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Plant Tissue Cultures as New Production Platforms for Terpenoid Based Anti-Cancer Drugs

Final Report Summary - DRUGTISSUECULT (Plant Tissue Cultures as New Production Platforms for Terpenoid Based Anti-Cancer Drugs)

The overall objective of DrugTissueCult was to provide ground-breaking fundamental understanding of sesqui- and tri-terpenoid biosynthesis and evolutionary dynamics of species specific metabolism, not only in Thapsia and Withania, but also within plants in general.
DrugTissueCult was established to promote the training of 2 ESRs in the use of Plant Tissue Cultures as New Production Platforms for Terpenoid Based Anti-Cancer Drugs in a collaboration between University of Copenhagen and Alkion Biopharma.
In DrugTissueCult (www.drugtissuecult.eu) we focused on two terpenoids with anti-cancer properties that are produced in very low amounts their original plant. Since both are of high pharmaceutical and economical value there is a need for higher production levels. The two compounds were the sesquiterpenoid Thapsigargin produced in Thapsia garganica and the triterpenoid Withaferin A produced in Withania somnifera. Subsequently, work on hairy roots for squash, Cucurbita pepo, and in watermelon, Citrullus lanatus, for the production of the anti-cancer terpenoids cucurbitacins has been established as an integrated part of DrugTissueCult.

DrugTissueCult has established a sustainable production of these complex anti-cancer molecules and also contributed to the understanding of their complete metabolic pathway in each relevant plant species. Accordingly, production of Thapsigargin and Withaferin A in plant based bioreactors is now an attractive opportunity to achieve a sustainable and industrial exploitable supply. The results from DrugTissueCult will enable future manufactures to establish production pipelines that can supply bulk scale of both chemicals, and possibly others.
The work initiated on C. pepo hairy roots has secured a Danish grant to pursue this promising area of biotechnological production of cucurbitacins. The new production platform for Thapsigargin has been secured through a patent, but the results for all three species are also being published as scientific peer reviewed manuscripts.
For all plants, the research has also lead to further understanding of the biochemical pathways of these valuable compounds. These results have been or are being published in peer reviewed scientific papers.

The goals of WP1 were to establish Thapsia garganica in vitro tissue cultures and study the production of thapsigargins. Shoot cultures have been obtained using TIBs (temporary immersion bioreactors) as a highly regenerative system to maximize and study shoot biomass production. The overall result was that the shoot cultures within 3 weeks could produce thapsigargin at a level of 0.4% of the d.w. and together with the chemical derivative nortrilibolide (which by two chemical steps can be converted to thapsigargin) the overall level was between 2.2 and 2.5% dw. This is much higher than in the wild population where a overall level of these compounds are approx. 1.5 % dw. This promising result has been patented and can be used for future production of the valuable compound.

The goal of WP2 was to establishing hairy root lines and shoot cultures of Withania somnifera producing Withaferin A. W. somnifera propagation is difficult through conventional methods of cultivation (seeds), due to several reasons, such as poor yield, poor viability and susceptibility of seeds from microbia. During the project, different in vitro W. somnifera tissue cultures have been established: Shoots, roots, calli and cell suspensions, of which withaferin A was detected only in shoot cultures. Using elicitation the level of withaferin A was increased up to 3 fold enabling production of 2 mg withaferin A/g dry weight in the best growing conditions.

The main goal of WP3 was to study the biosynthesis and regulation of thapsigargins in the in vitro tissue cultures developed in WP1 to enhance the production output. The lines from WP1 were used to gain a further understanding of the biochemistry in T. garganica. The expression and tissue localization of two key genes in the pathway, TPS2 and CYP76AE2 were studied in vitro roots, stems and leaves by RT-PCR and in situ PCR. These results confirm that the plants do not alter the biosynthesis in vitro when compared with data obtained from wild populations, and that the shoot cultures from WP1 harbored the cells needed to produce the thapsigargin in the same way as wild type plants.

The goal of WP4 was to elucidate at least one of the last steps in withaferin A biosynthesis pathway. This part of the project was not achieved mainly due to the difficulties in establishing an in vitro system where withaferin A was significantly induced. However, a transcriptomic dataset has been generated, analyzed and a list of gene candidates for witanolide/withaferin A biosynthesis has been generated.

The main goal of WP5 is the training of the two ESRs. They have been trained in Public-private partnership and exposed to practical Entrepreneurship through the industrial partner. They have received formal training as PhD students through enrolment at the PhD School of SCIENCE at UCPH for training activities such as courses. The training has given them knowledge of using multi-disciplinary approaches based on existing and new forms of collaboration and developed their individual curriculum of discipline specific and multi-disciplinary courses.