How Promising is CRISPR for Fragile X?

FRAXA sat down with Peter Todd, MD, PhD, Assistant Professor in the Department of Neurology in the University of Michigan Medical School. Dr. Todd was awarded a FRAXA Research Grant for gene reactivation with the use of CRISPR.

The first work using CRISPR in Fragile X syndrome was performed using the CRISPR-CAS9 system to cut out the CGG repeats. In patient derived stem cells and neurons, this worked. Two groups were able to use this system to remove the repeats, trigger FMR1 mRNA transcription and get enhanced FMRP protein expression (Park et al, 2015, and Xie et al, 2016).

However, not every cell targeted with CRISPR-CAS9, underwent the correction- likely due to inefficiencies in delivery of these complexes into cells and imperfections in targeting of the complex to the FMR1 gene. Neither group was able to demonstrate that these techniques worked in an animal model and no evidence was provided that the correction actually made the neurons behave normally. Yet, these studies were important in establishing that the gene could be targeted and corrected in patient cells using this technique.

This year, Rudolf Jaenisch and colleagues at MIT demonstrated that they could use the CRISPR-dCAS9 system to target an enzyme known as TET1 to the CGG repeats to demethylate the FMR1 gene (Liu et al, 2018). When they did this, they got transcriptional reactivation of the gene and a return of Fragile X protein production in patient derived cells. They were able to show that this reactivation of FMRP expression led the cells to behave more normally than the FXS cells from which they were derived. They could also show that the gene stayed on if they transplanted these Fragile X cells into mice.

Our own group independently generated data (with a FRAXA grant) that is complementary to the findings of the Jaenisch group. We utilized a different dCAS9 fusion protein, VP192, to try and directly reactivate transcription from the FMR1 gene. In both control and FXS human embryonic stem cells, targeting this CRISPR-dCAS9 complex to the CGG repeats leads to a reactivation of FMR1 transcription. However, in our stem cells and neurons with very large (>1000 CGGs) repeat expansions, this transcriptional re-activation does not lead to a significant increase in FMRP protein production.

We interpret this as indicating that in the setting of very large repeats, transcriptional re-activation alone may not be enough to correct the deficit in FMRP seen in Fragile X syndrome patients. This work is submitted for publication and currently under review (Haenfler et al, 2018 ,under review).

I think the studies performed to date are quite promising. They demonstrate that the gene can be turned back on- either by removing the repeats directly or by targeting the methylation and transcriptional silencing elicited by the repeats. Thus, in concept, I am quite excited about the prospects of these approaches.

However, it is still quite early days with this technology. At this stage we are still correcting problems present in cells in a dish. Improving the efficiency of these corrections is really an engineering problem, but one that many people around the world are working on addressing. Achieving reactivation of FMR1 expression from a human gene in live mice will be a critical next step in establishing that these approaches could be clinically transferrable. These studies will be hard given that our current animal models do not have this mechanism of FMR1 gene silencing that happens in patients.

The other key experiments needed are to establish that these approaches do not have significant off-target effects. CGG repeats are common in our genomes, so there may be impacts of this approach that are unintended. Gene editing with CRISPR-CAS9 is specific, but often not perfectly specific. Thus these issues need to be worked out on the technical side before anything could ever move into patients.

I see three key questions that must be answered as the research in this area moves forward:

1. How many cells must you reactivate FMRP expression in to get improvements in clinical symptoms in FXS patients- or even FXS model mice?
2. Is reactivating the FMR1 gene after brain development (i.e. after birth) sufficient to correct the problems? There are hints that getting more FMRP expression back will be helpful, but how much help remains unknown.
3. What is the impact of reactivating expression of a very large CGG repeat expansion in FXS patients or model systems? Transcribed CGG repeats can be toxic to neurons and can themselves be translated into toxic proteins. We think that enhanced expression of larger CGG repeats is what causes FXTAS. Thus, if we try to turn the gene back on with very large repeats still present, it may prove harmful. Efforts to correct these effects may thus be needed if gene reactivation becomes a viable therapeutic approach going forward.

These questions are important to answer regardless of the exact approach used to re-establish expression of FMRP.

I agree that we are a ways away from using CRISPR to correct the gene defect or to turn the gene back on in Fragile X patients. However, this technology is developing very quickly, so what seems like pie in the sky may be a real thing in the not-too distant future. The reason that these approaches are gaining so much interest is that if we can figure out the practicalities of delivery and specificity, then they can be used on many diseases, not just Fragile X. Thus, the technical advances will proceed quickly.

Also, I think that these tools will teach us a lot about how the gene gets turned off in the first place and how we might turn it back on using simpler approaches- with small molecules and drugs. Even if CRISPR is not the cure, it might point us in the right direction.

As stated above, I think this is not something we currently know and it is an important research question that I hope Fragile X researchers will address now that the concept of turning the gene back on in adults is more real. If reactivating the gene in adulthood makes things better, then we should be focusing more research and efforts on this front.

I started in Fragile X research when I was a graduate student in 1998. I worked in the labs of Ken Mack MD, PhD (now at the Mayo Clinic in Rochester) and Jim Malter, MD (now chair of pathology at UT Southwestern in Dallas). We were studying genes whose expression in the brain changed with experience and discovered that the Fragile X gene was translationally upregulated in response to environmental stimuli. We went on to demonstrate that FMRP expression was regulated by mGluR signaling in neurons and that it itself regulated translation of another gene, PSD-95, in response to mGluR activity. That early work was supported by a fellowship from FRAXA.

After my PhD, I went back to medical school and became an adult neurologist. I really thought at the time that I was done with Fragile X research. However, during the 6 years between when I finished graduate school and I became a clinical fellow in neurology, the adult onset disorder Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) was described by Randi and Paul Hagerman. So, when I started my own research lab in 2010, I decided to study FXTAS because I had already learned a lot about the Fragile X gene and I found the differences between the Fragile X syndrome and FXTAS disease mechanisms to be fascinating. In the past few years, however, we realized that our understanding of these repeats and their biology in FXTAS and emerging tools like CRISPR presented some really interesting angles for trying to correct the proximal cause of Fragile X syndrome- which is the silencing of the gene that prevents FMR1 RNA transcription and FMRP translation.

Working with FRAXA has been a real pleasure. This CRISPR based research was a completely new direction for our research group. It would not have been possible without FRAXA’s support. They have welcomed us back into the Fragile X syndrome research space with open arms and made sure we are well connected to the best research going on currently in the field. Now, with their support, we are invested in moving these and related therapeutic approaches forward.