genetic diseases

Move over, CRISPR: RNA-editing therapies pick up steam

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Two RNA-editing therapies for genetic diseases have in the past few months gained approval for clinical trials, raising hopes for safer treatments.

An artist's illustration of a messenger ribonucleic acid (mRNA) strand.
Tools to edit messenger RNA (artist’s illustration) are touted as being safer and more flexible than the CRISPR–Cas9 system, which changes the genome itself.

RNA editing is gaining momentum. After decades of basic research into how to manipulate this complex molecule, at least three therapies based on RNA editing have either entered clinical trials or received approval to do so. They are the first to reach this milestone.

Proponents of RNA editing have long argued that it could be a safer and more flexible alternative to genome-editing techniques such as CRISPR, but it poses substantial technical problems.

The launch of human trials signals the growing maturity and acceptance of the field, scientists say. “There’s a much greater understanding of RNA technology, and that’s been partially enhanced by the RNA vaccine and the COVID pandemic,” says Andrew Lever, a biologist at the University of Cambridge, UK. “RNA is now seen as a very important therapeutic molecule.”

Temp job

RNA has a crucial role in protein synthesis: the genetic information encoded in DNA is transcribed into messenger RNA (mRNA) before being translated into proteins. RNA molecules are composed of building blocks called nucleotides, each containing one of four bases, or letters.RNA therapies explained

RNA-editing techniques aim to compensate for harmful mutations by changing the sequence of RNA, allowing normal proteins to be synthesized. RNA editing can also increase the production of beneficial proteins.

Unlike CRISPR genome editing, RNA editing doesn’t change genes. Nor does it introduce permanent changes, because RNA molecules are transient. This means that the duration of the therapeutic effect could be shorter.

But that transience could offer safety advantages. One risk of CRISPR therapies is off-target effects, or unintended changes outside the target genomic region, notes Joshua Rosenthal, a neurobiologist at the Marine Biological Laboratory in Woods Hole, Massachusetts. “An off-target effect in DNA is potentially quite dangerous. In RNA, it’s less so, because it’s going to turn over.”

One letter at a time

One common RNA-editing approach, single-base editing, harnesses an enzyme that is already found in cells: adenosine deaminase acting on RNA (ADAR). This enzyme swaps a base called adenine in the RNA sequence for a base called an inosine.

Wave Life Sciences in Cambridge, Massachusetts, is exploring single-base editing to treat a genetic disorder called alpha-1 antitrypsin deficiency (AATD), which can damage the lungs and the liver. The disease reduces the production of AAT, a protein made in liver cells that protects lungs from damage caused by inhaling polluted air or other irritants.

Wave’s product is a short chain of nucleotides that directs naturally occurring ADAR enzymes to change a specific letter in each mRNA molecule to correct the mutation that affects AAT production. “By using the cell’s endogenous machinery to edit that single base, you now make a normal protein. And we’ve shown that the normal protein can be expressed at high levels,” says Paul Bolno, Wave’s president and chief executive.

In mice, the drug edited around 50% of the target mRNA in liver cells, which is enough to produce therapeutic effects, Bolno says.

The company’s clinical trial of the drug began last December in the United Kingdom and Australia, and will evaluate the drug’s safety and other features.

Editing whole paragraphs

Another approach, called RNA exon editing, changes thousands of genetic letters in an RNA molecule at once, as opposed to changing just one letter. Exon editing is akin to editing a whole paragraph instead of correcting one typo, says Lever. This technology is particularly important for disorders caused by multiple mutations in a person’s genome; such arrays of mutations are difficult to address with single-base changes, he adds.

The technique targets pre-mRNA, which is transcribed from DNA and then processed to make mRNA. Pre-mRNA includes both exons — parts of the RNA transcript that contain instructions for making proteins — and introns, which don’t contain such instructions. Through a mechanism called RNA splicing, the introns are cut out of the pre-mRNA, and the exons are stitched together to form the final mRNA, which is translated into protein.Collect more data from Africa to improve gene therapy

Companies such as Ascidian Therapeutics in Boston, Massachusetts, are leveraging the RNA-splicing process to remove mutation-containing exons and replace them with healthy ones. Last month, Ascidian received approval from the US Food and Drug Administration for a clinical trial of an exon editor to treat Stargardt disease, which causes vision loss. People with the disease have several mutations in a single gene, leading to the production of a defective protein that normally protects the retina.

Ascidian’s therapy relies on an engineered DNA segment that is delivered into cells and produces normal RNA exons. These replace the mutated ones during the splicing process, resulting in functional proteins. The DNA also produces RNA sequences that facilitate exon editing.

“With one molecule, [the therapy] is able to replace 22 exons at one time,” says biologist Robert Bell, head of research at Ascidian.

Cancer-quashing RNA

The potential of RNA-based therapies is not limited to genetic diseases. Rznomics, a biopharmaceutical company in Seongnam, South Korea, is testing an RNA editor to treat hepatocellular carcinoma, the most common type of liver cancer. In September 2022, the company started a clinical trial in South Korea, which it intends to expand internationally.

Rznomics’s approach involves mRNA splicing — but, unlike Ascidian’s method, it doesn’t use the cell’s own splicing machinery. Instead, the company co-opted a naturally occurring ribozyme, an RNA molecule that can induce splicing in target regions of mRNA. Researchers engineered the ribozymes to cut open mRNAs in tumour cells and insert a lethal cargo: an RNA sequence that is translated into a protein that generates a toxin that induces cell death. When surrounding cancer cells come into contact with these cells, the toxin spreads, promoting their death as well. This therapeutic molecule replaces an RNA sequence that is associated with tumour growth.

The use of the splicing approach against more than one disease is very exciting, says Lever, who is also the chief medical officer of Spliceor in Cambridge, UK, a firm that is working on RNA-splicing therapies. “It opens up a whole new range of possibilities of treatment for things which otherwise can’t be treated.”

CRISPR ‘will provide cures for genetic

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Scientists introduced CRISPR to the world as a gene-editing tool in summer 2012, when landmark papers from two independent groups demonstrated how the system could be wielded to make cuts in DNA. Now, less than 12 years later, we’re seeing CRISPR put to use in groundbreaking medical treatments.

“It’s really rewarding to see how fast the fundamental discoveries that were made in the lab actually are translated into the clinics,” said Šikšnys, who is chief scientist and head of the Department of Protein-DNA Interactions at the Vilnius University Institute of Biotechnology in Lithuania.

Prior to those seminal papers, other researchers had begun unraveling how CRISPR works inside microbes. Although best known as a gene-editing tool, CRISPR was first found in bacteria, and scientists realized that it acts as a kind of immune system — a defense against viruses. In this immune system, the bacterium has a memory bank full of virus’ genetic material. The bacterium will stash away this material after being attacked by a virus so it can guard against future invasions.

This memory bank is paired with tiny, molecular scissors called Cas proteins that snip through DNA, and a molecule that guides the scissors to their target. In bacteria, that target is a viral invader. But Šikšnys and his colleagues demonstrated that scientists could co-opt these scissors for their own purposes, targeting any DNA they want to edit. They specifically showed this with the protein Cas9.

Alongside Jennifer Doudna and Emmanuelle Charpentier — authors of the other groundbreaking CRISPR paper published in 2012 — Šikšnys was awarded the 2018 Kavli Prize in Nanoscience for the invention of CRISPR-Cas9, “a precise nanotool for editing DNA.” Nowadays, he and his team are investigating the diversity of CRISPR systems that exist in nature to see which others might be useful for engineering genomes.

Live Science spoke with Šikšnys about what it’s been like to see CRISPR enter clinical use and how he thinks the system might be applied and improved upon in the future.

Šikšnys studies the diversity of CRISPR systems found in nature. (Image credit: Vilnius University)

Nicoletta Lanese: Could you describe when you first began working with CRISPR-Cas? And could you give a sense of when you clued into the idea that this could be “a big deal” — a big-ticket technology that kind of shifts gene-editing as we know it?

Virginijus Šikšnys: We jumped into the CRISPR field, I would say, from the very beginning. It happened probably in 2007 when a paper appeared in Science, describing for the first time the possible function of the CRISPR-Cas system as an antiviral defense system in bacteria. And we decided to look, actually, at how this system functioned. This is how we started our CRISPR journey.

Of course, in the very beginning, we were very much interested in very basic biological questions. … It took us a while to understand the mechanisms behind the CRISPR-Cas systems. …

In [our 2012] paper, we showed that we can reprogram Cas9 protein and address it to any sequence in the genome. This was probably the moment where we understood that, indeed, this is a kind of really versatile system that could be employed for genome editing in different model organisms. And this is how, then, this kind of gene-editing field started.

NL: Did you envision right away that this might be applied in the treatment of genetic disorders? Did you see that as a possibility, even early on?

VS: If I recall, what we put in our paper at that time — we said that these CRISPR-Cas systems, or Cas9 protein programmed by CRISPR RNA, could be used for precise “DNA surgery.”

It means that, actually, you can direct Cas9 to any sequence in the genome, including the sequences where [there are] mutations that cause genetic disease.

NL: Having seen, within the last year, the first CRISPR-based therapies come to market — I’m wondering how it feels to have seen the progression of the field from that basic research to now seeing it applied at that level?

VS: Indeed, looking backwards, it’s really amazing to see that Cas9 made it into the clinics, nearly, in 10 years. I think it’s a really great achievement, and I’m sure that more therapeutic applications will follow in the near future and will provide cure[s] for genetic diseases that were incurable before.

And if you look at the list of clinical trials that are currently ongoing, where the Cas9 genome-editing tool is employed to treat different genetic diseases — the list is really very impressive. And it’s really rewarding to see how fast the fundamental discoveries that were made in the lab actually are translated into the clinics. 

Inforgraphic shows a labeled cas enzyme paired with an RNA strand. It then shows how the complex can cut through a length of DNA to either delete a gene or insert a new one in its place
CRISPR-Cas9 system uses Cas9 as molecular scissors and an RNA molecule as a guide for those scissors. (Image credit: VectorMine via Getty Images)

NL: Having seen the graduation into the clinic now, how do you anticipate the gene-editing systems borne of CRISPR-Cas might be refined in the future?

VS: Indeed, the CRISPR-Cas9 technology is a great tool that is rapidly advancing into the clinics. But still, there are several challenges that need to be overcome, and there are, of course, avenues for improvement of this tool. …

Recently, it made headlines that that CRISPR tool was used for treatment of SCD disease [sickle-cell disease]. In fact, [it] showed that this is really a tool that could be used in the clinics for the treatment of the patients.

But of course, this treatment has several limitations, because, in this case, the treatment is occuring ex vivo. It means that the cells that need to be treated are removed from the patient’s body, then Cas9 tool is applied to correct the mutation — or actually, trigger production of fetal hemoglobin. And then these engineered cells, they have to be delivered back to the patient’s body. And of course, this is kind of a challenging and time-consuming procedure.

So of course, it would be great if the CRISPR treatment could be done directly in [the] human body — we call it in vivo. But actually, to do that, you have to overcome several challenges: First, you have to deliver this CRISPR tool into specific tissues or organs in human body. And of course, there are many ways to deliver CRISPR tools, but after COVID, mRNA vaccines were approved as a therapeutic modality for treatment [prevention] of COVID. And currently, mRNA coupled with lipid nanoparticles became one of the key modalities that could deliver Cas9 into different cells and tissues in a human body.

[Other] delivery systems are also [being] explored, including virus-like particles and adeno-associated viruses. So AAVs are also used as delivery tools and they are approved as safe delivery tools into human body — but, for example, in the case of AAV, there is a packaging cargo limitation and you need to find smaller gene editing tools that could be packaged into a single AAV particle.

In my lab, in fact, we are looking at the avenues — how do you improve the existing tools or, actually, find new tools? To find new tools, we look at the diversity of CRISPR-Cas systems. These CRISPR-Cas systems [in nature] are very, very diverse, and we aim to understand this diversity of CRISPR-Cas systems from a fundamental perspective. And also, we hope that, looking at this diversity, we’ll be able to find new tools for genome editing applications.

Related: Meet ‘Fanzor,’ the 1st CRISPR-like system found in complex life

NL: Could you paint a picture of what it looks like to dig into the diversity of these systems?

VS: In my lab, we’re using a combined bioinformatics-based, biochemical approach. So we try to identify putative new CRISPR-Cas systems bioinformatically, and then, we try to characterize them biochemically using the tools available in the wet lab. …

First, we look at [microbial DNA] sequences that are present in really huge databases — you can just try to find new CRISPR systems there. Then, we try to express them in different bacteria, isolate them, characterize, and then move them to human cells to see whether they can be applied as new genome modification tools.

NL: We touched on the sickle cell treatments that have just been approved — I’m wondering if you, like others, anticipated that sickle cell would be one of the first diseases to get a CRISPR treatment? And what diseases do you see as the next frontier?

VS: I would say that it was clear from the very beginning that genetic diseases that are caused by a single mutation, like sickle-cell disease, will be the first target. It looked like low-hanging fruit, because you have to correct just a single mutation in the genome. And of course, I think part of the credit for this Cas9-based treatment of sickle-cell disease should also go toward the people who were studying sickle-cell disease for decades. They were providing us with understanding of the mechanisms of the disease that were harnessed into the treatment.

The other reason why SCD was a clear target was because, as I mentioned before, you can do the treatment [ex] vivo. You can remove cells that contain [the] mutation, actually engineer them in the lab, and put the cells back into human body. So that makes manipulations easier.

But of course, when you think about the next step — treatment of genetic diseases that are caused by multiple mutations, like cancer, for example, is still a challenge. But of course, scientists are trying to develop approaches how to tackle such complex genetic disease. And, for example, T-cell-based therapies are already in the clinics, and CRISPR [systems] are used there to facilitate engineering of these T cells … that could be used to treat cancers like lymphomas and solid tumors.

And of course, the CRISPR treatments in the human body, as I discussed before, is the next big step.

The beauty of CRISPR-Cas9 technology is that it’s a kind of versatile, or universal technology, because you can use this tool to engineer any living organism.Virginijus Šikšnys, Vilnius University

NL: This is somewhat tangential, but we’ve covered the idea of developing CRISPR as an antibacterial, as a kind of alternate antibiotic — do you see that as a fruitful research area?

VS: The beauty of CRISPR-Cas9 technology is that it’s a kind of versatile, or universal technology, because you can use this tool to engineer any living organism. You are just trying to engineer DNA, and DNA is the blueprint of every living organism. So instead of doing gene-editing in human cells, you can also think about doing editing of bacterial population — let’s say, that are present in [the] human gut. And these bacterial populations could be engineered. …

And as you mentioned, Cas9, CRISPR technologies could be used also as antiviral agents. Currently, the problem with antibiotics is pretty clear — we are probably losing our battle against bacteria using antibiotics. Novel antibiotics are always required and it’s really difficult to find them, and challenging and costly. So therefore, alternative technologies like viral therapies or CRISPR antibacterial systems are developed.

NL: Obviously CRISPR has so much potential, especially in the realm of treating genetic disease — I think people also have a lot of questions about the ethics of applying CRISPR in different contexts. Could you speak on that?

VS: I think it’s a very important question, and of course, CRISPR is a very important technology, and you can use CRISPR to do many things. But of course, you should keep in mind what you are doing and you need to be in touch and in conversation with society — Are these things acceptable? Or, what are the societal views on these technologies that scientists are developing in the lab? And I think it’s very important to communicate with people and explain, actually, what are these technologies, what they can achieve, and then what can be downsides, also, of these technologies.

We already heard about these stories that CRISPR was used several years ago in China for engineering human embryos — so that’s a line that scientists actually agree not to cross, because this could be a really dangerous thing.