A new gene-editing method tags fragile X mutation for repair

Minimization of mutations: Promoting the formation of specialized loops in the genome may help remove the genetic repeats associated with fragile X syndrome.
THOM LEACH / Scientific Source

Hun-Goo Lee didn’t set out to discover a new way to potentially treat fragile X syndrome. He just wanted to solve a mystery: why do some cells with a fragile X mutation remain unaffected?

Mutation of more than 200 copies of the CGG trinucleotide string in the FMR1 gene typically silences gene expression and prevents the production of the FMRP protein. Under some laboratory conditions, however, embryonic cells carrying these long CGG repeats still produce FMRP.

Production of FMRP occurs because culture conditions allow the cells’ DNA repair machinery to locate the CGG repeats and remove them, according to a new study by Lee and his colleagues. Inspired by this mechanism, the team developed a new gene-editing tool that allows other types of fragile X cells to remove the trinucleotide repeats as well.

This is exciting work, says Antonio Bedalov, a professor in the division of translational and therapeutic sciences at the Fred Hutchinson Cancer Center in Seattle, Washington, who was not involved in the study. This approach could, in theory, treat fragile X syndrome and other conditions with similar genetic repeats, he says. You have indeed attacked the root cause of the problem.

ANDEmbryonic fragile X cells can appear unaffected and express FMR1 when grown in a specific medium, and two drugs in that medium were responsible, Lees’ team found.

In fragile X syndrome, the CGG repeats in FMR1 accumulate epigenetic tags called methyl groups, which silence the gene. But these two drugs induce demethylating enzymes, which remove methyl tags and restore FMR1 expression in neural progenitor and stem cells derived from people with fragile X, they found. The treatment resulted in the excision of the CGG repeats. The contraction started after three to six days of treatment; within 12 days, it was complete.

The researchers achieved similar results when they used an existing version of CRISPR to target a methyl-clearing enzyme at the FMR1 gene.

But providing an inactive version of the enzyme as a control also accomplished the same thing, suggesting that demethylation wasn’t the sole driver of CGG shrinkage. That was most astounding, says lead investigator Jeannie Lee, professor of genetics at Harvard Medical School and interim chair of the department of molecular biology at Massachusetts General Hospital. Hun-Goo Lee is a research scientist in Jeannie Lee’s laboratory at Harvard.

The researchers then realized that the CRISPR-based approach and the demethylation process can both cause DNA to form a structure called an R-loop. These loops occur when double-stranded DNA is broken by a single-stranded RNA segment that looks like a piece of tissue that gets caught on a Velcro strip and creates a bump where the adhesive strips can no longer connect.

This genomic bump stabilized by the large number of CGG repeats in fragile X cells activates a cellular DNA repair mechanism, which removes the repeats.

The team then designed a catalytically inactive version of CRISPR that specifically prompts the CGG repeats to form R-loops. The tool could eventually be packaged in viruses and delivered to the brain, the researchers say. The findings were published in the journal in May Cell.

I like the idea of ​​using endogenous repair pathways because they come with millions of years of evolution to make things happen with the highest fidelity, says Jeannie Lee.

AWhile the work is preliminary, it does give a ray of hope in the fragile X-ray field, says Randi Hagerman, distinguished professor of pediatrics and medical director of the MIND Institute at the University of California, Davis, who was not involved in the study. It means that you could eliminate the methylation and therefore reduce the CGG repeats without having to use the gene therapy intervention.

But it’s not clear whether that treatment would have impacted people, Hagerman adds. For one thing, the method removes enough CGG repeats to put a cell below the 200-copy mark that silences FMR1, but not necessarily enough to put it in the typical range of less than 55. People with 55-200 repeats have a fragile X premutation one that may not result in intellectual disability but can cause other symptoms early in life, including fragile X-associated neuropsychiatric disorder, anxiety, and depression. These concerns about premutation involvement need to be addressed before doing a treatment that turns a full mutation into a premutation, says Hagerman.

The approach isn’t without risk, agrees Bedalov. However, he says, there’s likely an advantage to using the catalytically inactive version of CRISPR, which won’t produce genome-wide cleavages. And the tool is smaller than typical for CRISPR-based approaches, making it easier to deliver to the brain, he adds.

Before drawing any conclusions, the results need to be tested in non-dividing cells, says Bedalov. DNA repair is active during cell division, he notes, and so would easily occur in the stem cells and neural progenitor cells used in the study. Since neurons don’t divide, it will be important to demonstrate that R-loops can trigger repeated excision in non-dividing cells, he says.

The researchers plan to test it later. Eventually, says Jeannie Lee, the work could lead to a way to remove excess trinucleotides in people, which could help treat fragile X syndrome and other conditions that result from this form of mutation.

We started here, she says, pointing to the ground, referring to the initial puzzle she and Hun-Goo Lee set out to solve. And somehow we ended up in space.

Cite this article: https://doi.org/10.53053/YPXL8063