It is difficult to fix DNA mutations directly. But scientists suggest a different approach could be tried. In their opinion, RNA has a huge therapeutic potential, which has not yet been fully exploited.

The majority of American newborns will arrive home from the hospital and begin meeting developmental milestones. They will be upright in three months. They’ll be up by 6. And they’ll be walking by their first. But 1 in 10,000 won’t. They will be limp in their caretakers’ arms, unable to lift their heads or sit independently. Spinal muscular atrophy, or SMA, is a neuromuscular disorder in which particular motor neurons of the spinal cord gradually degrade. The condition is caused by a genetic abnormality called SMN2 (survival motor neuron 2), which causes misassembled proteins to produce gradual muscular weakening and paralysis.

This diagnosis was near-death five years ago. Most baby deaths were attributed to SMA. Many SMA babies died before their second birthday. Some survived toddlerhood but were never strong enough to run around or play with other kids and died of the sickness. In 2016, a novel FDA-approved therapy created by Adrian Krainer, a biochemist at Cold Spring Harbor Laboratory, in cooperation with Ionis Pharmaceuticals and Biogen, reversed that bleak prognosis.

The drug, Spinraza, solved the problem in an unusual way. Spinraza, which is administered through spinal tap, begins working as soon as the SMN2’s jumbled genetic code is transcribed into incorrect protein-making instructions—and corrects those instructions at the molecular level. Spinraza enters the picture shortly after DNA is translated into RNA, a workhorse molecule important for many cellular activities and, in this case, acts as a messenger transmitting DNA’s instructions.

“Spinraza is designed to bind to the messenger RNA, which enables the cell to handle it properly, and ultimately corrects the problem,” explained Krainer, who won a prestigious Wolf Prize in medicine this year, for his work explaining the molecular mechanisms behind this RNA process, which led to this new therapeutic.

Messenger RNA, or mRNA, reached the front pages of every newspaper last year when Pfizer and Moderna exploited it to develop COVID-19 vaccines. The medication makers use mRNA to transmit particular instructions to our cells in this new technology, which has never been used to vaccinate humans outside of clinical trials. The instructions instruct the cells to produce the spike protein used by coronavirus to infect us. Once inside the body, the spike protein enrages the immune system, which perceives it as a foreign intruder and prepares to combat the original coronavirus. After a period, the cells also degrade and eliminate any remaining mRNA from the vaccination. The mRNA technique appeared innovative, but it had been in the works for years, although in the shadows.

For decades, scientists’ primary focus has been DNA, with RNA considered as only a helper, a passive bearer of genetic instructions, merely an intermediary between DNA and proteins.

“When it came to RNA, even scientists weren’t clear what was so important about it,” said Lynne Maquat who heads the Center for RNA Biology at the University of Rochester and who shared the Wolf Prize with Krainer.

“People thought there were only three kinds of RNA, we already know what they do, end of story.”

However, that viewpoint has shifted. Not only did scientists find numerous varieties of RNAs, but they also discovered that a significant percentage of human DNA is devoted to their production.

“We now know that at best only 3 percent of our genome codes for proteins,” said Joan Steitz, professor of molecular biophysics and biochemistry at Yale University, and another co-recipient of the Wolf Prize, who has been studying RNA since the 1960s.

“And the other 97 percent is devoted to making all these different kinds of RNAs. We know what the most abundant and important of them do, but there are thousands of different ones that we still don’t have a full understanding of.”

Building this understanding holds the key to curing many genetic illnesses, which can be rectified by repairing the RNA or the processes in which this RNA is involved.

RNA, according to researchers, offers enormous undiscovered therapeutic potential. Pharmaceutical companies have traditionally targeted faulty proteins that cause disease while creating new drugs. According to Justin Kinney, a quantitative biologist at Cold Spring Harbor Laboratory who collaborates with Krainer to investigate the inner workings of this molecule, targeting the RNA allows the mistake to be fixed one step earlier, before the proteins are created. “RNA is a great target for drug development,” Kinney said, citing its versatility. His ambition is to provide the groundwork for a new generation of RNA-based medicines.

It’s not surprising that the DNA molecule has dominated its less glamorous cousin for more than a half-century. After all, the fascinating double-helix strand of DNA, neatly braided and snuggled inside the cell nucleus, contains the code for life. The DNA molecule, like a queen bee, governs its cellular kingdom, delivering commands for a plethora of cellular operations. Scientists attempting to discover the origin of genetic disease concentrated on DNA.

But no queen can rule her realm on her own. A queen’s messengers, maids, guards, and attachés are all necessary. And this is when the RNAs come into play. RNA molecules, like their queen’s ambassadors, carry out instructions for protein building, trigger reactions, and execute other functions to keep their cellular dominion healthy.

If you imagined each of your cells as a bustling kingdom, you’d see a million RNAs buzzing around all the time. The DNA would be translated, with its genetic instructions copied into messenger RNAs. These mRNAs would then carry these instructions on to ribosomes, the cellular protein and peptide-making machines, which would assemble them as needed. Transfer RNAs would provide amino acids to this protein building factory to keep the conveyor running. And the specific ribosomal RNAs would assist in the assembly of these amino acids into protein molecules. Meanwhile, additional mRNAs are being created—and as they are made, they are simultaneously being spliced and diced for reasons that scientists do not understand. This is only one of the mysteries that Kinney’s research could help to solve.

When an enzyme called RNA polymerase binds to the DNA and begins duplicating the DNA sequence into an RNA sequence, the process of transcribing DNA into mRNA begins. However, the result isn’t a particularly usable prototype. For starters, the resulting mRNA is around 10 times longer than it should be, so it must be trimmed—or spliced, a process in which certain sections are retained while others are discarded. Splicing is performed by molecular machinery known as spliceosomes, which remove unneeded nucleic acid sequences known as introns (from “intervening” snippets) and string the remaining portions, known as exons, together.

“You can think of the RNA polymerase as a newspaper reporter and the spliceosomes as a very, very stringent editor that cuts 9 out of 10 paragraphs the reporter writes,” Kinney explained.

“And it’s confusing why you would hire such a stringent editor to begin with—can’t your reporter just write less? So splicing seems like a very wasteful process. There are still debates about why it even evolved in the first place.”

According to Steitz, whose study elucidated the splicing mechanism, the prevalent hypothesis is that it allows for the creation of a broader diversity of proteins—and human bodies require all of those proteins to function. She discovered, among other things, that the splicing process is mediated by another another RNA player—the tiny RNA-protein particles known as snRNPs, or snurps. They locate and delete introns from mRNA molecules.

This procedure fails in patients with spinal muscular atrophy. As introns are deleted during splicing, one exon—exon 7—is likewise eliminated from the resultant SMN2 RNA. Without that exon, the proteins produced by these RNA instructions are faulty, resulting in spinal muscular atrophy.

Krainer compares the approach to a scribbled-up cookbook.

“Our genome is like a library where thousands of books contain recipes for protein-making, with every chapter spelling out precise instructions, and in the right order,” he said.

However, there are extra pages (the introns) in between the chapters that should not be there. Splicing removes such pages, making reading easier.

“If splicing is correct, you end up with perfect instructions. But in the case of SMN2, there’s a defect in Chapter 7, so splicing removes the entire chapter. Now a part of your instructions is missing, and you can’t follow the recipe.”

That’s where Spinraza comes in, with its splicing-level power. The treatment is essentially a brief portion of a DNA-like string that binds to SMN2 RNA prior to splicing. As it attaches, it prevents other proteins from interfering with splicing, allowing exon 7 to be included. The resultant mRNA includes the instructions for building the protein. Spinraza, the first registered RNA-based therapy, is paving the way for further RNA-based medicines, and for good reason.

RNA-based treatments may have a significant advantage over typical protein-based medicines. Currently, drug makers are focusing on dysfunctional proteins in order to correct their flaws. But, as Kinney pointed out, that’s a complicated and risky process in which three things must happen. First, the medicine must be able to bind to a specific location on the protein molecule, known as a site. Second, it must repair the protein’s rogue behaviour, such as turning off the active site and removing the protein’s potential to cause harm. Finally, it must not interfere with any other protein in the body in order to avoid clogging other critical functions.

“That’s a very difficult problem to solve,” said Kinney, as “most proteins don’t have a lot of potential binding targets.”

RNA, on the other hand, is densely packed with binding sites because it is designed to attract other molecules.

“The whole RNA is a target for drugs,” according to Kinney.

“The only limiting thing here is our understanding of how the RNA is controlled by various regulatory programs within the cell.”

In fact, our understanding is so restricted that scientists sometimes don’t know why a drug works. Kinney provided an example. Evrysdi, a new spinal muscular atrophy medication, was authorized by the FDA in 2020. It’s a smaller molecule than Spinraza, around the size of one base pair of DNA, and it’s easier to create and administer—it may be taken orally.

Kinney said: “It was essentially developed by trial and error.”

“Scientists took a few hundred thousand random molecules and tested each one in cells to see which ones increase SMN2 exon 7 splicing. The initial candidate molecule then underwent years of testing and tweaking.”

Although the final medicine, Evrysdi, is safe and effective, scientists are still disputing how it works at the molecular level, specifically how it distinguishes SMN2 exon 7 from the large number of other exons.

Splicing is one of the most complicated processes in human cells, and the spliceosome is the most sophisticated piece of cellular machinery discovered thus far. Aside from snRNPs, the spliceosome contains over a hundred other proteins. Surprisingly, at each RNA intron, all of these interlocking molecular pieces must reassemble. Decades of research have revealed how this sophisticated machine works once it is assembled, but nothing is known about how the spliceosome detects the exact pieces of RNA that must be cut out.

“Understanding this requires new quantitative approaches,” Kinney explained.

Kinney is working on something similar. Kinney’s goal is to solve these puzzles at the molecular level, using high-precision experimentation, mathematical modelling, and artificial intelligence—how the spliceosome analyses the RNA sequence and makes its cutting decisions, and how medications like Evrysdi zero in on their precise targets. Splicing causes numerous diseases, including cystic fibrosis and cancer, and even those to which it doesn’t contribute, Kinney said, “may be treated by modulating the splicing process.”

Once scientists understand the molecular cogwheels involved in splicing, they will be able to pinpoint where they go wrong and correct it.

“And that,” according to Kinney, “will open a lot of opportunities for making new and better drugs.”

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