From worms to cures

Emily B. Harrison, Ph.D
C. elegans, Image Credit: ZEISS Microscopy

Last month I attended the first RNA Therapeutics Conference held at University of Massachusetts Medical School. While I have attended several national conferences, this one beautifully encapsulated how basic science can lead to new drugs in only two decades. This conference inspired me to share with you the story of RNA interference (RNAi for short) from the discoveries that lead to the Nobel prize in Physiology and Medicine in 2006 to the imminent approval of the first RNAi drug by the United States Food and Drug Administration. At the end I’ll tell you a little about what this means for cancer treatment, but until then, enjoy the ride.

Illustration of a cell demonstrating the flow of genetic information from DNA to RNA to Protein. The purple circle represents the nucleus of the cell. Image Credit: OpenStax Anatomy and Physiology

RNA, the messenger

Before we delve in, a little background information. Deoxyribonucleic acid (DNA) is the genetic blueprint of life. DNA is held safely in a central compartment of the cell called the nucleus. Segments of this DNA code for functional elements called genes. Many genes code for proteins, which serve many roles in our cells. These proteins form many things from structural components of cells to the molecular machines that run nearly process in our bodies. Before DNA is turned into protein it is first transcribed into ribonucleic acid (RNA). This RNA, called messenger RNA, serves as the intermediate step between DNA and protein. It carries the information out of the nucleus, where it can be translated. The transcription of DNA to RNA and then translation to protein is such an important concept that it is called the “central dogma of biology.”

Humble beginnings

The discovery of RNAi came from a shortcut. Su Guo and Ken Kemphues at Cornell University were trying to understand the genes that control early embryonic development, specifically the very first cell division, using a simple model organism, C.elegans. This worm is one of the simplest multicellular organisms, so it is ideal for studying the basics of how all animals go from a single cell to a complex arrangement of cells with many unique functions. They started by mutating genes randomly, which often inhibits their function. This allows scientists to identify which genes are important for certain life processes, like cell division. Through this process, they discovered a gene they named par-1 (for partitioning defective). Trying to confirm the results, they wanted another method to test whether this gene was important. At the time, one way to do this was by microinjecting DNA into the worm. By producing the non-mutated gene they could “rescue” the divisions and make a functional worm. For whatever reason, they had difficulty making a copy of the entire gene to do this. But they still needed to prove the gene they discovered was important and the observations they made were not done to some other effect. So instead they tried to silence the gene in healthy worms, to see if it produced the same genetic defect they saw in the mutant worms. They injected a sequence of RNA that was complementary to the gene they wanted to silence, meaning it would bind to the sequence (this is also called antisense). They hypothesized that this would block the translation of the gene into a protein. Other groups had done this with DNA, but no one had tried directly injecting antisense RNA. This approach was effective, the gene was reduced and Gou and Kemphues were able to confirm that par-1 did indeed regulate cell division in early worm embryos. The ability to specifically inhibit any gene using antisense RNA was enticing. However, moving forward this approach did not work for every gene or for every organism and could not be replicated widely.

The world takes notice

The initial report of RNAi by Guo and Kemphues was expanded in the 1998 paper that earned Craig Mello and Andrew Fire the Nobel prize in 2006. Also working in C. elegans, Mello and Fire made several observations, the most critical to therapeutic translation of RNAi  being:

1) That gene silencing was much more effective if the RNA was double stranded. Meaning, two complementary RNA molecules bound to one another instead of a single RNA molecule. In fact, a few molecules per cell could almost completely silence the gene. In contrast, injecting only a single strand led to little or no gene silencing, explaining inconsistent results in earlier experiments.

2) The ability of small amounts of double stranded RNA to silence the expression of genes with 10s to 1,000s of copies of messenger RNA in a cell meant that this was not due to a 1:1 binding event, and led Mello and Fire to propose that there was probably a molecular machine, an enzyme, that could use the RNA as a template to silence many copies of a gene. This molecular machinery was later identified as the RISC complex.

3) The double stranded RNA could spread between tissues and even into progeny, suggesting that double stranded RNA could be administered systemically to treat disease.

The mechanism of RNAi. Double stranded RNA (dsRNA) added to the cell binds to messenger RNA (mRNA) and degrades it with the help of the RNA induced silencing complex (RISC). Image credit: PLOS

These three ideas laid the groundwork for RNAi as a feasible therapeutic. Silencing unwanted or mutated genes through RNAi could treat genetic diseases or cancer, and this potential attracted the attention of the Nobel committee, the scientific community, and pharmaceutical companies. Since this seminal work, a cornucopia of studies have identified the machinery involved and the biological importance of this system. However, there is no doubt there is still more left to uncover about how this native gene editing operates in health and disease. Mello presented work at the RNA Therapeutics conference on how the RNAi machinery can even encode a form of cellular memory.

Now, using RNAi (also called small interfering RNA, or siRNA) to study the function of a gene is routine, almost passé. However, there are no RNAi-based drugs currently approved…yet.

Hurdles to translation

The main problem with RNAi as a drug is its inability to overcome biological barriers and reach target sites. Naked RNA injected into the blood of larger organisms, such as mice and humans, is rapidly cleared by the kidneys. Also, our bodies are designed to detect and eliminate foreign RNA (many viruses have RNA genomes). RNases degrade RNA in the blood and RNA sensors trip an immune response causing systemic inflammation. Once RNA gets to the intended cells, it then has to enter the cell and break into the main compartment, the cytosol, to do its business. This, however, is easier said than done, as cells are very careful to keep foreign materials separate from their machinery, to prevent damage and infection. To enable RNA to traverse the dangerous landscape to and inside of target cells, several methods have been employed. Encapsulation in nanoparticles, such as those discussed in Sara Musetti’s recent post, is one solution. Another is modifying the very chemical structure of RNA to make it more stable and less recognizable to the immune system. Molecules that bind to receptors on the surface of target cells can also be linked to RNA or RNA carrying nanoparticles to increase cellular uptake.  For those interested, here are links to two thorough reviews on the topic of RNAi in the clinic, one in Nature Reviews Drug Discovery and Nature Reviews Genetics. Needless to say, the journey from basic discovery to clinical trials has not been trivial, but has happened at an impressive rate.

Fast forward to 2018

This year, the 20th anniversary of the seminal paper on RNAi, may well mark the approval of the first RNAi based drug. A major player in therapeutic RNAi is Alnylam, a biotech company dedicated to bringing the promise of RNAi to fruition. Vasant Jadhav, Senior Director of Research at Alnylam, presented results of a randomized, phase 3 clinical trial for their drug patisiran at the RNAi Therapeutics Conference, (phase 3 being the final phase required for FDA approval). The goal of this trial was silence the transthyretin (TTR) gene in patients with hereditary transthyretin amyloidosis, a rare and deadly genetic condition. In this disease, a mutation in the TTR gene causes the protein to misfold and build up in many tissues, leading to nerve degeneration, heart problems, and eventually death. TTR is produced in the liver and without a liver transplant, most patients die within 10 years of diagnosis. The results were impressive, and the following week, the results were published in the New England Journal of Medicine. TTR protein levels were reduced by 81% in the serum of patients treated with patisiran, which was sustained for 18 months. More importantly the patients had less nerve dysfunction, or neuropathy, than placebo. In fact, in 56% of the patients neuropathy was not only halted, but reduced, compared to 4% in placebo groups. Similar improvements were seen in patient quality of life. Some patients treated with patisiran were able to go from walking with assistance of a walker or cane to walking freely. Importantly, no serious side effects were observed.

Ok, back to cancer already

So what does all this mean for cancer therapy? RNAi therapies have been tested in early phase clinical trials for cancer, as of yet none have made it to phase 3. Currently, several groups are focused on using RNAi to inhibit the RAS family of oncogenes, particularly KRAS. The KRAS gene is mutated in 90% of patients with pancreatic cancer and at high rates in colorectal cancer, myeloma, and lung cancer. Overall, the RAS family is most frequently mutated oncogene family. Mutations that cause KRAS to be hyperactive drive cancer growth, so inhibiting KRAS with RNAi could stop tumors from growing. Even though the RAS family were the first human oncogenes discovered in 1982, no drugs have been found that can block KRAS. This is because the protein has no accessible divots or pockets for small molecules to bind it, leading some to call it “undruggable.” However, RNAi works by reducing expression of the KRAS gene, so the structure of the protein does not inhibit it’s success. One example of this is a biodegradable implant carrying siRNA against mutant KRAS that is now being tested in phase 2 clinical trials for inoperable pancreatic cancer after completion of phase 1 trials. As Manisit pointed out in the last OncoBites, this does not mean the therapy will work, but it is at least a step forward for RNAi cancer therapies. There are many other oncogenes that could be inhibited by RNAi, but there are limitations. RNAi will not be able to restore the function of tumor suppressor genes, whose loss leads to cancer. Also, the very nature of cancer cellsthat they divide rapidly and spread throughout the bodymakes them a difficult target for RNAi, unlike the liver cells targeted by patisiran. Only time will tell whether RNAi will become a successful therapy for cancer.

For me personally, the story of RNAi is an inspiration, it shows that with curiosity, perseverance, and a bit of creativity, science can make the leap from worms to cures and it can happen in my lifetime. The story of RNAi serves as a parable, teaching us how basic research, even on lowly worms, can change lives.


Guo, S., & Kemphues, K. J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 81(4), 611-620.

Fire, Andrew, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391.6669 (1998): 806.

Adams, D., Gonzalez-Duarte, A., O’Riordan, W. D., Yang, C. C., Ueda, M., Kristen, A. V., … & Lin, K. P. (2018). Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. New England Journal of Medicine, 379(1), 11-21.

Golan, T., Khvalevsky, E. Z., Hubert, A., Gabai, R. M., Hen, N., Segal, A., … Galun, E. (2015). RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients. Oncotarget, 6(27), 24560–24570.

Featured Image Credits

RNAi wordle | Wordle based on the titles of 288 matches for “rnai screen*” on PubMed:

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