Crop Yield Gains At The Flip Of A Gene Switch: RNAi

RNA background

Alongside DNA and proteins, RNA—or ribonucleic acid—is considered one of the three macromolecules essential to all forms of life. Whereas DNA (deoxyribonucleic acid) and proteins are the genetic blueprint and workhorses of the cell, respectively, RNA functions as a vital messaging system within the cell structure. RNA converts information stored in DNA into proteins: RNA, in the form of mRNA molecules, signals when a cell needs to create new protein (i.e. protein synthesis) by transmitting the protein-making instructions that are encoded in the DNA, from the chromosomes to the site of protein synthesis, and there provides those instructions to the ribosome (protein-synthesis machine).   

Until relatively recently, the primary function of RNA was understood to be in messaging and converting information from DNA into proteins. Now, however, research indicates that the role of RNA is much more complex, with the molecule also involved in the coding, regulation, and expression of genes. Although the full scope of RNA’s involvement in regulating cellular processes is still being researched, the key takeaway, at least within the scope of this discussion, is that RNA transmission plays a critical role in regulating protein activity within plants and insects. 

A very special petunia

In the 1980s, American and Dutch scientists accidentally stumbled upon one of the most significant genetic discoveries of our time. The researchers, in trying to make the purple petunia flowers “more purple,” introduced additional copies of the genes responsible for their coloring. But, what they got was the opposite—the flowers did not become more purple. In fact, quite the opposite happened: they turned white. 

The scientists, convinced that they had made an error, combed through their processes and repeated their experiments. The results, however, were the same. Their final product was not a mistake—rather, it indicated a cellular capability that had not been apparent. 

When the additional purple genes were introduced, the petunia cells tried to silence them—a reaction that appears to have evolved naturally as a defense against viruses. But in doing so, they did not just silence the additional purple genes—both the additional and endogenous (natural) purple genes were silenced via the RNAi process. The plant cells were doing this on their own by interfering in the plant cell’s RNA messaging. Thanks to the Nobel prize-winning research of Andrew Fire and Craig C. Mello, this process of RNA interference was discovered. 

RNA interference, or RNAi, refers to this process through which specific RNA messages are suppressed in order to prevent a particular protein from being made. Or, more succinctly—RNAi refers to the process through which genes are silenced. It can be triggered naturally when a cell encounters double-stranded RNA (RNA, unlike DNA, is single-stranded) which can be the result of a virus replicating their genetic code. The cell responds to the double-stranded RNA molecule by dissecting it and destroying the matching RNA messages. And so when RNA molecules sequenced with specific RNA messengers are introduced into an organism, scientists are able to silence those particular messages. 

To facilitate the commercialization of their RNAi research, scientists are increasingly relying on the field of Bioinformatics, the combined use of computer science, statistics, mathematics, and engineering to analyze biological data, to identify double-stranded RNA molecules that match specific messenger RNA within a target organism. Researchers have also begun testing for unintended sequences across a global set of non-target organisms, including bees ladybugs, and humans. 

Top seed companies are betting big on gene silencing

While most commercial research into RNAi thus far has been centered on the areas of human wellness, animal health, and food modification (i.e., allergy-free peanuts, virus-resistant livestock, and decaffeinated coffee beans), top crop chemical-seed companies are now following suit. Industry experts hope that RNAi technology will be able to, for example, adjust the oil composition within a soybean or to silence genes critical to pest survival (i.e., the silencing of genes essential to rootworm survival). 

To this end, the leading global seed companies have been steadily pouring money into RNAi development in the form of internal R&D activity, M&A, and/or industry partnerships. Syngenta, for example, purchased Devgen, a company focused on developing novel sprays for RNAi delivery, for $523 million in 2012. The company is also partnering with Nexgen, an Australian biotech firm, to develop RNAi mechanisms to resist the maize dwarf mosaic virus, sugarcane yellow leaf virus, and tomato yellow leaf curl virus.

At the same time, Monsanto formed a partnership with Alnylam Pharmaceuticals, a leading RNAi biotech company, in 2012 and spent $29.2 million to secure rights for associated intellectual property. The company has also invested $1.5 million in Tekmira, a RNAi delivery specialist. Prior to that, Monsanto acquired Beeologics, which has reportedly found a successful approach to more cheaply introduce RNAi into beehives in the hopes of reducing their vulnerability to harmful varroa mites. More recently, Monsanto established a new research company, Preceres, to develop delivery mechanisms for RNAi. Last but not least, BASF Plant Science also holds several patents and applications related to the use of RNAi technology in crop development and pest control.

Translating this invisible messenger into commercial gold

Today, engineering RNAi into a seed is typically the most commercially viable way to insert double-stranded RNAi molecules into crops. Monsanto, for example, has already introduced Vistive Gold soybeans that are altered with RNAi to produce lower levels of saturated fats and plans to introduce SmartStax Pro corn seeds by the end of the decade, which incorporate RNAi technology to combat western corn rootworms. At the same time, agrochemical companies are pushing to develop a viable way to deliver RNAi via chemical sprays or seed coatings. Global regulatory agencies appear willing to treat RNAi sprays as a biomicrobial (derived naturally from bacteria) or biological rather than a novel chemical compound or transgenic event (transgenic referring to containing artificially inserted genes)—a willingness that will likely help reduce the amount of regulatory data and environmental testing required for regulatory approval. 

While Monsanto has been the lightning rod for the anti-GMO (genetically modified organisms) movement in recent years, the company has been increasingly focusing its R&D efforts on improving crop yields vis-à-vis biologicals, including biomicrobial treatments and RNAi.  More importantly, potential breakthroughs from this cutting-edge research may allow some applications to be delivered through spray equipment and seed coatings rather than by inserting new genetic coding into a plant, which is the standard process in developing GMO seeds (Bacillus thuringiensis, or Bt, for example, is a gene toxic to insect larvae that is commonly introduced to pest-resistant GM crops). 

In an interview with the MIT Technology Review last year, Monsanto’s Chief Technology Officer, Robb Fraley noted that RNA sprays would represent new ways to use biotechnology without the “same stigma, intensive regulatory studies, and development cost associated with GMOs.” The shorter regulatory approval process and lower costs involved in the final stage of market approval make the return on investment (ROI) on RNA sprays particularly promising. 

We recently caught up with Monsanto’s vice president of global chemistry technology, Bob McCarroll, who told us that Monsanto anticipates that bringing a new RNAi application through the regulatory process in the form of a biological solution will take 3-4 years versus the current 6-7 years required by a new biotech seed.

Furthermore, he expects that the investment required to bring a new RNAi biological to market will be significantly below the average investment of $135 million associated with introducing a new biotech seed, owing again in part to the lower regulatory hurdle and to the fact that Monsanto will not have to breed a new RNAi event across multiple seed varieties as is the case for a novel seed trait. At the same time, the cost of producing double-stranded RNA molecules has dropped significantly: prices were in the thousands of dollars per gram relatively recently, and are now around $50/gram. Monsanto is confident that they can bring the cost down even more once it reaches manufacturing scale. 

Big gains at the flip of a gene switch

The option to deliver next generation agriculture solutions via transgenic seeds, seed coatings, or spray equipment could potentially give Monsanto the ability to tailor its technology offerings for different end-markets. For instance, countries strongly opposed to GMO seeds may, in theory, be more open to RNAi, given that the process is a natural one. And for the producers of such technology, RNAi could provide additional benefits beyond those of GM seeds, given that RNAi sprays would need to be reapplied every season—a fact which could help Monsanto and its peers navigate thorny seed royalty challenges in some countries. 

RNAi also appears to be a more economically viable option for specialty crops that cannot support the cost of biotech R&D, according to Monsanto.  As such, RNAi could open up yield gains for crops that have more limited commercial potential. The lower development costs related to RNAi could also eventually lower the technology cost curve for farmers in emerging markets. However, producers of inputs employing RNAi will have to first demonstrate that the technology can be delivered in a safe and effective manner. 

Spray-based RNAi compounds still face challenges

The US Environmental Protection Agency (EPA), in a 2014 white paper, concluded that current evidence suggests that new RNAi mechanisms are not an apparent threat to humans and animals, as they assume that the human digestive system, which is constantly exposed to RNA molecules already, would destroy double-stranded RNA before they could do potential harm and current bioinformatic systems can properly assess pre-triggers for non-target organisms. But the agency is still taking normal precautions towards RNAi agricultural products and is calling for further assessment of unknown ecological risks from RNAi technology and the study of dsRNA PIP (Plant-incorporated protectant-PIP) levels in mammalian blood and exposed tissues over a sustained period. 

Moreover, the companies developing RNAi solutions for agriculture are taking the stance that RNAi mechanisms, in and of themselves, are not permanently changing the genetic makeup of a specific crop. In layman’s terms, RNAi is regulating gene expression, but not permanently altering the genetic code of plant or, at least, not in ways that we know. As such, Monsanto and peers don’t anticipate that RNAi will receive greater scrutiny than a new biopesticide would receive during the deregulation process once official risk assessments are concluded. However, not all scientists agree with the EPA’s current stance and the industry’s view that RNAi in agriculture applications will not have an adverse impact on the environment or human health. 

In fact, several scientists, including Dr. Jack Heinemann, Professor of Molecular Biology and Genetics at the University of Canterbury, have voiced their concerns that government agencies have not constructed environmental tests rigorous enough to determine whether RNAi mechanisms in question can produce unintended effects. The MIT Technology Review pointed out that Monsanto’s own internal research has shown ways that double-stranded RNA can move between species; but the firm has stated that its internal Bioinformatics system can help pre-select RNAi triggers across target and non-target organisms in order  to prevent unexpected matches to non-target organisms in the ecosystem. Furthermore, the company is conducting experimental evaluations of safety and unintended non-targeted organisms (NTO) effects in order to incorporate further safety measures into the research and testing process. 

While the EPA has stated that more research should be conducted on the unintended ecological consequences of RNAi, the agency appears to be mostly siding with the commercial community at this stage. 

Companies such as Monsanto still face technological hurdles in bringing RNAi sprays to the marketplace. First off, the industry has to find an effective approach to inserting double-stranded RNAi into crops while limiting drifting to other non-target organisms. In some cases, the level of RNAi dosage required to ensure effective transmission to a crop or insect’s genetic coding is cost prohibitive. The industry must find a more precise way to insert RNAi molecules to mitigate not only cost but any potential ecological effects. 

According to Monsanto’s Bob McCarroll, the delivery mechanism that has shown the most promise in overcoming that hurdle is Lipid-Like Materials (LLM or lipid nanoparticles), which is being adopted from existing research conducted by MIT and cancer biotech companies. To overcome the resistance that mRNA molecules sometimes face when penetrating cell membranes (i.e., opposite charges), researchers discovered that it is easier to insert RNAi into a cell by wrapping the molecule with a substance that has similar characteristics to the targeted cells. In more precise terms, RNA is encapsulated in synthetic nanoparticles called lipidoids, which could then be slipped into a plant or insect via a chemical spray after which the coating would dissolve and release the RNA. Yet, some specialists, including Dr. Kassim Al-Khatib, the Melvin D. Androus Endowed Professor for Weed Science at UC-Davis, are skeptical that Monsanto and peers can ultimately build an RNAi delivery system that is both cost effective, stable, and ecologically safe. 

Nevertheless, Monsanto is moving full steam ahead in developing BioDirect Technology, a new spray delivery system for RNAi-infused molecules. The spray, which will likely initially target the Bee Health segment, is scheduled to hit the market early in the next decade. Monsanto is prioritizing its pipeline around solutions for bee mites, potato beetles, and canola flea beetles, as it appears that these insects are extremely sensitive to even small doses of RNAi molecules, eliminating the need for more advanced delivery systems. Further down the road, the company hopes to develop a RNAi spray product that would be an alternative to its widely used Roundup and Dicamba herbicides for corn, soybean, and cotton crops.  

And if it, or its peers, can perfect a broad-based delivery system for RNAi, the future of agricultural inputs and crop production are likely to look drastically different.  

Conclusion

While the implications of RNAi manipulation for agriculture are not fully clear yet, agrochemical and biotech giants show few signs of slowing down their commercialization efforts. 

In a way, technological hurdles may be the easiest to overcome when one considers the overall challenge of commercializing RNAi on a global scale. Although RNAi is a natural process and the regulatory environment so far has been supportive, the success of RNAi within agriculture will come down to companies reassuring farmers and global regulators that sufficient research has been conducted on the potential ecological or biological consequences—as well as convincing consumers that RNAi is a miracle of nature rather than the next version of “Frankenfood.” 

If agrochemical companies are successful on these fronts, and if the regulatory environment remains amenable, RNA technology could eventually unlock millions of additional acres of cropland currently restricted by GMO planting bans.

While 82 percent of soybean acreage and 58 percent of cotton acreage were planted with biotech seeds in 2014, only 30 percent of global corn was planted with such seeds, according to the ISAAA (International Service for the Acquisition of Agri-biotech Applications). With Monsanto stating at a recent investor day that they expect to drive earnings per share at a 20 percent CAGR (compound annual growth rate) from FY:16 to FY:19, it isn’t difficult to see why working towards access to another 100 million hectares of corn is a top priority for the company.