Carriers for RNAi

There is an urgent need for new pesticides that effectively combat pests and diseases without causing harm to other organisms. ‘RNAi offers a tremendous application for this,’ says Kristof De Schutter, crop protection researcher at the Faculty of Bioscience Engineering at Ghent University. Plants, insects, but also animals and humans, use RNA interference (RNAi) to help regulate genes. Within an organism, this works as follows: the cell’s RNAi machinery cuts double-stranded RNA (dsRNA) into small fragments of 19–21 nucleotides, separates the two RNA strands from one another, and uses one of the two strands as a guide. If the guide is complementary to messenger RNA, the RNAi machinery cuts that messenger RNA into pieces, thereby temporarily silencing a gene. Because RNAi operates on the basis of homology, it is highly specific. For the RNAi machinery, the origin of the dsRNA is irrelevant — it may come from outside the organism as well. This is precisely where RNAi pesticides, with dsRNA as the active molecule, come into play. One drawback is that outside the cell, dsRNA is rapidly degraded, which means the efficiency of RNAi pesticides is very low. To address this, researchers in Ghent are designing nanocarriers to deliver dsRNA into the cell intact.

‘We use proteins and peptides that are readily taken up by the cell’

Kristof De Schutter

Mating behaviour

RNAi pesticides can be used to silence specific genes in an insect. If the gene in question is essential, the insect dies. ‘The advantage of RNAi is that you don’t necessarily have to aim for lethality,’ says De Schutter. Behavioural modification is, in his view, also an option — for instance, a change in mating behaviour, resulting in fewer offspring. Moreover, the potential applications of RNAi are not limited to insect control. It is also possible, for example, to influence gene expression in plants in order to temporarily make them more resistant to stress. ‘Feeding’ dsRNA to insects is achieved by spraying plants with dsRNA, which then settles on the plant. When an insect feeds on that plant, it ingests the dsRNA and the RNAi process can be initiated. This already works well for the notorious Colorado beetle, which poses a serious threat to potato crops. ‘But those beetles are particularly sensitive to RNAi,’ says De Schutter. Not all insects take up dsRNA with equal ease. And if an insect sucks sap from a leaf, it may not even ingest the dsRNA at all. Another disadvantage of dsRNA is that it is highly unstable. Not only do nucleases break it down readily, but UV radiation and fluctuations in temperature and pH also cause rapid degradation. There are therefore still a great many obstacles to achieving efficient RNAi. De Schutter and his colleagues hope to overcome these barriers with nanocarriers that bind and protect the dsRNA.

Packages

These carriers can be synthetic polymers. ‘Everyone knows polymers from a plastic water bottle, or from packaging material,’ says Peter Dubruel, head of the Polymer Chemistry and Biomaterials (PBM) research group at Ghent University. But for the carriers, he is seeking advanced polymers capable of interacting with dsRNA and subsequently forming a polyplex — a composite molecule of the polymer and the dsRNA. By nature, dsRNA is a long, slender molecule carrying a large number of negative charges, which makes it difficult to enter the cell. The aim is therefore to reduce both the negative charges and the dimensions of the dsRNA by allowing the dsRNA and the polymer to form a compact package together — the polyplex. Such a package can be taken up by the cell far more readily. There are various ways of achieving this, but the most straightforward is to exploit opposing charges. Dubruel and his team therefore search the toolbox of monomers for molecules that carry a positive charge even under the physiological conditions found within an organism. For the carriers, he links these monomers together so that the positive charge is present along a long chain. In addition to synthetic polymers, researchers in Ghent are also investigating whether certain biological polymers — such as peptides and proteins — can smuggle dsRNA into the cell. ‘Here we use proteins and peptides that are very readily taken up by the cell,’ explains De Schutter. These include cell-penetrating peptides, which enter the cell with ease, and lectins, proteins that are naturally fairly resistant to degradation by proteases. ‘With biological polymers, we know that a single domain will not work for everything,’ says De Schutter. He is therefore searching for the optimal combination: a dsRNA-binding domain to bind the dsRNA, plus a cell-penetrating peptide to gain entry to the cell — and/or a lectin domain for stability. All in all, it is quite a puzzle.

‘If the biologists don’t indicate which direction we need to go, then as a chemist you’re stuck — and the same applies the other way round’

Peter Dubruel

Field trials

The first step towards an application is to test whether the carrier and dsRNA form a polyplex. A second crucial test is whether the carrier itself is not toxic to insect and plant cells. De Schutter: ‘We want the insect to die as a result of the RNAi effect. If it dies due to uptake of the carrier, then we do not want to release those carriers into the environment.’ Once these hurdles have been cleared, the researchers test the uptake of dsRNA in cell lines from both plants and insects. If these take up the polyplex more effectively than dsRNA alone, it is time to test on whole organisms — examining dsRNA uptake, whether an RNAi effect occurs, and whether that effect remains localised or manifests throughout the entire organism. The first carriers are now being tested at the organism level, though the group is also continuing to study them in cell lines in order to explore certain aspects in greater depth. ‘With that knowledge, we can provide feedback to Peter,’ says De Schutter. That feedback is essential, according to Dubruel. ‘We can make new materials, but if the biologists don’t indicate which direction we need to go, then as a chemist you’re stuck — and the same applies the other way round.’

Should the carriers also emerge unscathed from this round, it will be time for field trials. ‘Then we’ll see whether our technology can succeed in targeting specific pest insects,’ says De Schutter. That will reveal whether the nanocarriers genuinely bring efficient RNAi pesticides closer to reality.