Years After Playing Second Fiddle to CRISPR, RNA Editing Comes Into Its Own – An Interview with Dr. Joshua Rosenthal

(Photo Credit_Tom Kleindinst)

In recent times, RNA editing has emerged as a technology with a promise to replicate the achievements of gene editing. Eclipsed by the CRISPR frenzy for years, RNA editing is now beginning to draw its deserved recognition. “Thanks to COVID-19 vaccines, today, everyone is aware of mRNA. RNA is the superstar molecule now, but 10 or 20 years ago, people were not so familiar,” says Dr. Joshua Rosenthal, Neurobiologist at the Marine Biological Laboratory, an affiliate of the University of Chicago. He firmly believes the sky’s the limit for this technology. We interviewed him to obtain his views.

A renowned RNA and cephalopod scientist, Dr. Rosenthal has deeply studied the naturally occurring RNA editing phenomenon in this group of marine organisms. Realizing its therapeutic potential, he soon shifted some focus to its clinical applications as well and was instrumental in launching an RNA editing startup.

RNA Editing in Nature

Dr. Rosenthal worked on biophysics, cloning ion channels, and expressing them in heterologous systems in his formative years. His Ph.D. project was to clone the famous ion channels that Alan Hodgkin and Andrew Huxley discovered to explain the initiation and propagation of action potentials in the squid giant axon. It was when he first encountered RNA editing.

“I could never get a consistent sequence for the cDNAs while cloning the sodium and potassium channels. This was about the same time the first RNA editing paper came out from Peter Seeburg’s lab on the glutamate receptor. Eventually, on a hunch, we inspected the genomic sequence from the squid, and it was always an A. Yet, the cDNA was sometimes an A or a G residue! So we figured out this is RNA editing!” he recalled.

“I did a lot of work studying the function of RNA editing sites on different proteins. Later, at a Gordon conference, I met Prof. Eli Eisenberg, a gifted bioinformatician from Tel Aviv University, and teamed up to work on cephalopods”.

Together they found over 100,000 RNA editing sites in the neural transcriptome of squids, just from the protein-coding regions alone! “This was already several orders of magnitude more than any other organism,” he quipped.

Shift Towards Practical Applications

Dr. Rosenthal noted that much of this early work was performed at a molecular level with expensive techniques and modest enthusiasm from funding agencies. So, in the early 2000s, he shifted some focus to the practical applications of RNA editing. He observes that even before the arrival of CRISPR, it was evident that a technology capable of editing genetic information could be developed into a therapeutic.

Inspired by the contemporaneous discovery of RNA interference, he initiated a challenging project while serving as a Professor at the University of Puerto Rico. There, he designed systems for RNA-guided editing to edit a specific region of interest for therapeutics.

“For years, we worked in isolation when people weren’t aware of RNA editing. Later, it was overshadowed by the CRISPR revolution. However, people have begun to recognize the potential of RNA-level editing. So what started as a project to obtain more funding turned into a fun endeavor,” he stressed.

Applying the Programmability of RNA Beyond Genetic Diseases

Dr. Rosenthal says that the medical community focuses predominantly on curing rare genetic diseases and permanent disorders. Therefore, innovations like CRISPR are quickly lapped up. Although RNA editing is following CRISPR’s footsteps, he believes they have the potential to go beyond genetic diseases. “The transient nature and programmability of RNA are its two very valuable properties,” he asserts.

Many medical conditions today are short-term. Hence, it is redundant to opt for a permanent solution for a temporary problem. He believes that RNA editing approaches could offer more advantages in such cases.

“People typically suffer from pain for moderate periods of time, days to weeks, although sometimes it can be chronic. Like an aspirin, small-molecule drugs require repeat dosing because they offer short-term relief. In addition, effective pain treatments such as opiates can cause addiction issues. Therefore, it would be valuable to have a non-addictive treatment that lasts in the system for a couple of weeks, as long as the RNA and the protein it encodes are present,” he said.

RNA Editing vs. CRISPR

Dr. Rosenthal then proceeded to enlist the advantages of RNA editing over CRISPR. He indicated that the Cas9 enzyme used in CRISPR has a bacterial origin, and many of the CRISPR-based gene editing endeavors are predominantly performed ex vivo.

In contrast, RNA editing enzymes, the Adenosine deaminases acting on RNA (ADARs), are naturally present in almost all multicellular organisms. Therefore, one need not express an external cutting enzyme alongside a guide RNA.

“The simpler the system, the greater the chance for regulatory approval. So, just expressing small guide RNAs to edit genetic information is a very attractive prospect as compared to co-expressing bacterial proteins,” he reiterated.

Another issue one has to confront in CRISPR gene editing is off-target effects. Although RNA editing isn’t devoid of off-target effects, it wouldn’t be as harsh or create long-term problems as in the case of DNA. Whereas DNA edits are much more effective in dividing cells, RNA editing can be used to edit even post-mitotic cells like neurons.

Dr. Rosenthal also explained the advantage of RNA editing over gene therapy, saying that single-cell sequencing has revealed great variability in gene expression between cell populations. Adjusting expression levels across whole tissues using gene therapy is a difficult task. With RNA editing, one could work within the dynamic range of expression without worrying about over-expression or under-expression.

Emerging Companies in the RNA Editing Space

Dr. Rosenthal said, five years ago, there weren’t many RNA editing companies that he could recall besides Dutch firm, ProQR Therapeutics. Today, the field is considered a parallel track to CRISPR, attracting good funding from investors. He believes more exciting innovations are yet to burst into the scene.

Dr. Rosenthal’s own work on RNA editing led to the launch of Cambridge, MA-based RNA editing startup, Korro Bio. He was instrumental in pitching the potential of the technology to Atlas Venture and other investors. The company has now grown considerably, raising $91.5 million in Series A financing last year.

Beam Therapeutics, another Cambridge startup, has a unit that focuses on RNA editing research. Other notable companies include Seattle-based Shape Therapeutics and ADARx Pharmaceuticals from San Diego.

Current Challenges with ADARs

Although RNA editing offers numerous benefits, Dr. Rosenthal concedes that it is not devoid of challenges. He stated that people are increasingly trying to produce therapeutics which are smaller in size. In fact, he anticipates very few oligonucleotide-based therapeutics that are bigger than 20-something nucleotides to be approved by the FDA in the future.

“I think the major hurdle would be to create effective small guide RNAs for ADAR recruitment or find a way to design larger ones without causing problems,” he said.

“Another approach is engineering a system where the catalytic domain of ADAR is attached to an antisense RNA. This can serve as a guide RNA and also create a double-stranded structure for facilitating ADAR activity,” he added.

Dr. Rosenthal also mentioned that humans have two ADAR genes but not every cell type expresses them at desired levels. Furthermore, endogenous ADARs don’t edit every adenosine similarly. However, with engineered ADARs, one can overcome these limitations and edit sites more efficiently. He cited the work of Dr. Peter Beal, a structural biologist from UC Davis who is working on engineered ADARs.

When asked about the current development, Dr. Rosenthal mentioned a couple of engineered ADARs. Of them, E488Q mutation is a noted example that has improved the catalytic activity of ADARs. However, it leads to more off-target edits. To overcome this, Peter Beal and coworkers mutated ADARs at a different residue, which lowered the overall editing efficiency but effectively got rid of off-target edits.

Future Directions

“ADARs aren’t the only enzymes capable of RNA editing. There are several other enzymes like AID/APOBEC-like cytidine deaminases and tRNA adenosine deaminases that could be redirected to edit messenger RNAs. So, there is a ton of stuff to do on the basic level”, Dr. Rosenthal observes.

In the future, he would like to see people who develop enzyme systems also get involved. “Presently, people are mostly thinking about A to I or C to U editors because that could have implications in curing several genetic diseases. Ideally, we must be able to change any base into any other base. I am curious to see who gets the first thing approved,” he said.

“I look at the marine environment a lot, and I believe nature holds a lot of clues to novel DNA or RNA editing systems. I still am amazed at how RNA editing in squid is so prolific. Once we figure that out, I think we will use those lessons in developing human therapeutics. The lesson here is, if you stumble upon something odd, there is probably a gold mine in there,” he concluded.

Interview & Edits: Rajaneesh K. Gopinath, Ph.D.

Author

Rajaneesh K. Gopinath

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