LUCIANO MOREIRA/FIOCRUZMosquito with green fluorescent protein transgenesis markerLUCIANO MOREIRA/FIOCRUZ
A medicine in capsule form packaged in a blue and white box similar to many other products sold in drugstores and hospitals has recently been launched in Europe. The drug is used to treat thrombus-embolism in patients with hereditary anti-thrombin deficiency, a condition that causes clots within blood vessels. There is nothing unusual about launching a new drug. The main difference is that this one is made from a substance extracted from the milk of transgenic goats developed by US company Genzyme Transgenics Corporation (GTC) Biotherapeutics, which also manufactures the medication. The drug is distributed in Europe and in Canada by Denmark’s LEO Pharma. The aforementioned substance is human antithrombin III (AIII), a protein introduced into the genome of a goat through a technique called recombinant DNA. Using biotechnological means, this molecule is placed in the embryo at the very start of its formation, in the form of a codifier gene of the same protein. Later, the AIII is produced in the goat’s mammary cells. During the nursing period, each goat produces three liters of milk a day, which is equivalent to the production of some three kilograms of purified protein a year.
The animal transgene technology strategy is used by many companies and research institutes, especially in Europe and the United States. Various drugs are going through trials prior to being released in the market. In Brazil, this kind of research is limited to research teams at universities and research centers. Approximately ten research teams in the country produce transgenic animals, such as genetically transformed mice, to obtain some protein of therapeutical interest or for use in scientific experiments. Many of these researchers attended the First Brazilian Symposium on Transgenic Technology, held at the Federal University of São Paulo (Unifesp) in March and listened to the opening lecture delivered by 2007 Nobel Prize Winner in Physiology and Medicine, Oliver Smithies (read more about this in Pesquisa FAPESP nº 146).
“I was quite surprised by the Brazilian research teams. I had no idea there were so many and I noticed that many of them met for the first time at the symposium. This is very important for the country’s industry,” said Spain’s Lluís Montoliu José, president of the International Society of Transgenic Technology/ ISTT, created in 2006, and a professor at the Autonomous University of Madrid in Spain. In Montoliu’s opinion, the use of transgenic animals has a broad scope for science and for other economic sectors. “There’s a lot to be researched, but we already know, for example, that purifying proteins in milk is relatively easy, whereas in the blood it’s more complicated (because of the possibility of diseases being transmitted). Besides the production of human proteins and future vaccines from animal milk, we can also avail ourselves of specimens to study diseases, transgenic animals to improve production and prevent the onset of diseases in cattle, cure diseases in fish and transplant pig organs, for example, to human beings.”
The transgenic animals used to produce proteins, called bioreactors, are becoming the solution to obtain pharmacological compounds that act as repositories of substances produced by most human beings, yet sometimes lacking, or barely present, in some individuals. “This system is less expensive than the production techniques of recombinant proteins used by enormous lab structures with the help of bacteria, yeasts, or mammal cells, which require an expensive reactor or human plasma to be produced,” says professor João Bosco Pesquero, director of Cedeme the Center for Development of Experimental Models for Medicine and Biology Unifesp and the symposium’s coordinator. The products manufactured by using the recombinant DNA technique with bacteria or with the cultivation of ovular cells from Chinese hamsters, also referred to as CHO, include the Human Growth Hormone (hGH) and the blood clotting Factor IX, needed by hemophiliacs, that is also produced from human plasma.
A hemophiliac patient in Brazil can cost more than R$ 70 thousand a year, because the necessary drugs must be purchased abroad and paid for by the federal and state governments. Hemophiliacs need these proteins to avoid hemorrhages. Two announcements made last April promise that traditional technology will be used to supply Factors VIII and IX, made in Brazil, at low cost. The first factor will be manufactured by the Hemobras (a Brazilian hemoderivates and biotechnology company), that is linked to the Ministry of Health, in partnership with the Federal University of Rio de Janeiro (UFRJ), while the other factor will be produced by the Butantan Institute, which is beginning to prepare its Plasma Processing Unit.
Despite the promise of being low cost drugs, these new transgenic medicines are still expensive. In the case of Atryn, the commercial name of the antithrombin manufactured by GTC, each ampoule costs – 2,5 thousand in the European market. In the United States, Atryn is at the final stage of clinical approval. “The development of transgenic animals is linked to biotech companies that have invested a lot in this field. Transgenic animals make it easier and cheaper to test new drugs because an active principle can be tested against a given human protein in a mouse, for example,” says Pesquero. “This eliminates some stages of clinical tests. A gene that is important for diabetes can be tested in a mouse prepared with a human gene instead of the animal’s gene. If the drug bonds with the human protein, this means the drug will work. This reduces the testing time for new drugs and checking for side effects. In addition, fewer animals are used in the lab.”
Flamínio Araripe/Office of the Secretary of Science and Technology of CearáTransgenic kid goats of the canindé breed in CearáFlamínio Araripe/Office of the Secretary of Science and Technology of Ceará
In view of the help that they provide for studies on diseases and the development of new drugs, most of the transgenic animals are those used in labs. “There are thousands of transgenic animals, especially mice, worldwide. Most are produced by laboratories linked to universities.” Some companies, such as Australia’s Ozgene and Switzerland’s Polygene, also produce customized animals. A group of mice with a knocked-out gene – a technique that switches off a gene and thus prevents the production of a protein – a situation that can cause a disease, for example, can cost up to US$ 45 thousand.
Pesquero’s team at Unifesp develops mice for the researchers from Unifesp and other institutions, such as the Heart Institute at the Medical School of the University of São Paulo. One of the research team’s achievements was the production of a female goat that expressed the Factor IX in milk (read more about this in Pesquisa FAPESP nº 117). In 2005, the idea was to express a protein in the mouse; if everything went smoothly, the system would be transferred to generate bovines at the Embrapa Genetic Resources and Biotechnology in the capital city of Brasília. “The goal at the moment is to generate cows or goats able to transform grass into milk inexpensively. This option is cheaper, even if the purification phase of the protein in the milk is included, – says Elíbio Rech, a researcher at Embrapa. “We need to keep in mind that monoclonal antibodies, which are molecules produced by biotechnological engineering, studied mostly for cancer treatment, can be used as a means of diagnosis in vaccines and can also be produced in mammary glands,” he adds.
Research on and production of transgenic animals is very important for Brazil. If we do not produce transgenic animals here, we will soon have to import them,” predicts Pesquero, who is currently also working on transgenic rabbits to express commercially interesting therapeutic proteins, such as Factor IX and the follicle-stimulating hormone (FSH) for bovines. “Once we establish the technique to be used in rabbits, we will be able to produce different proteins.” The symposium showed that Brazil has research teams producing at different levels. The latest news in this respect was the birth of two goats – a male and a female. The animals were born between March 11 and 20 in the city of Fortaleza, State of Ceará. The genome of these two transgenic animals contains a gene that produces the protein of the human granulocyte colony stimulating factor (hG-CSF), used in cases of immunodeficiency, such as AIDS, and in the treatment of cancer patients undergoing chemotherapy or of people who have had myocardial infarctions or strokes. The experiment was conducted by a team from the School of Veterinary Medicine at the State University Ceará (Uece), coordinated by professor Vicente José Freitas in partnership with the team of professor Antonio Carlos Carvalho, from the Federal University of Rio de Janeiro (UFRJ), in addition to researchers Irina Serova and Ludmila Andreeva, from the Russian Academy of Sciences. At the beginning of April, Freitas got the confirmation that two kid goats, plus a stillborn, – out of a batch 23 newborn kid goats – had the gene for the hG-CSF in their genome.
The tests were conducted at the State University of Ceará and confirmed at UFRJ. This was the research team’s second attempt. The result of the first attempt, conducted in 2006, was one transgenic kid goat, which died 19 days later from an infection unrelated to the transgenesis procedure. The method used by Freitas is the standard method used by researchers worldwide – the pro-nuclear injection, in which a copy of the DNA and the protein gene to be expressed are injected into a recently fertilized ovum. Then the embryo is transferred to the uterus of a procreating female that is not necessarily the ovum’s donor.
The transgenic couple will make it possible for the goat to mate with the nanny goat so that approximately 75% of the kid goats contain the human protein. “Our joy is twofold, because the couple belongs to the canindé breed, an endangered species in Brazil’s Northeast region. The breed stemmed from goats brought by the Portuguese who colonized Brazil. “Thanks to the transgene technique, we added value to the breed because the transgenic goat can mate with a non-transgenic female of the same breed, with a 50% chance that the offspring will be transgenic producers of the hG-CSF protein.” Another possibility is cloning these animals. The mating would result in 100% transgenic specimens. Freitas, as GTC, believes that the goats are at an advantage in comparison to other animals in the role of bioreactors to produce many of the proteins for medical therapy purposes. “Goats often give birth to three kids after five months of pregnancy, while a cow only gives birth to one calf after nine months of pregnancy, rarely two.”
Miguel boyayanMouse with human factor IX geneMiguel boyayan
In Latin America, Brazil ranks behind Argentina in terms of studies on the transgene technique in animals. Argentina’s Bio Sidus company had already announced in 2004 that it had produced a cow that produces human growth hormone in its milk, even though the company is not selling this product yet. In April of last year, the company also announced the birth of four transgenic Jersey cows – specialized in milk production – able to produce human insulin in the milk. The company informed that a batch of 25 cows would be enough to supply Argentina’s insulin needs – Argentina’s diabetic population is 1.5 million people. But Bio Sidus correctly states that the production of transgenic animals with this potential is only part of the technological path to achieve the end product. The technique for extracting and purifying a human protein from cow’s milk has to be refined to eliminate possible contaminations.
In Brazil, another research team – this one from the Federal University of Pelotas (UFPel), in the State of Rio Grande do Sul – has begun research on transgenic hens. By diluting the codifier gene of green fluorescent protein/GFP in the sperm of the rooster, followed by insemination, the researchers, coordinated by Professor João Carlos Deschamps, were able to obtain a stillborn chicken, but nonetheless, it had expressed the aforementioned protein. “The experiment was useful to show how the technique works,” says professor Denise Bongalhardo, a member of this research team. “Experiments conducted in Europe show that transgenic eggs are feasible, though they express very little protein and are not commercially feasible yet,” Denise explains. “The advantage of the hens is that they produce approximately 330 eggs a year with a short interruption between generations; in addition, the egg whites are naturally sterile. The point is that eggs come packaged and are easy to store.” The next step of this research team from Rio Grande do Sul is to express a human blood coagulation protein, factor IX, in chicken eggs. Denise also pointed out future uses for transgenic chicken, such as the manipulation of production characteristics, genetic improvement and resistance to diseases. In addition to the production of drugs via animal milk or eggs to obtain human proteins, one can treat illnesses such as dengue and malaria by means of the genetic manipulation of the mosquitoes that transmit these diseases.
An alternative still at the early stages of feasibility studies is the introduction of a gene into the carrier mosquito (the Aedes aegypti), which causes dengue, or the Anopheles spp., which causes malaria; the objective is for the gene to block the virus of the former and the protozoan of the latter, thus blocking disease transmission. With this objective in mind, the research team, coordinated by researcher Luciano Andrade Moreira, from the René Rachou Research Center, of Fiocruz (the Oswaldo Cruz Foundation) in the city of Belo Horizonte, State of Minas Gerais, developed a strain of the Aedes fluviatilis mosquito that can be a carrier of the parasite for avian malaria.
Action against the Plasmodium gallinaceum
Inside the mosquito is activated by means of the phospholipase A-2 protein, in muted and inactive form, found in bee poison. This protein has already been extensively researched. “The protein has to provide a barrier in the mosquito’s intestine to bar the plasmodium from penetrating the intestinal wall and, after forming the cyst, reach the insect’s salivary gland and be transferred to another bird at the moment of the sting,” says Moreira.
Moreira and his team used the DNA construction micro-injection technique in the embryo phase of the mosquito eggs to produce the transgenic insect. “We have reached our thirtieth generation of lab-created transgenic mosquitoes,” he says. A similar study on the dengue mosquito is being conducted under the coordination of Professor Margareth Guimarães, from the University of São Paulo (read more in Pesquisa FAPESP nº 131).
The animal transgene technique first appeared in 1982, when US researchers from the Universities of Washington, Pennsylvania and California produced a mouse that expressed the growth hormone of a rat. The result was that the mouse grew bigger than normal. “Nowadays, many people are working with transgenics, which makes it imperative to patent research results,” Pasquera points out. “In biotechnology, any development can be patented, but a transgenic animal cannot be patented in Brazil. The patent has to be related to the project for the genetic construction of the animal,” says chemical engineer Ricardo Amaral Remer, who is an intellectual property expert. Remer, who is a partner of consulting firm Atem&Remer, gave a lecture on this at the symposium organized by Unifesp. “What can be patented in Brazil is the expression system or the gene construction, or, further, the content and the form of introducing the gene into the animal,” he explained. Even in the case of expressing a protein that exists in another animal, such as Factor IX, it is possible to patent another production process related to this substance.
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