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In Vivo RNA Editing Repairs Rett Mutation in Mouse Hippocampus

In a mouse model, researchers were able to use in vivo RNA editing to repair the toxic protein implicated in Rett syndrome.

Scientists who demonstrated in 2017 they could edit an RNA mutation that causes Rett syndrome by targeting the mutant RNA in vitro have now done so in the hippocampus of living mice.

The scientists, from the Vollum Institute at Oregon Health and Science University (OHSU) in Portland, injected adeno-associated (AAV) virus containing an engineered RNA editing enzyme and guide RNA specific for methyl CpG binding protein 2 (MeCP2) into the hippocampus of mice bred to have a Rett syndrome patient mutation.

One month after the injection, half of the MeCP2 RNA measured from three different types of hippocampal neurons carried the corrected sequence.

The enzyme was also found to have edited off-target sequence areas of the RNA transcriptome, but a majority of sites were edited at rates of 30 per cent or less, considered to be promisingly low by the authors and commentators.

Describing it as a proof-of-principle study, the researchers did not test whether behavioural deficits associated with Rett were corrected in the mice.

“There were a lot of questions about whether or not this approach could be used in vivo,” said John R. Sinnamon, PhD, first author of the paper and a postdoctoral fellow in the laboratory of the paper's senior author, Gail Mandel, PhD, senior scientist at the Vollum Institute and professor in the department of biochemistry and molecular biology in the School of Medicine at OHSU.

“One question was whether we could deliver enough of this enzyme in vivo. Another was whether it would work in multiple neuronal types. What we found was that we saw about 50 per cent editing in each of the three neuronal fields we tested. That was surprising to us because functionally they're very different neuronal types.”

Timothy Benke, MD, PhD, the Ponzio Family Chair in Pediatric Neurology Research, and Rett Clinic medical director, and director of research at the Neuroscience Institute at Children's Hospital Colorado, called the Cell Reports paper “very exciting. Other approaches are addressing important clinical aspects of the disorder. This group is seeking to correct the genetic cause.”

“We are still far from this preclinical work being translated into a clinical trial, but these types of disease-modifying therapies hold tremendous promise in genetic neurodevelopmental disorders.”—DR. SHAFALI SPURLING JESTE The next step, Dr Sinnamon said, is to test whether RNA editing can be delivered throughout the brain and correct the behavioural phenotype in the mice.

“For behaviour studies, you need a large number of animals to get statistical power,” Dr Sinnamon said. “We had been gearing up for the next step before COVID-19. We're a little further behind than we would like to be because of restrictions on non-essential research.”

The Scottish scientist who first demonstrated in 2007 that the Rett phenotype could be rescued in both immature and mature mice by activating Mecp2 expression said he hopes to see an RNA or gene therapy trial in humans within five years.

“There are going to attempt to improve this disorder pretty soon,” said Adrian Bird, FRSE, FMedSci, the Buchanan Professor of Genetics at the University of Edinburgh. “I like to think that within five years it will definitely have been tried. Everything that's happened preclinically says it stands a very good chance of working if you can get it into enough cells of the brain.”

Study Details

Rett syndrome is caused by mutations in the MeCP2 gene; a subset of these mutations are guanosine-to-adenosine (G>A) mutations. In a 2017 paper in the Proceedings of the National Academy of Sciences, the Oregon team delivered an AAV and found that it corrected the defect in 72 per cent of cultured neurons from a mouse model containing one such G>A mutation using what is called “site-directed RNA editing.”

For the in vivo study, Dr Sinnamon and colleagues engineered an AAV to express an editing enzyme, or “editase” containing the hyperactive catalytic domain of adenosine deaminase acting on RNA 2, or ADAR2. The editase switches the A to inosine, which is translated by the RNA machinery as a normal G, thereby fixing the mutation. But to make the switch only where needed and nowhere else, they added a MeCP2 RNA guide designed to link only to the mutated RNA sequence.

One month after injecting the AAV, they found that 50 per cent of the MeCP2 RNA had been corrected in three different hippocampal neuronal populations and that the corrected MeCP2 protein associated with chromatin, its primary function, was restored in neurons to 50 per cent of wild-type levels. Off-target editing rates were largely limited to 30 per cent or less and were influenced by editase levels, suggesting that lower amounts of the editase might result in fewer off-target edits.

The paper noted that other groups are already identifying editase molecules with higher specificity and efficiency for in vivo testing.

The similar rates of expression in the three neuronal populations achieved by injecting into the hippocampus, the paper concluded, suggests that the next study could use peripheral injections into the bloodstream or into the cerebrospinal fluid (CSF) in order to repair neuronal populations across the entire brain.

“With peripheral injections,” the paper stated, “comprehensive behavioural testing combined with quantitative measurements of Mecp2 protein function and gene expression are possible and will need to be performed in male and female Rett syndrome mouse models. How much repaired MeCP2 per cell is necessary and how many neuronal and glial cells need to be repaired to reverse Rett syndrome phenotypes in mice is not known.”

Although 50 per cent repair per cell is unlikely to result in a wild-type mouse, the paper noted, “this level of repair may be reasonably expected to result in significant improvement in Rett syndrome-like phenotypes in treated mice.”

Because overexpression of MeCP2 can be as harmful to neurodevelopment as under-expression, a significant advantage of RNA editing over gene therapy is that it can never result in excess levels.

“It's a Goldilocks gene,” said Dr Sinnamon. “You can't have too much and you can't have too little. Our strategy of fixing the RNA means it will never result in too much.”

Expert Commentary

Shafali Spurling Jeste, MD, FAAN, associate professor in psychiatry, pediatrics and neurology at the David Geffen School of Medicine at UCLA, said, “This is an exciting study, where they are showing us in preclinical models that this new RNA technique does work.

We are still far from this preclinical work being translated into a clinical trial, but these types of disease-modifying therapies hold tremendous promise in genetic neurodevelopmental disorders.”

With some of the drug treatments under development, she noted, “you would have to bring a kid in every month for an injection. This RNA technique would involve a single injection. It's exciting to work.”

Huda Zoghbi, MD, the Ralph D. Feigin Professor in the departments of pediatrics, molecular and human genetics, neurology and neuroscience at Baylor College of Medicine, said she was “pleasantly surprised” by the amount of corrected RNA found in the mouse hippocampus after the treatment.

“Fifty per cent is a good, decent amount of editing to make a difference,” said Dr Zoghbi, who first described the Mecp2 mutations as the cause of Rett syndrome in a 1999 paper in Nature Genetics.

Dr Zoghbi said: “Whether they might be able to lower the number of off-target edits by lowering the dosage of editase designed to make the change has to be tested. That is what has to be tested to show efficient editing while minimizing the off-target effects.”

Dr Bird said he was less concerned about the off-target edits than he would be if the study had involved gene therapy, which involves changes to DNA.

“DNA is forever,” Dr Bird said. “If you mess up, that's it. RNA is different. If you get a few wrong, cells have ways to handle that. Nevertheless, it is important that this group has looked at off-target edits seriously.”

Dr Bird's greater concern, he said, is whether an AAV can be found that will spread far enough in the human brain.

“The weak link in all of this is the AAV vector,” Dr Bird said. “In this particular study they used a variant that hit many neurons, but it only works that well in mice, not humans. But the fact that such an efficient vector exists for mice does encourage the belief that somewhere out there is an AAV that will solve this problem for humans. People in this field are going all out to find such a variant.”

In 2019, a randomized clinical trial of trofinetide, a synthetic analogue of a growth factor produced by cells in the brain, found that the highest dose, 200 mg/kg for 42 days, resulted in statistically significant and clinically relevant improvements over placebo on the Rett Syndrome Behavior Questionnaire and other measures in 82 children and adolescents.

Whether they might be able to lower the number of off-target edits by lowering the dosage of catalyst designed to make the change has to be tested. They're going to have to do the experiment to show that.”—DR. HUDA ZOGHBI

A number of other trials testing therapies are in progress against Rett syndrome—trofinetide is in phase 3, ketamine is in phase 2, and the oral cannabidiol solution Epidiolex is in phase three.

“If COVID-19 hadn't happened, those trials would be moving along,” said Dr Benke. “Both of the companies supporting the phase 3 trials are looking for approval in the next year or two.”

He pointed to ongoing studies of treatments for other neurodevelopmental genetic disorders, including Angelman syndrome, Dravet syndrome and spinal muscular atrophy.

“The next couple of years are going to be big,” Dr Benke said. “Clearly companies have decided that the rare disease space is interesting to them. For those of us in the rare disease space, this is hugely exciting.”


Drs Sinnamon, Benke, Bird, and Jeste disclosed no relevant disclosures. Dr Zoghbi has received fees from Regeneron, Denali, and the Column Group.

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