Gene Therapy Appears to Improve Behavioral Symptoms in Fragile X Mice

Lindsey Shapiro, PhD avatar

by Lindsey Shapiro, PhD |

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An oversized hand holds a mouse next to a trio of vials of blood in this laboratory illustration.

A gene therapy using a modified form of an enzyme called diacylglycerol kinase kappa (DGKk) led to the long-term rescue of behavioral symptoms in a mouse model of fragile X syndrome, according to a recent study.

Lysogene, which was involved in the studies, is currently developing the potential treatment in collaboration with Hervé Moine, PhD, a researcher at the University of Strasbourg in France.

“These preclinical results confirm the validity of our innovative approach targeting DGKk for the treatment of [fragile X], a [central nervous system] pathology with high unmet medical need,” Ralph Laufer, PhD, chief scientific officer of Lysogene, said in a press release. “We are looking forward to expanding these results in further preclinical studies.”

The study, “AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model,” was published in EMBO Molecular Medicine.

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Gene Therapy Shows Promise in Fragile X Rat Model

Fragile X is caused by mutations in the FMR1 gene which leads to impaired production of FMRP, an important regulator of other proteins involved in nerve cell function. A lack of functional FMRP in fragile X leads to the behavioral and cognitive symptoms characteristic of the disease.

Dysfunction of a number of FMRP’s products could be involved in the mechanisms underlying fragile X. Previously, Moine and his colleagues found that mice with fragile X had lower brain levels of one of these proteins, DGKk, which appeared to be associated with brain and behavior changes in the mice.

While the results suggest that DGKk could be a promising fragile X treatment, its function depends on FMRP signaling, thus limiting its potential in patients who do not have enough functional FMRP.

The team now investigated whether modifying DGKk to enable its independence from FMRP might be a promising therapeutic approach in fragile X.

In human brain tissue from fragile X patients, the researchers found that, much like in the mouse model, DGKk levels were reduced compared to tissue from people without the disease.

In cell cultures, the reduction of FMRP also led to a significant reduction of both human and mouse DGKk protein, confirming the notion that levels of DGKk are regulated by FMRP.

What is the DGKk enzyme?

DGKk is one in a family of enzymes that perform a similar function (the DGK family). DGKk converts diacylglycerol to phosphatidic acid, two lipids used by cells for key molecular processes. The enzyme basically serves as a switch that terminates the signaling of one lipid while simultaneously activating signaling by another.

The researchers found that only DGKk, and not other members of the family, seemed to interact strongly with FMRP. They suspected that one particular region of DGKk, which is lacking in the other DGK forms, may be responsible for its relationship with FMRP.

Indeed, when the team disrupted that part of the protein, DGKk levels were no longer dependent on FMRP activity. Importantly, removing this part of DGKk did not influence its normal activity in the brain.

This modified version of DGKk, called delta-N-DGKk, “could represent a potential therapeutic candidate for [fragile X] gene therapy by bypassing the need for FMRP,” the researchers wrote.

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How does delta-N-DGKk work in a fragile X mouse model?

Normally, FMRP acts to block the production of many proteins. Without it, excessive protein synthesis is often observed, which is thought to be a major contributor to brain dysfunction in fragile X.

In cell cultures, delta-N-DGKk showed an ability to lower the rate of protein synthesis. Additional experiments showed that it likely does this by restoring a more normal conversion of the diacylglycerol molecule to phosphatidic acid, a process which is known to be disrupted in fragile X.

To assess the effects of delta-N-DGKk in a mouse model, the researchers packaged it into an adeno-associated virus (AAV), which helped deliver it into the nerve cells of live animals.

When delivered directly into the brains of mice lacking FMRP — a fragile X model — this gene therapy was able to be taken up by the nerve cells, and showed signs that it lowered protein production and normalized phosphatidic acid levels.

One month after the treatment was delivered, the team performed a number of behavioral tests to determine whether delta-N-DGKk could improve fragile X behavioral symptoms.

Overall, these tests showed signs that the treatment restored more normal behaviors in the mice lacking FMRP. Specifically, treatment led to improvements in hyperactivity and social abnormalities that were observed in untreated animals. While some other behavioral changes, including repetitive and anxiety-like behaviors, tended to be counteracted with delta-N-DGKk, high variability prevented a firm conclusion to be drawn, the researchers said.

These behavioral effects were largely the same when tested again at eight weeks after the treatment. The fragile X mice also showed weight gain, which was reduced with delta-N-DGKk treatment.

“Our data suggest that DGKk is an important factor in [fragile X] pathogenesis and provide preclinical proof of concept that its replacement could be a viable therapeutic strategy in [fragile X],” the researchers wrote.