Proteins are the building blocks of life, and yet if they misfold or accumulate they can lead to disease or death (1). Many of the commercially available drugs inhibit harmful protein functions or ensure that they fold correctly (2). But it might be useful for drugs to stop problematic proteins from being made in the first place. 

It's just been a wonderful experience contributing to what I consider a historical introduction of a new modality. 
– Phillip Sharp, MIT

It turns out that nature evolved a way to do just that. In 1998, biologists Andrew Fire, now at Stanford University, and Craig Mello of the University of Massachusetts Medical School discovered that cells in plants and worms can silence genes and shut down protein production by degrading mRNA with double-stranded RNA in a process called RNA interference (RNAi) (3). Phillip Sharp, a cell biologist at the Massachusetts Institute of Technology (MIT) who won the Nobel Prize in 1993 for the discovery of RNA splicing, said that he remembers being stunned by the paper when it first came out. “It offered an opportunity to do something that I've been hoping to be able to do since I began working in mammalian cells: have a method to inactivate a gene,” he said. 

Shortly after encountering the paper, Sharp developed the first in vitro system for studying RNAi with collaborators Thomas Tuschl, Phillip Zamore, and David Bartel, who were then at MIT (4). In 2001, Tuschl, now at the Rockefeller University, showed that RNAi also exists in human cells (5). Based on these early RNAi discoveries, Tuschl, Sharp, Zamore, and Bartel joined with Paul Schimmel of Scripps Research to found Alnylam Pharmaceuticals.

“The company was set up with one mission, one purpose, and that was to turn RNA interference into a medicine,” said Paul Nioi, senior vice president of research at Alnylam Pharmaceuticals. “If you could silence any gene in the genome, the idea of what's druggable and what's not goes away. Everything is druggable.”

In 2018, one of Alnylam Pharmaceuticals’ drugs, Onpattro, became the first RNAi drug approved by the FDA (6). Onpattro treats hereditary transthyretin-mediated amyloidosis (hATTR), a rare genetic disease that leads to the buildup of amyloid proteins in the heart and other organs. Alnylam Pharmaceuticals has since developed four more FDA-approved RNAi drugs to treat high cholesterol and rare genetic disorders.

Today, Alnylam Pharmaceuticals has 15 RNAi drugs — some of these in partnership with other companies such as Regeneron and Roche — in both early and late stages of clinical trials to treat a variety of genetic diseases, cardio-metabolic diseases, infectious diseases, and central nervous system (CNS) and ocular diseases. 

“It's really been a remarkable story. It's taken a long time, but you must realize from the day of the discovery of the whole phenomenon, the company was started four years later,” said Sharp. “It's just been a wonderful experience contributing to what I consider a historical introduction of a new modality.”

Developing the first RNAi drugs

The core of the RNAi mechanism rests on a protein complex called the RNA-induced silencing complex (RISC) (7). Small interfering RNA (siRNA) molecules load into the RISC, and then the RISC searches for a target mRNA that matches that siRNA. When it finds this mRNA, the RISC degrades it, which leads to drastically reduced levels of protein synthesis. Alnylam Pharmaceuticals’ RNAi therapeutics involve synthesized siRNA that encode a target mRNA related to a disease. The major advantage of this platform, Nioi said, is that “Your universe of targets is the whole genome.”

Sharp pointed out that targeting the protein expression of a single gene also eliminates other lengthy steps in the drug discovery process. “Once you encode the target you want, you can design the drug. You don't have to screen; you don't have to hope something happens,” he said.

There were two major hurdles to overcome before scientists could turn RNAi drugs into reality: enhancing siRNA stability and determining delivery. “The first 10 years or so of Alnylam was all about solving that [stability] problem and figuring out how to deliver siRNA to the right cell type,” said Nioi. 

To improve siRNA stability, researchers modified the sugars that make up the backbone of the RNA so that nucleases in the blood could no longer recognize and chew them up. Then they investigated how to deliver the siRNA safely to specific sites. Their first target protein was the transthyretin (TTR) gene, which encodes for the TTR protein, which transports thyroid hormones in the blood. They aimed to shut down production of TTR to treat neuropathy symptoms in hATTR amyloidosis, which is caused by a mutation in the TTR gene. Most of the TTR protein is synthesized in the liver, so they had to find a way to deliver siRNA there. 

According to Nioi, the researchers first decided to move forward with a conjugate approach that would allow them to administer the drug subcutaneously but they ran into safety issues in the clinic. As a result, they pivoted back to a lipid nanoparticle (LNP) approach that they had tried earlier, which encased the siRNA in a fatty protective molecule and delivered them intravenously. “It was anything but smooth. You can imagine how the stock price was behaving during that turmoil,” said Nioi. The LNP approach led to significant improvements in neuropathy symptoms on assessments compared to a placebo in a Phase 3 clinical trial (8). That drug became the first FDA-approved RNAi drug in 2018 (6).

The researchers went back to developing a safe and effective conjugate approach that would allow for a subcutaneous, self-injectable delivery to the liver. They developed GalNAc conjugates, which are sugar molecules that bind to the asialoglycoprotein receptor found on liver cells. Adding GalNAc conjugates to siRNA therapeutics led to four additional FDA-approved drugs to treat acute hepatic porphyria, primary hyperoxaluria type 1, hereditary ATTR amyloidosis, and high cholesterol.

“What's really kind of cool about it is that if you look at the indications, none of them are for liver disease, but the targets are all in the liver. So it goes to show the biology of the liver is an incredible thing,” said Nioi. 

Nioi also emphasized his goal to move beyond targets in the liver to reach the brain, muscle, and adipose tissues. To reach the brain, scientists recently demonstrated success using lumbar punctures that introduce siRNA attached to C16 conjugates into the spine and cerebrospinal fluid. After observing gene silencing in the brains and spines of rodents and nonhuman primates, Alnylam Pharmaceuticals signed a collaboration deal with Regeneron in 2019 to advance RNAi therapeutics targeting the CNS. 

Currently, Alnylam Pharmaceuticals has an ongoing Phase 1 clinical trial studying ALN-APP, an RNAi drug that targets the amyloid precursor protein (APP) gene, to treat patients with early-onset Alzheimer’s disease (9). “ALN-APP is a unique approach to treating Alzheimer's disease because it hits the target, which is a genetically valid target for Alzheimer’s disease at the source, at the mRNA level,” said Kirk Brown, vice president of research and director of the CNS Early Development program at Alnylam Pharmaceuticals. “[By] cutting the target at the source, you're essentially shutting off any intracellular fragments that are causing this toxicity from the inside the cell.”

Nioi said that developing the technology that will allow the siRNA to reach more organs and tissues is still their biggest challenge. “We imagine that there's a whole host of other tissues that we would love to be able to get to where we think there're great targets for RNAi,” he said.

RNAi drugs of the future

Aiming Yu, a pharmacologist at the University of California, Davis who is not involved with Alnylam Pharmaceuticals, said that the development of RNAi drugs over the last two decades has been full of ups and downs. Many companies invested in RNAi therapeutics and then backed out. “Alnylam, of course, is a great example of the success [of RNAi therapies],” he said. “It’s encouraging for me working in this field.” 

Yu noted that future studies should address how to improve the ratio between the small amounts of siRNA that bind to the RISC compared to the large amounts that accumulate in a tissue, which could lead to toxicity. Yu’s group works on the development of biologic RNA molecules, or BioRNA, that are produced in living cells. It’s possible that these BioRNA could be better tolerated and more efficiently loaded into the RISC, said Yu.

Over the next five to ten years, Yu would love to see more RNAi drugs approved for common diseases that affect a large number of people. Alnylam Pharmaceuticals currently has several ongoing trials studying RNAi drugs to treat common cardiometabolic diseases like type 2 diabetes, nonalcoholic fatty liver disease, and hypertension. “Despite the medicines that are on the market already to treat cardiometabolic disease, it is still the number one killer,” said Nioi. “There’s an incredible amount of unmet need remaining. I think we have, with our platform, a big opportunity to make a difference there because we can go after any target. We’re not limited.” 

 “You can project [a new drug] once every year to almost any gene, and in time, to almost any tissue … That's where the science is leading us,” Sharp said. “There's a lot to be done, and a lot of people will benefit from it.” 

References

1. Gregersen, N., Bross, P., Vang, S. & Christensen, J. H. Protein misfolding and human disease. Annu Rev Genomics Hum Genet  7, 103–124 (2006).
2. M. Gomes, C. Protein Misfolding in Disease and Small Molecule Therapies. Curr Top Med Chem  12, 2460–2469 (2012).
3. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature  391, 806–811 (1998).
4. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev  13, 3191–3197 (1999).
5. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature  411, 494–498 (2001).
6. Alnylam Reports Additional Positive Interim Phase 1 Results for ALN-APP, in Development for Alzheimer’s Dise. Investor Relations | Alnylam Pharmaceuticals, Inc.
7. Dana, H. et al. Molecular Mechanisms and Biological Functions of siRNA. Int J Biomed Sci  13, 48–57 (2017).
8. Adams, D. et al. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med  379, 11–21 (2018).
9. FDA approves first-of-its kind targeted RNA-based therapy to treat a rare disease. FDA (2020).