Restoring Fragile X function with gene editing

Fragile X syndrome is the most prevalent genetic form of intellectual disability. There is no cure or treatment due in part to the complexity in the Fragile X syndrome neuronal circuitry. In this webinar, Dr. John Graef (Fulcrum Therapeutics), demonstrates how using CRISPR gene-editing and patient-derived cells Fulcrum can create the Fragile X syndrome phenotype in a dish. Moreover, this approach has enabled an estimate of the level of FMRP protein expression required to correct the observed Fragile X syndrome phenotype.

Transcript of webinar:

Thank you for joining us on today's coffee break webinar!

Today's topic is Restoring Fragile X Neuron Function with Gene Editing.

Fragile X Syndrome is the most common genetic form of intellectual disability with an incidence of 1 in 4,000. Patients with Fragile X Syndrome display a broad spectrum of autistic phenotypes such as intellectual social and reasoning deficits. These characteristics are attributed to the loss of the Fragile X Mental Retardation Protein or known as FMRP encoded by the fmr1 gene during brain development. FMRP is an RNA binding protein which is essential for the maintenance of normal synaptic function. Currently there is no cure or treatment for Fragile X Syndrome due in part to the enormous complexity of the Fragile X Syndrome neuronal circuitry.

In this webinar Dr. John Graef principal scientist at Fulcrum Therapeutics demonstrates how using patient-derived cells and CRISPR gene editing can create and reverse the Fragile X Syndrome phenotype observed in a dish.

Thank You, Melissa for that introduction. What I would like to talk about for this webinar is how at Fulcrum we are using patient iPSC-derived neurons and Axion's multi-electrode array platform to elucidate a functional phenotype for Fragile X neurons and how we went about correcting that phenotype by restoring levels of FMRP, the deficient protein, underlying this neurodevelopmental disorder.

First I'd like to tell you about Fulton therapeutics we are a drug discovery company located in Cambridge, Massachusetts and our focus is on treating the genetic root cause of diseases. Our fundamental premise is that by working on monogenetic diseases caused by disrupted gene regulation we can focus our drug development strategies on normalizing the aberrant gene expression. One of the diseases we are working on is Fragile X. Fragile X Syndrome is a neurodevelopmental disorder with an incidence of 1 in 4,000. It is the major genetic cause of intellectual disability and mental retardation. Patients with Fragile X also display a variety of autistic spectrum phenotypes including; anxiety, avoiding, and verbal disability. Almost four decades of Fragile X research has identified the X-linked gene FMR1 as the disease-causing gene. The majority of Fragile X patients have an expanded CDG repeat at the five prime untranslated region of the FMR1 gene, which leads to the epigenetic silencing of FMR1 and therefore no expression of the protein FMRP. FMRP is an important RNA binding protein localized in the synapses and therefore plays a critical role in a neuronal communication.

To date the most relevant preclinical trial for Fragile X is based upon the mGluR5 theory. This theory argues that FMRP plays a negative role in activity dependent local protein synthesis associated with a metabotropic glutamate receptor, mGluR5. However several clinical trials targeting mGluR5 have failed. These could have failed for a number of reasons but the mechanisms are all acting downstream of FMRP acting at proteins whose expression is themselves controlled by FMRP. Fulcrum strives to correct the root cause of Fragile X by reactivating FMR1and FMRP to physiologically meaningful levels. We feel that by restoring enough FMRP to neurons we can correct the functional deficits, however, before we can do this we need to identify a functional phenotype in Fragile X patient-derived neurons and then determine the FMRP levels needed to correct this phenotype. In order to identify a functional Fragile X phenotype we have used Axions Maestro MEA platform to measure differences in spontaneous activity between isogenic pairs of iPSC-derived neurons, where FMRP expression is the dependent variable.

We are utilizing the MGN2 method previously described by Marius Wernig and Thomas S├╝dhof to differentiate iPSCs to neurons through overexpression of the transcription factor in neuro gen2. An example of spontaneous spiking activity from sixteen different electrodes in one well can be seen on the left hand side of the slide. On the right is an example of spontaneous activity shown on a 48 well MEA plate as depicted by a real-time heat map. We use three different isogenic cell lines to assess the FMRP dependent effects on functional activity, on the MEA these three isogenic lines are shown here. The first pair of cell lines denoted as SWFs X and s WC12 were generated by Steve Warren through CRISPR edited removal of the CGG repeats in a Fragile X patient line.

The immunofluorescent images for these neurons are shown on the top left where the nuclei are shown in blue, the neuron-specific microtubule component, beta-tubulin, is shown in green, and FMRP is shown in red. As can be seen from the images, both cells form viable neuronal networks, with the Fragile X neurons having no FMRP expression and the CRISPR corrected neurons showing high levels of FMRP located in the cell body. The second line used was a wild-type line purchased from Thermo Fisher denoted as epi 49. A FMRP knockouts cell line was created from this wild-type line by Dr. Hal Wu, a senior scientist in the biology group here at Fulcrum, by inserting a premature stop codon in fmr1 in which fmr1 mRNA levels are still measurable, but no FMRP is detected. Images for the wild-type neurons which show detectable FMRP levels, and for the FMRP knockout neurons which don't are shown to the left. The final isogenic pair used was also generated from a Fragile X patient line denoted as 135.3. This line was also generated by Dr. Wu through CRISPR editing of the CGG repeats in order to produce a clone with a truncated CTG repeat region.Images of this isogenic pair also show detectable levels of FMRP and the CRISPR corrected isogenic control neurons and a lack of FMRP expression and the Fragile X patient drive neurons.

On this slide is a figure from a recent self-publication demonstrating the method for deleting CGG repeats in the fmr1 locus with CRISPR cas9 in order to reactivate fmr1 expression in the c1 - line. Panel B shows fmr1 mRNA levels measured by qPCR, in a control line the Fragile X line and the isogenic c12 generated by CRISPR cas9 editing. Panel C shows representative immunofluorescent images of these three lines with Dappy stains shown in blue, beta-tubulin stains shown in green, and FMRP shown in red. Panel F demonstrates that re-expression of the FMRP and the isogenic control neurons with truncated CGG repeats, show reduced excitability as measured by spikes per minute, over a two-week period on the Axion Maestro MEA system when compared to the Fragile X neurons.

In follow up experiments we use the three previously described isogenic lines to confirm this increase in spontaneous activity on the MEA. We were able to demonstrate hyperactivity in neurons lacking FMRP that manifested after three weeks in culture for all three isogenic pairs tested. These experiments established a functional phenotype for Fragile X iPSC-derived neurons, where reduced FMRP levels leads to neuronal hyperexcitability. In these graphs the neurons lacking FMRP are shown in blue and the neurons expressing FMRP are shown in gray. The x-axis denotes the time in culture and the y-axis shows the average weighted firing rate per well, which was calculated as the product of the spontaneous spikes per minute and the number of active electrodes in the well.

Once we had established a functional phenotype and knew that restoration of the FMRP to near full wild-type levels was enough to normalize this increased spontaneous activity, we wanted to determine if partial restoration of FMRP was sufficient and if so, what partial FMRP levels are needed to correct the functional phenotype. We did this two ways first we asked a full restoration of FMRP levels and only a subset of cells was enough, we used fmr1 mRNA transfection experiments along with experiments where we titrated in FMRP expressing cells into in varying percentages into Fragile X cultures, these experiments essentially created an FMRP mosaicism in a dish.

Next, we asked if partial restoration of FMRP and a majority of Fragile X neurons was enough. We did this by titrating and varying MOIs, the virus containing the caste 9 Test 1 and CGG guide RNA constructs, to achieve varying levels of fmr1 demethylization reactivation. This slide shows representative amino fluorescent images demonstrating FMRP mosaicism in a dish. The top three images are from fragile x neurons 24 hours post transfection with fmr1 mRNA, as can be seen from the top middle image only a subset of Fragile X neurons took up the fmr1 mRNA, and are expressing FMRP at near control levels. Additionally the bottom three images show examples of Fragile X and control neuronal cultures where FMRP expressing neurons were mixed with Fragile X neurons the bottom middle image shows a culture where 20% of the neurons were expressing FMRP.

This slide summarizes the data from our FMRP mosaicism experiments starting in the top left-hand corner part of the slide, this graph shows the average number of FMRP positive neurons 24 hours post fmr1 mRNA transfection and Fragile X neurons as measured by immunofluorescence. The data are normalized the isolated controls shown in dark gray which was set at 100%, this shows that when neurons were transfected with 4 to 12 nanograms of fmr1 mRNA depicted as the green, blue, and orange bars, that this leads to around 5-10% FMRP positive neurons. When neurons were transfected with 16 to 24 nanogram fmr1 mrna, shown by the brown and gold bars respectively, this resulted in around 20-25% FMRP positive neurons. The corresponding MEA data is shown below in the bottom left-hand corner, this graph shows increased spontaneous activity for Fragile X neurons in light gray, as compared to the isogenic control neurons shown in dark gray, the spontaneous activity of Fragile X and neuronal culture's transfected with fmr1 mRNA levels that lead to 5-10% FMRP positive neurons, shows a trend towards reduced activity, however, Fragile X in neuronal cultures transfected with fmr1 mRNA amounts that lead to 20 to 25 percent levels of FMRP positive neurons show full normalization of hyperactivity.

The data from experiments in which FMRP expressing neurons were mixed with Fragile X neurons is summarized in the upper right portion of the slide, here the increased spontaneous activity and Fragile X neurons in the gray bar over control neurons in the black bar shows a partial normalization when less than 10 % FMRP expressing cells are mixed into the culture. Only when 20 percent or greater FMRP expressing neurons are added is the hyperexcitable Fragile X can type fully rescued. Therefore transient transfections of fmr1 mRNA and titration of control neurons into Fragile X cultures suggest that 20% of neurons need to be expressing high levels of FMRP to normalize hyperactivity.

This slide shows another figure from our recent self-publication demonstrating the method used to demethylate the CGG repeat of the fmr1 gene an inactivated cast nine enzyme was tethered to a tet1 enzyme and targeted to the hypermethylated CGG fmr1 repeat region with CGG guide RNA this led to demethylation of the fmr1 CGG repeat region reactivation of fmr1 levels and normalization of hyperactivity as measured on the MEA. This was done in IPSCs followed by fax sorting in order to capture successfully transduced cells, then differentiated into neurons to assess fmr1 expression shown in the lower left and spontaneous activity shown in the lower right.

The MEA data shows that over time the increased spontaneous activity seen in the Fragile X neurons shown as the red line as compared to the control neurons depicted as the blue line can be rescued following near full restoration of fmr1 levels.

In follow up experiments we transduce post differentiated Fragile X neurons with the same dcas9 tet1 construct to determine if low levels of FMRP expression in a large portion of neurons could also normalize the increased spontaneous activity. The immunofluorescent images in the upper right hand corner show that after transduction with the dcashman tet one construct low levels of FM RP could be detected in the majority of Fragile X neurons this is quantified in the lower left as around 5 to 7% FMRP levels. The MEA data is shown in the upper right hand corner of the slide this data demonstrates that Fragile X neuronal cultures that are expressing 5 to 7-percent ever repeat levels begin to reduce this increased spontaneous activity. Therefore demethylation of the CGG repeats region through transduction of dcas9 tet 1 suggests that at least 5% FMRP levels in a large proportion of neurons is needed to normalize the fragile x hyperexcitability phenotype in summary reduced FMRP levels lead to neuronal hyperexcitability as measured by the Axion MEA system.

Close to full restoration of FMRP levels and around 20% of Fragile X neurons is sufficient to normalize this hyperexcitability and then greater than 5% FMRP restoration and a majority of Fragile X neurons is sufficient to normalize the increased spontaneous activity seen.

In closing I would like to acknowledge everyone who is instrumental in generating this data, thank you for your time and I hope you enjoyed this webinar.

And that is the conclusion for today's coffee break webinar, if you have any questions you would like to ask regarding the research presented or if you are interested in presenting your own research with microelectrode array technology please forward them to for questions submitted for Dr. Graef, he will be in touch with you shortly. Thank you for joining in on today's coffee break webinar and we look forward to seeing you again