. They usually sequence a gene’s DNA in separate chunks, examining each chunk for mutations. But people have two copies of almost every gene, and if a chunk is entirely missing from one copy, current tests will detect the intact chunk from the other copy. To find out how many mutations conventional tests miss and so determine how useful new genetic tests could be, Graham Casey of the Cleveland Clinic and colleagues separated copies of genes to look at each individually.

Methods and Results: The researchers studied cells from 89 colon cancer patients, each of whom appeared to have a mutation in one of three genes. They examined the cells’ DNA using both traditional sequencing and another technique called “conversion analysis.” In conversion analysis, researchers create new cells, each of which contains only one copy of the gene being studied; they then compare the RNA messages coming from both copies.

Sequencing analysis found 28 mutations deemed likely to have ill effects; conversion analysis found all these and 14 more. Sequencing also found 42 mutations that could not be classified as either harmful or harmless; conversion analysis identified 21 of these as harmful (and the rest as innocuous). Thus, traditional sequencing identified 28 harmful mutations, while conversion analysis found 63.

Why it Matters: Genetic deletions likely contribute to breast cancer, neurofibromatosis, Duchenne’s disease, and Parkinson’s. Researchers and physicians know that traditional tests do not detect large genetic deletions. As a result, doctors often advise preventive procedures to at-risk patients, no matter what the genetic tests say. Not only may patients be unnecessarily worried, but they may also undergo expensive and painful procedures, like colonoscopies, that are unlikely to benefit them. Even healthy patients cannot know how likely they are to pass genetic mutations to their children. Casey and colleagues have, for the first time, rigorously examined how frequently such mutations are missed and shown that, for some diseases, the more difficult genetic tests might be worth the investment.

Source: Casey, G., et al. 2005. Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer. Journal of the American Medical Association 293:799–809.

Beaming Biodata
Mutation detection goes wireless

Context: DNA chips for detecting genetic variations abound. Typically, a processed biological sample is placed on a chip, which then must be loaded into a separate, expensive device for reading. Now, Yoshiaki Yazawa and colleagues at Hitachi have designed a tiny chip that not only detects DNA variation but can report it wirelessly from the inside of a sealed sample container. These chips could be dropped directly into a solution containing copies of patient DNA and should be cheap enough to be disposable.

Methods and Results: The chip packs a biosensor, radio transceiver, and antenna coil onto 2.5 by 2.5 millimeters of silicon. An off-the-shelf external unit powers the chip with radio waves and reads its transmissions. To detect a particular DNA ­sequence, researchers add the complementary sequence to the sample along with the chip. If DNA in the sample binds to the probe sequence, an enzyme emits light. When the sensor on the chip detects the change in light, the radio unit sends a signal to the external unit.
This is a simpler technique than the one used by most other chips, which requires fluorescent dyes, lasers, and microscopes. Additional chips can be added to the sample to boost accuracy or to detect different kinds of variations. Hitachi researchers believe that up to 100 variations could be measured at one time. Also in development are wireless sensors that use the same technology to monitor temperature and pH, which could enable better control of experimental conditions and thus more reliable readings.

Why it Matters: DNA analysis promises to make medicines more effective, if it can be made easy and cheap enough. Hitachi’s chip is the first that can both detect mutations (albeit so far only the simplest and most common kind) and report them wirelessly. Since this kind of chip can transmit data from inside a sealed container, samples tested with it are less likely to be contaminated by researchers or the environment, and samples containing pathogens are less likely to infect workers. Assuming patient samples can be prepared easily for chip analysis, the chip could also make it easier to detect DNA variations in settings less controlled than a research lab. Though the research is still in its initial phases, Hitachi expects that the chips could be used in clinics or small hospitals to help doctors decide which drugs to prescribe for patients.

Source: Yazawa, Y., et al. 2005. A wireless biosensing chip for DNA detection. Paper presented at 2005 IEEE International Solid-State Circuits Conference. Feb. 6–10. San Francisco, CA.

Xenotransplant
Pig hearts in humans?

Context: When he was a heart surgeon, David Cooper would have 140 patients referred to him for transplants each year; because of the shortage of donor organs, only about 25 would receive them. Now an academic researcher at the University of Pittsburgh Medical Center, Cooper is part of a broad effort to search for ways to use pig organs for human transplants.

Without extremely high levels of immunosuppressive drugs, pig organs seldom last for half an hour in, say, baboons. The organs swell up and turn black and must be removed quickly, or the animals may die. Cooper led a team of scientists from Harvard Medical School and Immerge Biotherapeutics in determining what would happen when hearts from pigs genetically modified to seem less “piglike” to a foreign immune system were transplanted into baboons.

Methods and Results: The baboon’s immune system targets the pig organs for attack mainly because of a particular sugar that covers porcine blood vessels. Three years ago, scientists created pigs unable to make this sugar by deleting a gene for a certain enzyme. Cooper’s team transplanted hearts from the genetically modified pigs onto the abdomens of eight baboons, where the researchers could tell how strongly the pig hearts were beating. Three baboons died for reasons other than organ rejection, and the hearts remained viable. In the other five baboons, the hearts stopped beating between 59 and 179 days after transplantation, at which time they were surgically removed. The small blood vessels in the organs were full of tiny clots, probably caused by a mismatch between tissues and blood-clotting systems. But the researchers found evidence that this clotting process can be at least slowed with anticlotting medicines, like aspirin. None of the baboons suffered serious infections as part of the study.

Why it Matters: Cooper and colleagues’ study marks the first transplant using pigs engineered to lack a gene and the first time xenotransplanted organs have survived for months when immunosuppressive drugs were administered in doses similar to those used in humans. It also marks the longest time a pig heart has survived in another species. In previous work, organ rejection has been inhibited, though not as dramatically, by using pigs engineered to contain human genes that protect their organs from the human immune system. Experiments using additional pigs with these human genes and without the pig sugar gene are planned, but the first pig-to-human transplants are years away, says Cooper. First, xenotransplants must be deemed as likely to help patients as other available treatments, like mechanical heart-assistive devices. Even then, concerns about ethics and infectious disease must be addressed.

Source: Kuwaki, K., et al. 2005. Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nature Medicine 11:29–31.