Abstract
Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.
Highlights
Recombinant human antithrombin ATryn, in Europe[5] and the USA6 and, more recently, the commercialization of Ruconest, an esterase inhibitor for the treatment of dermal swellings[7]
When choosing an expression system, two main questions have to be addressed: Does the host system yield functional protein? Is the production of the recombinant protein economical or are there cheaper alternatives? To date, most biopharmaceuticals that require complex posttranslational modifications are produced in mammalian cell culture but the production and commercialization of recombinant proteins from milk of genetically-modified animals has highlighted the potential for this less-costly in vivo approach; restricting the genetic modification to only the protein-producing organ would be cheaper, simpler and faster
We extended the concept of transducing the mammary epithelium with recombinant adenovirus, to test rAAV efficacy for the production of recombinant proteins in the mammary gland
Summary
Recombinant human antithrombin ATryn, in Europe[5] and the USA6 and, more recently, the commercialization of Ruconest, an esterase inhibitor for the treatment of dermal swellings[7]. An alternative strategy is to directly introduce gene constructs into the secretory epithelial cells of the mammary gland. Such somatic gene transfer can be applied directly to a lactation-competent animal with the potential to provide rapid production of a recombinant protein. Two studies were able to demonstrate expression of the recombinant protein for up to three weeks[11,21] This poor persistency is generally believed to be caused by the induction of a potent immune response against the virus[22]. The gene construct introduced by rAAV vectors stably persists episomally and is capable of mediating long term transgene expression in mammalian somatic cells[27]. To date no AAV serotype has been used to successfully transduce mammalian epithelial cells of the mammary gland of pregnant or lactating mammals
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