All roads lead to milk: transgenic and non-transgenic approaches for expression of recombinant proteins in the mammary gland
Castro, Fidel OvidioToledo, Jorge RobertoSánchez, OlibertoRodríguez, Lleretny
Background: With the advent of transgenic technology to farm animals, it became possible to express recombinant proteins of high complexity in the body compartments and fluids of these animals; the term "pharming" was coined. This was a tremendous achievement taking into consideration the high costs associated with conventional (cell-based) production methods and the incapacity of lower organisms to adequately process complex proteins. The mammary gland had been the organ of choice and milk the appropriate vector for successful expression of many recombinant drugs of high added value. Review: While theoretically the mammary gland is able to carry out all the complex post-translational changes related with glycosylation or others, in the practice, not all proteins can be actually processed in a way that closely remembers the wild protein, thus making difficult the production of some proteins in full biologically active form. This is especially true for complex (branched) forms of glycosylation as it is the case of human erythropoietin (hEPO), or gamma carboxylation of blood clotting factors, to mention a few examples. These cases are discussed in this review, with special emphasis in the glycosylation of hEPO. In spite of the imperfectness of the mammary gland to accurately add some sugar residues, it continues to be the most desirable organ to which target gene expression, due to its potent biosynthetic machinery and the possibilities to amend the said incapacity to glycosylate appropriately all kinds of proteins. In line with this, the European Medicines Agency first (in 2006) and the Food and Drug Administration later (2009) approved the first milk-derived recombinant protein for human use, (ATryn; human anti-thrombin-III) after more than two decades of thorough reviews and test, thus opening the way for future massive production of blockbuster drugs using the mammary gland as bioreactor. In this job we reviewed briefly the state of the art of mammary gland-based production of recombinant proteins with emphasis in two different systems to target it. In the first approach, a transgenic mammal carrying appropriate mammary specific gene promoter linked to a transgene is made, then grown, mated, its milk tested for the presence of the protein, if expression levels and biological activity of the proteins meet the requirements, then a production flock is created from the founder(s) and milk collected and processed. In this way, most of the recombinant proteins produced in the milk had been created, including the leading drug ATryn. We developed an alternative method for transient viral vectors-mediated transduction of the mammary gland, using constitutive viral promoters linked to the transgene, thus producing very quickly high amounts of the desired protein. The drawback of this method is its transient nature; the advantage is the fastness and easiness to produce grams of recombinant proteins in the milk of otherwise non-transgenic mammals. In this way several drugs had been produced. Notably one of them, the E2 antigen of classic swine fever (CSF) had been secreted in biologically active form at high levels in goats´ milk; a veterinary vaccine formulation was established and tested successfully in clinical trials that included viral challenging with CSF. It is foreseen that this vaccine could be in the market this year and became the first recombinant drug produced in the milk of non-transgenic animals to get regulatory approval. In this article, we also reviewed the state of the art of different body fluids as vectors for recombinant protein production Conclusions: At least for the next coming years, all the animal-based recombinant protein production ways will lead to the milk.
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