Expression systems

Biopharmaceutical production in transgenic plants is counting on technological platforms whose basic elements have been developed by Transactiva, independently or in collaboration with public research centers. Specifically, Transactiva has focused on two expression systems, namely the tobacco leaf and the rice seed systems. The system adopted in the specific case depends upon the type and amount of the pharmaceutical molecule to be produced. Other relevant factors are the need for a swift production or the level of interference in the cell metabolism expected for or shown by the recombinant protein.

The tobacco leaf system

Tobacco is a highly responsive species in vitro and can be easily transformed; many cultivars with a high leaf biomass production are available. These characteristics, combined with a relatively fast vegetative growth, make tobacco an almost ideal species for the rapid production of pharmaceutical proteins which are normally needed such as drugs for a personalized healthcare (mg quantities).

Transactiva has developed a platform for the swift production (e.g. few months) of patient-specific vaccines to contrast non-Hodgkin lymphoma relapse; vaccine quantities largely sufficient to treat one patient are synthesized within a single selected plant. In order to set up this technology, Transactiva has designed a highly performant expression vector in which the sequence coding for the protein of interest is under the control of a light-inducible promoter; the best combination of a patented leader and a suitable terminator is disclosed in this vector. Further research has demonstrated that the same technology can be effectively used for the industrial production of antibodies.

Transactiva has also optimized transformation and regeneration methods, modifying the protocols for the micro-manipulation of plant tissues in vitro and setting up a hydroponic-based cultivation system with led lighting. With this facility, a high leaf biomass production is achieved in few weeks under standardized growth conditions, full traceability and confinement of transgenic plants. The whole cultivation system can be easily implemented and scaled-up by industry. Since plant growth occurs independently from the season, several productive cycles can be completed within a year. 

The rice system

Rice transformation is a well-acquainted technique in Transactiva. For transformation purposes, the CR W3 variety (selected by the public authority Ente Nazionale Risi, Milan) is used as genetic background donor; it is a variety unfit for human consumption, harboring a range of genetic markers and morpho-physiological traits which make it clearly distinguishable from all other rice cultivars, irrespective of their geographical origin.

Transactiva gained the exclusive right on CR W3 in 2007 and since then has developed molecular vectors for the endosperm-specific expression of recombinant proteins at levels adequate for industrial exploitation.

Endosperm is a triploid tissue of the seed, almost dehydrated at harvest, whose main constituents are starch and a narrow set of storage proteins; it can be easily and economically isolated from seed through industrial processing. The removal of glumes, pericarp, seed embryo, aleuronic and sub-aleuronic layers greatly favors the extraction and purification of heterologous proteins because the lipid content and the protein contaminants are dramatically reduced. The low water content associated with the presence of proteinase inhibitors reflects in a lack of degradation processes and the possibility to produce large amounts of recombinant proteins as well as to preserve them directly in the biomass without prior extraction. Therefore, a more flexible management of the production cycle is achieved together with the possibility to store product provisions at an intermediate stage of processing. Moreover, endosperm is a physiologically permissive tissue, hence it is an ideal site for expression, accumulation and long-term storage of phytotoxic proteins.

Using this system, Transactiva has developed a production platform for human lysosomal enzymes that is extremely competitive compared to current standards.

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