Rett Symptoms in Mice Alleviated with RNA Editing

RNA editing, one of six genetic-based strategies that RSRT is investing in to cure Rett, has seen explosive growth in the last handful of years and has generated attention from many academic labs and companies – and with good reason. A new paper published online this week in Proceedings of the National Academy of Sciences finds that RNA editing can improve symptoms in a mouse model of Rett syndrome. Gail Mandel, PhD, and post-doctoral fellow John Sinnamon, PhD, authored the paper, and RSRT has been funding the Mandel lab at Oregon Health & Science University for more than a decade.

This study provides an essential proof of concept for improving Rett syndrome symptoms using RNA. Potentially 45% of Rett syndrome mutations are repairable by RNA editing, including the common mutations R168X, R255X, R270X, and R294X. Now that it has been established that RNA can affect symptoms in models of Rett, future studies can focus on improving the efficiency of editing and of symptom rescue in models of these mutations.

Not only is this a pivotal discovery for a Rett syndrome cure, this study is also the first demonstration of symptomatic improvement in a mouse model of any human disease by RNA editing. Needless to say, this is a very exciting moment as we drive progress to correct the root cause of Rett syndrome: mutations in the MECP2 gene.

What is RNA editing?
As a quick refresher, genes make proteins through a two-step process. The first step is to make a copy of the genetic instructions that are in the gene. That copy is called RNA. The next step is to convert the RNA into a sequence of amino acids that make up proteins, such as the MECP2 protein.

As we discussed in a previous webinar, RNA editing is a naturally occurring process in human cells where a group of enzymes called Adenosine Deaminases Acting on RNA (ADARs) change the RNA base adenosine (A) to an inosine (I) base. The inosine is interpreted by the cell as guanosine (G) as it makes the protein.

The editing of A-to-I(G) in RNA then causes coding changes in the resulting protein, altering protein function. Targeted, or site-directed, RNA editing utilizes the naturally occurring activity of ADARs to correct mutations, either by using engineered ADAR enzymes or by recruiting the enzymes that are naturally present in cells. One of the advantages of using targeted RNA editing for correcting mutations is that the repaired protein is expressed at the same level as the normal protein. This is particularly important for Rett syndrome, where MECP2 over-expression can result in a distinct but related disease.

Putting RNA editing on the map for Rett
The lab of Gail Mandel, PhD, from Oregon Health & Science University, was the first to publish on RNA editing for Rett syndrome and in so doing put this curative strategy for Rett squarely on the map. With funding from RSRT and the NIH, the Mandel lab first demonstrated targeted RNA editing in MECP2 protein-deficient neurons in culture and then in multiple neuronal populations in the brain of a mouse model of Rett syndrome.

Dr. Mandel and John Sinnamon, PhD, a senior researcher in her lab funded by RSRT, have extended this work in the paper published this week and answered the longstanding question of whether RNA editing of the MECP2 gene could actually improve Rett-like symptoms in mice.

What happened in this newest RNA editing study?
The authors made a new mouse model of Rett syndrome that contains a patient mutation that has an A in place of the G in a critical place for MECP2 function. Rett mouse models like this one are essential to test various curative approaches. To repair the mutant A back to the normal G, the authors injected into the bloodstream an adeno-associated virus (AAV) encoding an engineered ADAR enzyme, referred to as Editase, and a guide RNA that acts like a GPS to bring the Editase to the mutation in the mouse MECP2 RNA.

After injection of virus into young male Rett mice, they observed editing in multiple brain regions, with the highest amount of editing, 18%, in the brainstem. By looking at individual cells in the brainstem, they found that about the same percentage of cells showed repair of the mutation. The repaired MECP2 protein was expressed, on average, to 70% of normal levels. Importantly, some cells even showed protein levels equal to those observed in normal (non-mutated) cells, indicating no inherent ceiling to the level of repair that is possible in some neurons in vivo (in live animals). This is very encouraging.

Looking closely at breathing and other measurable symptoms
Many of the Rett-like behaviors involve multiple brain regions, or the specific neuronal circuits underlying the behavior are not well defined. However, the brainstem, which is the region with the highest level of MECP2 RNA editing, is known to be essential for the control of respiration, and respiratory dysfunction, such as apnea (the temporary cessation of breathing) is a hallmark symptom of Rett syndrome patients that is repeated in Rett mice. Untreated mutant male mice and those injected with the Editase and a guide RNA that did not target MECP2 messenger RNA showed an expected high number of apneas and irregular breathing patterns. However, the mutant male mice injected with a virus targeting MECP2 RNA had the same number of apneas and breathing pattern as untreated normal mice.

In addition to the improvement in respiration, the mutant mice with edited MECP2 RNA exhibited a longer life span compared to the nontreated and control-treated mutant mice but did eventually die. Since we do not know the underlying cause of this phenotype, it is difficult to infer why the survival was not completely rescued, but the authors hope that targeting more cells with next-generation viruses will further improve this symptom as well.

Factors that limit RNA editing efficacy
Importantly, Dr. Mandel and her colleagues identified a limiting factor to maximizing editing of cells in the brain: expression of the guide RNA that targets the Editase enzyme to MECP2 RNA. While both the Editase and guide RNA were packaged in the same virus, the pattern of expression of these two essential components was distinct and inconsistent across different brain regions, with the brainstem showing the most cells showing expression of both enzyme and guide. This key finding affects all guide-based therapies used in the brain, including DNA base editing, which typically uses the same viral component as this study to express guide RNAs. Improvements in the design of the virus, and the elements controlling the Editase and guide within the brain, will likely result in more cells exhibiting guide RNA expression and a correspondingly higher number of cells showing MECP2 repair, and, hopefully, improved behavioral outcomes.

Congratulations are in order!
We congratulate Dr. Gail Mandel and Dr. John Sinnamon, and the rest of the Mandel lab members who contributed to this very important work.

We also thank the members of the Rett syndrome community who have donated to RSRT, thereby allowing us to support the Mandel lab for the last decade, including this latest paper.