Dried tobacco leaves (Nicotiana tabacum) have with good reason fallen out of favour in western industrialised countries. Cigarette consumption has been declining for years, and with it, the cultivation of tobacco plants. Newcotiana is a European project that aims to show the tobacco industry a route into a sustainable future. “Molecular farming” are the magic words. This refers to modifying the composition of tobacco plants to replace cigarette nicotine with added value substances used in medicine and cosmetics.

Newcotiana is a large-scale project with 7.2 million euros of EU funding from the Horizon 2020 programme over four and a half years, involving 19 academic and industrial partners from eight European countries and Australia. Plant biotechnologist Prof. Dr. Holger Puchta from the Karlsruhe Institute of Technology is working on a crucial key technology known as CRISPR/Cas: "We want to apply the gene-editing technology CRISPR/Cas to the targeted and efficient modification of the tobacco genome. In this way, we hope to produce medically relevant proteins such as antibodies or growth factors.”

"Molecular farming" methods have been studied for several years. “Old genetic engineering” methods have been found unsuitable for plant breeding as they are relatively complicated and it takes a long time before suitable plants are available. One plant cell-based recombinant therapeutic protein drug has already been approved for application in human patients: genetically engineered carrot cells produce an enzyme that is absent in patients with Gaucher disease, a rare genetic disorder that affects an enzyme that normally breaks down materials for reuse in the cells. The drug, which is marketed under the name Elelyso, has been approved for treating patients with Gaucher disease in the USA, Israel and several other countries since 2012.

Research into CRISPR/Cas is progressing at a tremendous pace. Scientists worldwide are regularly coming up with new results on the use of the CRISPR/Cas gene-editing tool in plants. The first CRISPR/Cas-edited plants, e.g. soybeans with an altered fatty acid composition, are already being cultivated in the USA.

Currently, CRISPR/Cas is mainly used to selectively switch off individual genes. The gene-editing tool has been used in soybeans to block two genes, resulting in a higher-than-normal fatty acid content. CRISPR involves making a cut at a desired location in the genome, leading to a double-strand break. The double-strand break is repaired by the cellular machinery (non-homologous recombination). Errors in linking the two strands together again can occur, so that the targeted gene loses its function as the translation of the correct protein is disabled.

With CRISPR/Cas it is also possible to insert foreign DNA at specific genome loci. This is a prerequisite for turning tobacco into a biofactory. "So far, however, this so-called homologous recombination has been difficult to perform and is inefficient in plants," says Puchta highlighting that plant genomes are very large and that the ability for homologous recombination is probably downregulated in order to ensure the stability of the genome. "In our test plant, the thale cress, the technology has already been improved significantly," explains Puchta, who is confident that this hurdle will be overcome in the medium term.

Tobacco would have some advantages over conventional protein production systems. Above all, the use of tobacco would enable rapid production of large quantities of even complex proteins, such as antibodies or vaccines. These can currently only be produced in mammalian cell cultures or chicken eggs. Compared to bacteria, these systems are difficult to cultivate and have only a low yield. Moreover, a chicken egg only delivers one flu vaccine dose. This is expensive, and in the case of a pandemic, the supply would quickly dry up. A tobacco production system would be cheaper and easily scalable.

Bacteria cannot be used as production systems because they are unable to attach sugar residues to proteins, a process known as glycosylation. The latter occurs only in eukaryotic cells and is essential for the functioning of many proteins such as antibodies. "Eukaryotic tobacco plants equip proteins with sugar residues, but these are different to those in humans, which is why we would have to modify the enzymes," says Puchta.

Tobacco plants need to produce high enough quantities of protein in order to make them suitable for producing pharmaceuticals. However, extensive protein production induces a protective reaction that leads to the degradation of the protein produced. CRISPR could also be the remedy in this case, e.g. for impeding the function of the protein-degrading enzyme.

A gene-edited tobacco plant that produces complex human proteins carries foreign genetic information and is as such defined as a genetically modified organism (GMO) that automatically falls under the Genetic Engineering Act. "GM plants have to undergo numerous safety tests and initially have to be grown in closed greenhouses," explains Puchta. Field trials are now being planned in Spain in compliance with existing GMO regulations.

"However, tobacco plants have biosynthetic pathways that can easily be altered by introducing point mutations. This approach can be used to produce specific secondary plant substances,” says Puchta who, like many other scientists, had hoped that CRISPR-modified plants, which do not carry any foreign material and have only undergone minor and precise modification, would not be subject to the strict EU rules on GMOs.

However, in July 2018 the European Court of Justice (ECJ) ruled that the new plant breeding methods, all of which are grouped under the term "genome editing”, fall under the Genetic Engineering Act and plants created with the novel genome editing methods are subject to GMO regulation. "Mutations are something natural and occur naturally all the time. Traditional breeding is also based on mutations. We use chemicals and radioactive radiation to induce large numbers of mutations without knowing how many mutations are created and where they occur. Such plants are considered safe. With CRISPR, we produce single point mutations at defined loci, which could also have been created in a natural way. But our CRISPR-edited plants are considered potentially dangerous. It's absurd,” says Puchta.

The ECJ rule also has an indirect influence on the Newcotiana project. From now on, CRISPR-edited tobacco plants with point mutations will also have to go through the expensive and time-consuming GMO approval process. “This makes it very difficult to apply our research,” says Puchta. “Europe has long been lagging behind the USA due to its restrictive plant biotechnology legislation. The latest decision has worsened the situation further.”

Following the EU’s decision, the German Bioeconomy Council, an independent advisory body to the German government, urged the German government to modernise genetic engineering laws. "Otherwise, Germany will not be part of this "biological revolution” and neither will it be involved in shaping the necessary international regulations," the Council wrote in its statement. "This is a crucial issue,” says Puchte. “But at present I cannot see any political will. That said, plant biotechnology is an extremely promising technology.”

References

https://newcotiana.org/
https://www.gesetze-im-internet.de/gentg/BJNR110800990.html
http://biooekonomierat.de/aktuelles/stellungnahme-zu-genome-editing/