Gene Therapy Shows Promise in Fragile X Rat Model

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by Steve Bryson, PhD |

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Gene therapy rescued several behavioral and brainwave abnormalities seen in a rat model of fragile X syndrome.

Findings from the study support further development of gene therapy for treating people people with the condition, the scientists said.

The study, “Gene Therapy Using an Orthologue of Human Fragile X Mental Retardation Protein Partially Rescues Behavioural Abnormalities and EEG Activity in a Rat Model of Fragile X Syndrome,” was published in the journal Molecular Therapy, Methods & Clinical Development.

Fragile X syndrome is caused by mutations in the FMR1 gene, which provides instructions for making FMRP, a protein involved in nerve cell communication. FMR1 mutations result in a deficiency in FMRP, leading to developmental, cognitive, and behavioral symptoms.

Gene therapy is a therapeutic approach to treat fragile X. A functional FMR1 gene is delivered to cells using a harmless, modified adeno-associated viral (AAV) vector to induce the expression (production) of FMRP protein.

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Previous attempts at gene therapy in fragile X mouse models using the full-length FMR1 gene were limited to a partial reversal of symptoms. However, studies suggest cells express other forms of the FMRP protein, including a highly abundant and stable form called FMRP isoform 17.

Researchers based at the University of Toronto in Canada, collaborating with scientists at the company Regenxbio in Maryland, evaluated the impact of AAV gene therapy to deliver genes that encode the rat form of FMRP protein isoform 17 in animals bred to lack the Fmr1 gene.

“Isoform 17 is an excellent candidate for a single isoform expressing therapeutic vector for [fragile X syndrome] treatment,” the team wrote.

Rats were treated by injection into the cerebrospinal fluid (CSF), the liquid that surrounds the brain and spinal cord, two or three days after birth. As a control, both healthy and fragile X rats were exposed to an empty AAV vector without the Fmr1 gene.

Initial experiments confirmed the expression of this FMRP protein in various regions of the brain, and its distribution across tissues was similar to normal healthy mice. Treatment did not activate an immune response against the AAV vector.

Brainwave activity was assessed using an electroencephalogram (EEG) during awake and sleep-like states. Of note, electrical activity in the brain can be measured as brainwaves (oscillations) with different frequencies. Brainwaves are produced by electrical pulses from nerve cells (neurons) communicating with each other. They are divided into different bandwidths, specifically infra-low, delta, theta, alpha, beta, and gamma, that change according to what an individual is doing and feeling.

An increase in the gamma brainwave by EEG has been shown to be a common abnormality in both Fmr1 mice and fragile X patients.

There were significant differences in gamma waves following treatment between control rats, normal and fragile X, and healthy controls and treated fragile X rats during awake and sleep-like states. However, in treated and untreated fragile X rats during sleep-like states, higher gamma waves were found with no significant differences, suggesting “treatment had no effect on the abnormal gamma oscillations found in the [Fmr1] rats,” the researchers wrote.

An assessment of another brainwave called delta, which is associated with deep sleep, showed gene therapy restored delta wave activity during the sleep-like state in fragile X rats,  and “suggested a restoration of the mechanism(s) that normally induces the increase in delta wave activity during sleep by [gene therapy] treatment and may suggest an improvement in sleep quality in these rats,” the researchers added.

A wood-chew test was conducted in male rats, as male fragile X rats engage in repetitive chewing behavior. Fragile X rats injected with gene therapy had a lower level of chewing than healthy rats injected with the empty AAV. There was an intermediate effect in fragile X rats also treated with empty AAV.

Additionally, wood-chewing behavior was higher in healthy rats treated with empty AAV compared to non-injected normal rats, suggesting that the empty AAV “may have an effect on wood-chewing repetitive behavior and could be a confounding factor in this test,” the researchers noted.

Both male and female fragile X rats were more socially submissive than sex- and age-matched healthy rats. To test the impact of treatment on this behavior, two unfamiliar rats were placed in the ends of tubes such that they were unable to turn around or pass each other. The match ended when one rat put both hind paws outside the tube, and the one that remained in the tube was deemed the winner.

Male and female fragile X rats treated with gene therapy had the same percentage of wins as healthy rats treated with empty AAV. In comparison, female fragile X rats treated with empty AAV had significantly fewer wins against their ordinary opponent, which was not seen in male rats, suggesting that “treatment reversed the submissive behavioral phenotype [characteristic] in female [Fmr1] rats,” the scientists wrote.

Fragile X rats have been shown to display reduced motor activity than healthy controls in the open field test, a measure of exploratory behavior and general activity. Gene therapy-treated fragile X male rats showed no differences compared to normal rats treated with empty AAV, indicating a partial rescue of symptoms. In contrast, treated female fragile X rats showed no change in behavior.

Finally, a fear conditioning test was conducted in which rats first were placed in a soundproof chamber and exposed to mild electrical shock to the feet along with a sound tone as conditioning. Compared to normal rats, female fragile X rats show increased freezing behavior in response to the conditioning sound tone one day later.

Regardless of treatment, fragile X rats froze more than normal rats within the first minute of sound exposure. Notably, female fragile X rats injected with gene therapy showed a reduced freezing rate by the third minute of sound tone.

“Our findings demonstrate that AAV-mediated expression of the rat [form] of a major human isoform of FMRP (isoform 17), rectified several behavioral and brain wave abnormalities in a rat model of [fragile X syndrome],” the authors wrote. “These results together with previous proof-of-concept studies of AAV-FMRP in the mouse model of [fragile X syndrome] strengthen the validity of the FMRP replacement strategy for treating [fragile X syndrome].”