Scientists Milk Animals For Malaria Vaccine

In their quest to mass-produce an effective malaria vaccine, scientists might one day replace expensive manufacturing facilities with a goat. In a study reported December 18 in the Proceedings of the National Academy of Sciences online, researchers developed mice that could secrete an experimental malaria vaccine into their milk. When the purified candidate vaccine was injected into monkeys, it protected four out of five animals from a lethal dose of the malaria parasite. If the process can be scaled up to larger animals such as goats -- and early experiments indicate it can -- livestock might prove to be inexpensive, high-yield malaria vaccine factories. “A vaccine must not only be effective, it must be cheap to manufacture if it is to be used in those countries hit hardest by malaria,” says lead author Anthony Stowers, Ph.D., a malaria researcher at the National Institute of Allergy and Infectious Diseases (NIAID). “Using transgenic animals to achieve both ends is an exciting possibility. If it works, a herd of several goats could conceivably produce enough vaccine for all of Africa.”

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Transgenic animals are so named because they contain a gene, called a transgene, from another individual or organism. For years transgenic animals, particularly mice, have been used to help scientists understand how genes work and interact with one another. More recently, researchers have introduced genes encoding specific proteins into animals to produce large quantities of those proteins for medical use. Dr. Stowers and his colleagues investigated whether transgenic animals could produce proteins for use in malaria vaccines.
Dr. Stowers and Louis Miller, M.D., chief of NIAID’s Laboratory of Parasitic Diseases and the director of its Malaria Vaccine Development Unit, joined other investigators from NIAID and Genzyme Transgenics Corporation, Framingham, Mass., to produce two transgenic mouse strains. Each strain carried a form of the gene for a surface protein from the deadliest malaria parasite, Plasmodium falciparum. The researchers designed the transgenes to be switched on by the cells that line the mammary glands, such that the resulting proteins would be secreted into the animals’ milk.
Both mouse strains produced large quantities of the desired vaccine protein, which was used to vaccinate monkeys against malaria. Only one of the five immunized animals contracted the disease. By comparison, six out of seven unvaccinated animals had to be treated for virulent malaria.
The high yield of the protein and its ability to stimulate protective immunity in mice offers promising evidence that the technique could also be used in goats or even cows. The researchers had anticipated future studies in goats by designing the transgenes’ on/off switch using regulatory elements from goat DNA. Preliminary experiments, which have not yet been published, suggest the procedure works well in the larger animals. That possibility offers a far more practical option for large-scale vaccine production.
Anthony S. Fauci, M.D., who as NIAID director guides an extensive research program on global infectious diseases, believes this approach is promising. “Malaria causes untold suffering in the poorest regions of the world, so we cannot restrict our focus simply to finding a vaccine that works,” he says. “Rather, we must look for innovative strategies that will bring effective vaccines to regions where economic conditions preclude the use of costly alternatives. Transgenic animals could be one way to accomplish that goal.”
The study involved many researchers from the two participating institutions. Among these, Drs. Li-How Chen and Harry Meade directed the studies conducted at Genzyme Transgenics. B. Fenton Hall, M.D., from NIAID’s Parasitology and International Programs Branch, also helped initiate the project and played an active role in its development.
NIAID is a component of the National Institutes of Health (NIH). NIAID supports basic and applied research to prevent, diagnose, and treat infectious and immune-mediated illnesses, including HIV/AIDS and other sexually transmitted diseases, tuberculosis, malaria, autoimmune disorders, asthma and allergies.

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World's First Cloned Transgenic Goats Born

The world's first cloned transgenic goats have been born as part of a research program conducted by LSU Agricultural Center and Genzyme Transgenic Corp. While much of the research was done at LSU in Baton Rouge, the goats made their appearance last fall at the Genzyme farm in Massachusetts.
LSU's resident transgenic goat is Millie. Though she's a million-dollar goat, she doesn't look any different from the other goats she hangs out with. She is different, though, because her milk contains a therapeutic protein that could be extracted to make a drug for patients undergoing coronary bypass surgery. The drug works in conjunction with heparin, which prevents blood from clotting.
The protein, anti-thrombin III (AT III), is now in the third phase of human clinical trials. Pending FDA approval during the next 12 months, the product could be on the market in the next couple of years.

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"The technology used to clone the three Genzyme goats is one of the first applications of the nuclear transfer (cloning) procedure to produce transgenic goats for the pharmaceutical industry, said Richard Denniston, a researcher with the LSU Agricultural Center. An article in the May issue of Nature Biotechnology will announce the breakthrough. Researchers at Tufts University School of Veterinary Medicine also participated in the research.
LSU researchers have for the past six years carried a collaborative research program with Genzyme, which may become the first company to have a transgenic product on the market. "The FDA is being very careful because there's no precedent," Denniston said. Transgenics is the process of taking DNA from one species and implanting it into the genetic structure of another.
"Genzyme takes the gene for anti-thrombin III. The DNA is like a computer code. Once researchers identify the code, they can punch it into a DNA sequencer. Four different molecular building blocks of the DNA material are put in a vial, and a gene is built synthetically. Then you can make millions of copies of that," he explained.
The AT III gene is attached to a promoter gene, usually the gene for casein, a milk protein, and then microinjected into the male pronucleus of the newly fertilized egg. During the first cell divisions, the gene may become attached to the genetic material of the embryo. If this happens, the new gene, or transgene, will be incorporated into every cell of the developing goat embryo. The embryo is cultured and then transfered to the goat surrogate mother, and researchers wait for normal fetal development. If a female is produced, she will produce milk with the AT III protein, which can be extracted from the milk for pharmaceutical use.
"When you breed the AT III female offspring, 50 percent of her offspring will have this gene. Now you've got an animal that is very valuable. How do you produce these animals as quickly as possible? Genzyme does the molecular work. We are trying to develop technologies to reproduce these transgenic animals as quickly as possible," Denniston said.
"The idea of cloning arose in the past two years. Cloning is desirable because it would increase the efficiency rate. When you insert the AT III protein into 1,000 fertilized eggs and transfer 100 embryos, you can expect one transgenic offspring. There's a 50 percent chance of its being female," he said.
Denniston explained that there are three possible sources of genetic material for cloning. One source is the adult animal; this is how Dolly the sheep was produced. "That's what really made big news, taking a cell from an adult animal," he said. "Another source is the 16-32 cell-stage embryo, which was first done about 15 years ago. A third source is a developing fetus. In each case, the donor cell containing all the genetic material is fused with an enucleated egg (an egg that has had its genetic material removed). The resulting cloned embryo is then transferred into a recipient female that carries the clone to term."
The transgenic goat clones born from the joint LSU/Genzyme project were produced by taking fibroblast cells from a 30-day female goat fetus. These cells were grown in an incubator in media containing the gene for AT III. The growing cells then underwent a procedure called electroporation, which allows the gene to cross the cell membrane and enter the host cell's nucleus.
The resulting transgenic fibroblast cells were then fused with an enucleated egg. These cloned embryos were then transferred into a recipient goat. The result was three genetically identical transgenic female goats that can produce the valuable AT III protein in their milk. Using the cloning process in conjunction with transgenics improves the overall efficiency of producing transgenic animals, Denniston said.
Not all pregnancies are successful, but 100 percent of the births will be females with the gene for AT III," Denniston said.
The agricultural applications of this technique are widespread. For example, researchers could insert a gene for bruccilosis resistance and clone a herd of bruccilosis-resistant cattle.
"What we are doing for Genzyme is to test procedures or techniques and develop in vitro fertilization. Six years ago, nobody was doing in vitro fertilizationin goats. They asked us to, and we did," he said.
"The Massachusetts farm has more than 1,500 goats under the watchful eye of the FDA. They produce the gene in a lab in Framingham. They get a 30-day fetus, culture fiborblast cells, insert the gene for AT III into fiborblast cells, do nuclear transfer, send embryos to us, and we transfer them into recipients."
"The market for AT III is $200 million. That amount of protein can be produced by fewer than 100 goats. Even with the cost being a half to $1 millon per animal, the potential earnings from the pharmaceutical product are enormous," the LSU researcher said. Genzyme has received a patent on the protein.
It is cheaper to produce transgenic goats than cows; the goat's gestation period is shorter (5 months to a cow's 9 months), and the protein is not required in huge quantities, Denniston said.
LSU's contract with Genzyme has brought LSU approximately $850,000 in research funds, and LSU's reproductive physiology laboratory will continue working with the nuclear transfer process as a tool for pharmaceuticals, agricultural applications and propagation of endangered species.

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Medicine From Milk: Gene Therapy Could Transform Goats Into Pharmaceutical Factories

Researchers have used gene therapy to reduce the time it takes to breed goats capable of producing therapeutic proteins in their milk, such as insulin or those that fight cancer. (Credit: iStockphoto)

University of Pennsylvania researchers have used gene therapy to reduce the time it takes to breed large animals capable of producing therapeutic proteins in their milk, such as insulin or those that fight cancer. This represents a significant milestone in drug development, as current methods involve cloning, which takes more time and generally costs more.

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"Having an easier way to harness nature's power to produce large quantities of specific proteins in milk could increase the availability of drugs for people who could otherwise not afford these treatments," said Ina Dobrinski, one of the researchers on the study.
The study also is significant because it may also be a new way to eliminate diseases in future generations of animals, such as those used for livestock. Here's why: To get the goats to produce specific proteins, the researchers used radiation to kill a portion of a male goat's germ cells (the cells that produce sperm). Then they used a modified adeno-associated virus (a well studied and tolerated gene therapy vector) to insert a gene in the remaining cells. Once the new gene took hold in the germ cells, a predictable number of offspring carried the gene necessary to produce the desired protein in their milk.
The advance is immediately valuable for pharmaceutical development and biology research, but a similar approach could be used to bolster the food supply by eliminating genetic disorders in animals over several generations. It is also possible that once perfected, this technique could eliminate disease genes in humans over several generations, assuming ethical concerns can be resolved adequately.
This study is published in the February 2008 print edition of The FASEB Journal.
"For thousands of years, people have domesticated cows and goats to make milk, butter and cheese. And for thousands of years dairy products have been used as folk remedies for practically every human illness. Most have been completely ineffective." said Gerald Weissmann, MD, editor-in-chief of The FASEB Journal. "So it is reassuring that modern science would find a way to use the milk we drink to yield of drugs that actually work."

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