UNIFORGluca: UNIFOR’s transgenic goat whose milk contains a human protein that can be used to treat Gaucher’s diseaseUNIFOR
Gluca the goat lives in a special refuge at the University of Fortaleza (UNIFOR) in the state of Ceará. She is the first Latin American transgenic goat produced through the technique of cloning using genetically modified cells. Her name comes from a protein she carries in her milk, glucocerebrosidase, which works to process glucocerebrosides, a type of cell lipid. People who are unable to produce it experience problems with organs such as the liver, spleen and central nervous system, in addition to bone pain. The symptoms are part of the clinical description of a rare genetic disorder known as Gaucher’s disease. Gluca is part of a UNIFOR experiment so that transgenic goats have glucocerebrosidase in their milk that once extracted and purified can be made into a biologic to fight the disease. In October 2015, the transgenic herd housed at the university’s Center for Experimental Biology (NUBEX), consisting of Gluca and a goat cloned from Gluca named Beta, could get bigger. The eldest is now pregnant with two or three offspring—ultrasound was unable to determine the exact number — and there is a 50% chance that each of the offspring will be transgenic. This is because the father is not a transgenic animal.
When Gluca, who was born in March 2014, gives birth, it will be the first time that she experiences normal lactation. When she was six months old, researchers induced lactation through the use of hormones to determine whether glucocerebrosidase are present in her milk. “When Gluca is lactating, there will be a lot more milk available to determine its composition and functionality and run purification tests,” says Marcelo Bertolini who coordinated the project at UNIFOR, where he worked for six years. Since July 2015, he has been a professor at the Federal University of Rio Grande do Sul (UFRGS). “During induced lactation, the presence of glucocerebrosidase in Gluca’s milk ranged from 4 to 8 grams per liter (g/l). If we get an average of 5 g/l with four goats, we’ll be guaranteed to have the number of animals needed to supply all of the nearly 700 patients suffering from Gaucher’s disease in Brazil,” Bertolini says. With Gluca and Beta, and if the two or three offspring are female and have the protein in their milk, the full herd would be able to produce the biologic.
Availability of the Brazilian medication will not be immediate however. The protein molecule found in the milk would first need to be purified and then an injectable drug would have to be produced in a process that could take five years or more. The group of researchers plans to wait for the initial analyses of Gluca’s natural milk before seeking partnerships with companies and research institutions, a critical requirement in conducting clinical trials and submitting medication to the Brazilian Health Surveillance Agency (ANVISA) for approval. The Quatro G Company, a partner of the UNIFOR group, will get Gluca’s milk in order to purify the protein. At the start of the project, the company, headquartered at the Technology Park of the Pontifical Catholic University of Rio Grande do Sul (PUC-RS) in Porto Alegre, handled production of the gene sequences for inserting the glucocerebrosidase gene into the genome of the goat.
Gluca’s development was the result of the technique of cloning using genetically modified cells. The first step is to introduce a complex gene sequence containing the human gene of the glucocerebrosidase protein into the cells of a goat. When the gene is absorbed into the animal’s genome, researchers select the best cells for insertion into the ovocytes, which are the female reproductive cells from which the maternal DNA has been removed. Then the cloned transgenic embryo is transferred to a non-transgenic goat to establish gestation. “The efficiency of this method is still relatively low, but the result has been positive given how extremely valuable the transgenic goat is to science,” explains veterinarian Leonardo Tondello Martins, a professor at the Health Sciences Center at UNIFOR. “In the experiment that resulted in the birth of Gluca, we initially transferred 858 embryos among 60 recipients. Of the 11 pregnancies identified, one healthy transgenic animal was born: Gluca,” says Martins. The project received R$2.4 million from the Brazilian Innovation Firm (FINEP), under its Economic Support Program secured by Quatro G in partnership with the UNIFOR researchers. The Anglo-Nubian goats were made available by Esperança Agropecuária, part of the Edson Queiroz Group, which is also associated with the foundation that goes by the same name and supports UNIFOR.
Different origins
Producing a medication to treat Gaucher’s disease in Brazil could save the government more than R$140 million annually – R$200,000 per patient – over medications imported for patients who receive them free of charge. “From the market data currently available to us, we estimate that using animals as bioreactors for complex drugs could be up to 80% less expensive than using other biotechnology techniques,” Bertolini says.
One of the medications used to treat Gaucher’s disease is Cerezyme, which is produced using a recombinant DNA technique characterized by the insertion of a gene from a protein found in the ovarian cells of Chinese hamsters, developed by the Genzyme Corporation of the United States, currently a subsidiary of the French company Sanofi. The other is Uplyso, from the Israeli company Protalix, which uses cells from transgenic carrots to produce the drug. These medications do not contain glucocerebrosidase itself, but rather substances that promote the same effect.
There are currently two examples on the market of medications that are produced using animals, as reported by the International Society for Transgenic Technology (ISTT). The first is ATryn, approved in 2006 in Europe and in 2009 in the United States. It was developed by Genzyme Transgencies Corporation (GTC) Biotherapeutics, now owned by the American firm rEVO Biologics, which makes antithrombin alfa from transgenic goat’s milk. The substance is used in the treatment of thromboembolisms in surgery by patients suffering from hereditary antithrombin deficiency, a disorder that causes abnormal blood clots in the blood vessels. The second medication, approved in 2013, is Ruconest, used to treat hereditary angioedema, an ailment that affects people born with a deficiency of C1 esterase inhibitor (C1-INH). The disease causes painful swelling in soft tissues of the body, especially in the abdomen, face and genitals. The solution found by the Dutch company Pharming, which developed the medication was to express and purify the enzyme in the milk of transgenic rabbits. According to agronomist Elibio Rech, a researcher at Embrapa Genetic Resources and Biotechnology in Brasília, pharmaceutical companies are very interested in using animals to make drugs . “We think the product developed [by the group at UNIFOR] is extremely important for Brazil from both a scientific and technological standpoint,” Rech says.
UECEA 10-month-old female Canindé transgenic goatUECE
In Ceará as well
In Brazil, the groundbreaking experiment in the field of transgenic animals also took place at the State University of Ceará (UECE) in Fortaleza. In all, seven transgenic goats have been born since 2008 in a project led by Vicente José Freitas, a professor in the School of Veterinary Sciences at UECE. The animals carry the gene encoding the human granulocyte colony stimulating factor (hG-CSF), which is critical for strengthening the immune system of patients such as those with AIDS who are vulnerable to opportunistic infections while undergoing chemotherapy. There are presently medications produced abroad that have the same effect, offering similar proteins as those synthesized by recombinant bacteria or Chinese hamster ovaries. The proteins found in the transgenic goats at UECE and UNIFOR are the same as those in humans, thus they may adapt more easily to the body.
The UECE experiments were carried out in partnership with the team led by Professor Antonio Carlos Carvalho at the Federal University of Rio de Janeiro (UFRJ) along with two Russian researchers: Irina Serova of the Institute of Molecular Genetics of Moscow and Lyudmila Andreeva of the Institute of Cytology and Genetics of Novovsibirsk. “We were able to obtain 600 micrograms (mcg) of the protein per liter (l) of goat’s milk. The only reference we have was from a project in South Korea carried out by two research institutes and the firm Hanmi whose experiments rendered 50 mcg/l,” Carvalho says.
“Our problem is finding a business partner to purify the milk protein and run the clinical trials for us. Years have gone by and this still has not come to pass,” Carvalho laments. “My hope is for a partnership with the Vital Brasil Institute in Rio de Janeiro which last year expressed interest in using our transgenic animals for [protein] purification and drug production.” The researcher adds that because there already are various medications produced using bacteria and hamster ovaries, although not containing 100% human proteins, the pharmaceutical industry is not interested in changing its system of production. He adds that the cost of fabrication in animals is less than in cells and cites the scientific article “Expression Systems and species used for transgenic animal bioreactors,” by researchers from one Japanese and two Chinese universities. Their report shows that in general, 50 kilograms of protein per year would cost $147 per gram (g) for cell culture production but only $20/g if produced in transgenic animals. To Carvalho, despite all the difficulties involved in investing in animal bioreactors, the experiments conducted thus far have shown that there is still time for Brazil to become a reference in this field.
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