© ISTOCK.COM/KTSIMAGECold Spring Harbor Laboratory molecular geneticist Adrian Krainer was at a National Institutes of Health workshop in 1999 when he first learned about the crippling neurodegenerative disease spinal muscular atrophy (SMA)—the leading genetic cause of death in infants. The disease has no treatment, and more than 90 percent of infants born with SMA die before the age of two. At the workshop, Krainer recalls, researchers presented their findings on two genes associated with the disease, SMN1 and a duplicate gene, SMN2, both coding for survival motor neuron (SMN) protein, an essential component in the production of spinal motor neurons.

Despite the apparent similarity of the genes, SMA researcher Christian Lorson, then of Tufts University School of Medicine in Boston, and colleagues had found that a single nucleotide difference was causing the RNA transcripts of each gene to be processed differently, Krainer says. While SMN1—which is usually absent or defective in SMA sufferers—produces functional protein, SMN2 contains a mutation that causes exon 7 to be regularly left out of the transcript during splicing. The resulting messenger RNA (mRNA) is unstable and quickly degraded, resulting in low levels of SMN.

The research piqued Krainer’s interest. He had been studying general mechanisms of splicing and exon skipping and saw the potential to restore proper splicing of SMN2 transcripts as a way to compensate for SMN1 loss in infants who suffer from SMA. After the workshop, he and his colleagues took up the challenge. In the early 2000s, they developed a splicing enhancer and a synthetic strand of nucleotides that bound close to exon 7 in SMN2 transcripts. On binding, this complementary, or antisense, compound would promote splicing of the nearby exon into the final mRNA, boosting levels of SMN protein.

A decade of improvements, modifications, and collaboration with antisense therapeutics company Isis (now Ionis) Pharmaceuticals resulted in the development of nusinersen—a drug delivered via spinal injection that is expected to become the first therapeutic for SMA. In August, Ionis halted its Phase 3 trial in SMA patients after interim analyses showed that infants receiving a spinal injection of nusinersen were better able to kick, stand, and walk than infants undergoing a sham procedure. “Obviously, we’re extremely excited,” says Krainer. “It’s sort of beyond anyone’s most optimistic expectations.”

Nusinersen is more than just a breakthrough for SMA. It’s among a handful of late-stage therapeutics in a class of molecules being hailed as the third major drug-development platform after small molecules and biologics: oligonucleotides. These short, chemically synthesized nucleic acids—between 10 and 30 nucleotides in length—have served as vital research tools for more than half a century, playing central roles in DNA sequencing, polymerase chain reaction (PCR), and molecular cloning. Oligonucleotides have also long been recognized as potential therapeutics thanks to their ability to modify gene expression, and in recent years, the number of clinical trials testing oligonucleotide therapies has spiked. Many believe that the platform will soon be ready to treat a wide range of genetic diseases, including those, such as SMA, that were previously undruggable.

“The promise is already here,” John Rossi, an RNA biologist and cofounder of Dicerna Pharmaceuticals, wrote in an email to The Scientist, adding that he sees “the next few years as becoming the era [of oligonucleotide therapeutics].”

A slow start

The first oligonucleotide technology explored for therapeutic purposes was antisense technology, which relies on single-stranded sequences of nucleotides that are complementary to RNA transcripts in human cells. Antisense therapies bind to an mRNA or a pre-mRNA—a transcript awaiting splicing and other modifications—and either block protein translation to eliminate a gene product or, as in the case of nusinersen, alter splicing to restore stability or function to a protein. The technology led to the first oligonucleotide therapeutic marketed in the U.S.: fomivirsen, a drug approved in 1998 that is injected into the eye to treat cytomegalovirus retinitis, an infection afflicting immunocompromised patients.

In the early 2000s, researchers also began to take therapeutic advantage of a recently discovered mechanism of gene silencing: RNA interference (RNAi). By employing double-stranded small interfering RNAs (siRNAs) to hijack this natural pathway in cells and degrade target mRNA, researchers hoped to block the translation of proteins associated with a range of genetic conditions, from cancers to macular degeneration. Unfortunately, lack of efficacy and immune reactions to the treatments led to a series of disappointing clinical trials in the late 2000s. (See “The Second Coming of RNAi,” The Scientist, 2014.)

Besides fomivirsen, which was discontinued in the U.S. a decade ago following improvements in HIV medications, only two oligonucleotide drugs had reached the market prior to 2016. The first, pegaptanib, is a protein-blocking RNA known as an aptamer that was approved in 2004 to treat age-related macular degeneration, but soon met the same fate as fomivirsen, being overtaken by a more effective monoclonal antibody treatment. The second, mipomersen, is an antisense oligonucleotide that was approved in 2013 for the treatment of a genetic form of high cholesterol but floundered in the face of poor marketing and a failure to gain regulatory approval in Europe.

Oligonucleotide therapeutics are being hailed as the third major drug-development platform after small molecules and biologics.

“[These drugs] were never commercially successful,” says Dirk Haussecker, an independent biotech consultant and author of The RNAi Therapeutics Blog. “They’re approved, but nobody’s taking or prescribing them.”

Despite the slow start, oligonucleotides have both therapeutic and commercial promise. In addition to current antisense and RNAi-based therapies in clinical trials, researchers are now developing oligonucleotides that target microRNAs—small, noncoding RNAs that regulate gene expression posttranscriptionally. These include anti-miRs that block the activity of specific microRNAs and miRNA mimics that upregulate it (see table on following page). As of August, there were more than 70 oligonucleotide therapeutics in ongoing or recently completed clinical trials in the U.S. alone, and a recent analysis by a market research firm predicted that the global antisense and RNAi therapeutics market will reach $4.58 billion by 2022.

Researchers, pharmaceutical companies, and investors now anxiously await results from several late-stage trials, anticipated in 2017, as well as nusinersen’s approval, for confirmation that oligonucleotide therapeutics are finally reaching maturity. “Every year it seems like we’ve gotten closer,” says Phillip Zamore, director of the RNA Institute at the University of Massachusetts School of Medicine and cofounder of RNAi therapeutics company Alnylam Pharmaceuticals. Even RNAi drugs, which joined the game later than antisense, are catching up, he adds. “Now there are Phase 3 clinical trials.”

Delivering success

One of the main difficulties in developing oligonucleotide therapies is that nucleic acids are broken down by endonucleases in the bloodstream and within cells, making systemic delivery of naked molecules ineffective. While chemical modifications can help antisense oligonucleotides resist this degradation, siRNAs used for RNAi-based therapies are much less stable, and challenges delivering these molecules were blamed for the disappointing drug trials a few years ago. After that, the industry realized that, “to be a successful RNAi company, you had to become a drug delivery company,” says Zamore. “When that was solved, then things took off.”

Early methods for packaging oligonucleotides used liposomes or lipid nanoparticles that, like unpackaged oligonucleotides, localize to cells in the liver. This natural hepatic affinity pointed the way to some of the most successful drug development in the field, says Barry Greene, president and COO of Alnylam. “It turns out that what some view as a shortcoming—that is, the ability to deliver to hepatocytes—is a treasure trove of opportunities,” he explains. “We’re only at the tip of the iceberg in terms of the kind of genetically valid targets that are in fact produced in hepatocytes.”

Consequently, most oligonucleotide therapeutics companies have focused on the liver. Ionis has antisense oligonucleotide therapeutics in more than 10 clinical trials targeting diseases in the organ, from thrombosis to hepatocellular carcinoma. And Alnylam’s most advanced therapeutic is an intravenous, nanoparticle-carried drug called patisiran, an RNAi treatment that knocks down the translation of a mutated protein made in the livers of patients with a rare disease called hereditary amyloidosis. Results from Phase 3 trials of the drug are expected in 2017 and, if successful, could pave the way for the first approval of an RNAi therapeutic.

More recently, Alnylam has conjugated its siRNAs to the sugar molecule N-acetylgalactosamine (GalNAc) to give the drugs even greater affinity for hepatocytes, improve potency, and reduce off-target effects. Early-stage testing of fitusiran, a GalNAc-conjugated siRNA targeting a liver-produced protein associated with hemophilia, is ongoing, the company says. Meanwhile, Ionis has already adopted a similar method to conjugate GalNAc with antisense oligonucleotides destined for the liver—an advance that Ionis claims improves delivery and provides a more than 30-fold increase in potency over unconjugated approaches currently in trials. RNAi company Arrowhead Pharmaceuticals is also using GalNAc to help deliver oligonucleotides to the liver, but instead of conjugating it directly to the siRNA, the company’s dynamic polyconjugate (DPC) technology links the sugar to the siRNA via a polymer that helps protect the cargo until it enters a cell. A recent clinical study on hepatitis B therapeutic ARC-520 found that the drug inhibits the production of viral protein in infected patients by up to 99 percent after a single dose.

“For anything where the target is made by hepatocytes, the problems are largely solved,” says Zamore. “I think the next big challenges are delivering RNAs to a wider range of tissues and cell types with similar specificity.”

Although targeting other tissues is challenging, some companies are now making strides to do just that. Ionis, for example, has developed modified antisense oligonucleotides that show effective targeting to cells in the kidney. Arrowhead, meanwhile, is working on DPCs that will target cells in non-liver tissues such as tumors by incorporating ligands that target cell type–specific surface proteins. “It’s a matter of finding the right receptor-ligand combinations and to figure out how to conjugate the proper ligands,” explains Krainer. “Down the line, I think one could have a set of conjugates that would allow you to target particular organs.”

The waiting game

Despite considerable progress in oligonucleotide delivery, clinical successes have been balanced with setbacks that serve as reminders that the path to commercialization is never guaranteed. In August, for example, biopharmaceutical company and Ionis partner OncoGeneX announced that its antisense drug custirsen failed to improve survival of patients with prostate cancer in late-stage clinical trials. And earlier this year, Ionis reported dangerous reductions in platelets among patients in Phase 3 trials for two unconjugated antisense therapies, one for a rare cardiac condition, and one for elevated triglycerides. Although most drugs in Ionis’s pipeline are now conjugated, which the company hopes will avoid such side effects, the announcement triggered a 40 percent plunge in share prices in May. (Shares are now recovering as the company makes progress with nusinersen.)

More worryingly for the development of RNAi therapeutics, Alnylam halted development of its GalNAc-conjugated therapeutic revusiran for hereditary amyloidosis this October after reporting more deaths among patients receiving the drug in a Phase 3 trial than among those on a placebo. Despite attempts to reassure investors that other drugs in its pipeline use a newer GalNAc technology, the company saw its market value nearly halve overnight.

Even for drugs that make it through trials without major complications, there’s still the slog of obtaining regulatory approval. Antisense drug eteplirsen, developed by Sarepta Therapeutics as a treatment for certain Duchenne muscular dystrophy sufferers, attained priority FDA review in late 2015. But the clinical data supporting the drug’s approval—based largely on a trial including just 12 people—came under fire from regulators. The FDA finally granted approval this September, but the decision has proved controversial. “This has been delayed numerous times,” Krainer notes. “Clearly, the drug is safe and there’s no other treatment, but there are questions about efficacy.”

With these sorts of ups and downs, the commercialization of oligonucleotide therapeutics is risky, Krainer says. “You could have a good drug that fails a clinical trial just because the right outcome measures weren’t selected,” he explains. “It’s expensive enough that I don’t think one gets to play the game too many times. Perhaps you have one shot or two—luck is involved and there’s no guarantee.”

But as more companies report the results of late-stage trials, a little good news could go a long way, says Ron Renaud, CEO of gene-upregulation biotech RaNa Therapeutics. “There’s so much science and so much knowledge and so much benefit to be gained by everybody. The whole notion of a rising tide lifting all ships definitely resonates with a lot of us in these companies.”

Just like previous drug platforms, which all took time to reach maturity, oligonucleotide therapeutics may now be on the brink of delivering on the promises of the last 20 years. “People are pretty impatient, but when you look at technologies like these, just the general platform can take decades to develop,” says Krainer. “It was no different for making monoclonal antibodies; . . . it took a really long time. These therapeutics encompass a wide variety of technologies and each of them has to be given a chance to prove its worth.” 

ORDERS OF OLIGONUCLEOTIDES: Multiple therapeutics using small, synthetic nucleic acids, or oligonucleotides, have been designed since the first antisense drugs of the 1990s and early 2000s. Although only four therapies have been approved by the US Food and Drug Administration to date, recent and expected approvals from regulators in the U.S. may give the field the boost it needs to launch this class of molecules as the third major drug development platform after biologics and small molecules.