PORTLAND, Ore. - Researchers at Oregon Health Sciences University report the production of the world's first genetically modified nonhuman primate as an important step toward designing and perfecting new treatments for human genetic disorders. This research was conducted by Anthony Chan, Ph.D., a staff scientist at Oregon Regional Primate Research Center and colleagues. Scientists were directed by Gerald Schatten, Ph.D., a senior scientist at the ORPRC; research director of the Center for Women's Health; and professor of obstetrics and gynecology, and cell and developmental biology, in OHSU's School of Medicine. These results will be published in the Jan. 12 issue of Science.
Currently mouse models are utilized for investigating many genetic disorders ranging from muscular dystrophy to cancer. However, many of these mouse models do not accurately mimic symptoms found in patients affected by these diseases. This need has prompted development of improved disease models in monkeys.
ANDi, (inserted DNA, spelled backward), is the first genetically modified rhesus monkey produced using techniques commonly utilized for genetic modification in other animals as well as in human gene therapy. DNA was introduced to monkey eggs through a neutralized retroviral vector, which carried a foreign gene, GFP (green fluorescent protein). After the foreign DNA was introduced, eggs were fertilized by intracytoplasmic sperm injection (ICSI). A total of 20 embryo transfers resulted in five pregnancies and three healthy male offspring, including ANDi. The presence of the foreign gene in ANDi has been verified by DNA analysis.
Scientists chose GFP due to its common use and documented safety in animals. While genetic testing has revealed the presence of this foreign DNA in accessible cells throughout the monkey, ANDi is healthy and is being reared in dedicated group housing with other rhesus monkeys at OHSU's Oregon Regional Primate Research Center.
"ANDi's birth along with last year's birth of Tetra, the first rhesus cloned by embryo splitting, is another incremental step in accelerating the discovery of innovative cures for devastating diseases. These diseases range from diabetes and Alzheimer's to breast cancer and HIV," said Schatten. "By developing cloned, genetically modified and stem-cell-derived primate models, scientists will be able to carefully and rigorously test the most innovative therapies, using the fewest animals, so that these treatments are perfected and optimized before being used to treat humans."
Judith Vaitukaitis, M.D., director of the National Center for Research Resources at the National Institutes of Health, notes the significance of this research toward developing and using animal models that are similar to humans. "While the mouse model can be credited with countless breakthroughs, scientists are increasingly turning to higher research animal models in order to develop new human gene transfer therapies. The modified monkey model helps bridge this gap. It provides a much needed animal model of a human disorder that allows scientists to test whether or not the level of expression of the transferred gene can correct for the patient's missing or malfunctioning gene and can assess whether the gene transfer vectors have any significant side effects." The NCRR and the National Institute of Child Health and Human Development, two institutes of the National Institutes of Health, funded this research.
Background information on genetic modification of primates
Currently scientists have an arsenal of mouse models available to study the genetic basis of numerous diseases and disorders, some of which are being discovered by deciphering the human genome. Many of these mouse models have been produced through the introduction of new genetic material, or DNA, to the animal and express such diseases as cystic fibrosis, muscular dystrophy and cancer. Although these mouse models have been invaluable in furthering the understanding of the genetic basis of diseases, many of these models do not accurately mimic the symptoms seen in patients.
The intent of this research is to develop and optimize techniques currently used to produce mouse disease models in an animal more closely related to humans, the monkey. Once optimized techniques are available to produce genetically modified monkeys, relevant disease models can be produced, which will lead to improved therapies and ultimately cures.
In order to produce a genetically modified monkey, Anthony Chan, Ph.D., and colleagues introduced the GFP gene (green fluorescent protein) to monkey eggs, which expressed a green protein. The GFP gene was used due to the ease in detection, it is commonly used throughout research labs, and it's been proven safe in animal models. After the GFP gene entered the egg, fertilization was accomplished by injecting a single sperm into the egg, known as intracytoplasmic sperm injection (ICSI).
The GFP gene was transmitted to the monkey egg using a neutralized retroviral vector. Most commonly, viruses are associated with illnesses. However, scientists and physicians utilize neutralized vectors to both produce transgenic animals for disease models and use similar viruses to treat diseases in humans. Viruses have the unique ability to introduce DNA into cells, thus scientists take advantage of this ability to introduce genetic information to produce relevant animal models or clinical treatments to patients. These viruses have been modified with no ability to replicate or cause disease.
Chan began with 224 monkey eggs, and while using two versions of the viral vector (each varied in their control switch, also known as a promoter), he chose 40 embryos to transfer to surrogate monkey females, at two embryos per monkey surrogate. From these transfers, five pregnancies were established, consisting of one blighted pregnancy (no fetal development), one set of twins and three single pregnancies.
At approximately halfway through the pregnancy, the twins were unfortunately lost to miscarriage, but each of the three single pregnancies led to healthy offspring, one carrying the GFP reporter gene, who is named ANDi. The name ANDi was chosen due to the method in which the reporter gene (GFP) incorporates into the monkey's DNA. The reporter gene is first copied in a reverse orientation and then is inserted into the monkey's DNA (inserted DNA backward = ANDi). The miscarriages also were carrying the GFP gene.
This work demonstrates that through the use of a modified virus, scientists now can produce genetically modified monkeys to use as improved models of human disease. With the availability of improved animal models, treatments and cures will more quickly move from laboratories to patients.
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