Gene editing stops Huntington’s disease mutation from expanding
Study finds technique effective in patient-derived cells, mouse model of disease
Introducing small interruptions into the abnormally long CAG repeat expansion that causes Huntington’s disease can stabilize or even reduce the length of that DNA stretch, which would be expected to slow disease progression.
These are the findings of a study that tested base editing, a technique that changes a single DNA building block at a time without cutting the DNA itself, in Huntington’s patient-derived cells and a mouse model of the disease.
Researchers observed some off-target changes — a known bottleneck of base editing — but most occurred in regions of the DNA that do not contain genetic instructions for producing proteins, reducing the risk of unwanted side effects in patients.
“A lot more studies would be needed before we can know if disrupting these repeats with a base editor could be a viable therapeutic strategy to treat patients,” David Liu, PhD, the study’s senior author at the Broad Institute in Massachusetts, said in an institute press release. “But being able to illuminate the biological consequences of interrupted repeats is a really useful and important milestone.”
The study, “Base editing of trinucleotide repeats that cause Huntington’s disease and Friedreich’s ataxia reduces somatic repeat expansions in patient cells and in mice,” was published in Nature Genetics.
Natural changes mimicked in lab
Huntington’s is caused by mutations in HTT, which contains the genetic instructions for producing huntingtin, a protein needed for the normal function of nerve cells. The HTT gene includes a segment in which a trio of DNA building blocks — a cytosine (C), an adenine (A), and a guanine (G) — typically appears 10 to 35 times in a row.
In Huntington’s, the CAG repeat is expanded to 36 or more copies, resulting in a longer-than-normal version of the huntingtin protein that is toxic to nerve cells. This can lead to a range of symptoms, and the longer the CAG stretch, the earlier and more severe the symptoms usually are.
CAG repeat expansions are not stable, and they can grow longer in the course of a person’s lifetime. These somatic expansions are believed to contribute to disease onset and progression.
In some patients, single-letter changes — known as interruptions — occur naturally within the repeat sequence, and are linked to reduced risk of somatic expansion and mutation transmission to offspring, delayed disease onset and progression, and milder symptoms.
“These interruptions are proposed to stabilize repeats and suppress somatic repeat expansion,” wrote the researchers, who set out to mimic these changes in the lab using base editing.
This technique was used to change some of the CAG repeats to CAA, a trio of DNA building blocks that codes for the same protein’s building block as CAG. While this change does not affect the resulting huntingtin protein, it can make the DNA more stable and less likely to undergo somatic expansion.
Interrupting DNA sequences has stabilizing effect, researchers say
The researchers tested different versions of a base editor, guided by an RNA molecule designed to direct it to its target, to measure the number of gene copies where at least one CAG had changed to CAA in lab-grown human cells. On average, 44% to 62% of gene copies were edited with their top six base-editing strategies.
To reduce the risk of unintended changes, the researchers further tweaked one of these strategies, making it the best-performing editing tool. It was subsequently called CAG-CBE.
In lab-grown cells from people with Huntington’s — containing one mutated HTT gene copy with 48 to 180 CAG repeats — CAG-CBE successfully interrupted CAG repeats in the mutated gene of 66% to 82% of cells.
Unlike in untreated cells, where the number of CAG repeats continued to expand, in edited cells, the length of the CAG repeat region remained stable or even became shorter.
“These findings demonstrate that inducing interruptions in [disease-causing] CAG repeats by base editing can prevent somatic repeat expansion and promote contraction of the [disease-causing] repeats,” the researchers wrote.
CAG-CBE caused off-target edits, meaning it resulted in CAG repeat interruptions in other DNA sequences, but most of these changes occurred in regions that do not code for proteins. However, more testing is needed to understand the safety of CAG-CBE.
Not only does this study show for the first time that inducing interruptions has a profound stabilizing effect on repeats, but that the base-editing approach we’ve used can also be applied to study any of over a dozen repeat [expansion] disorders.
The team then used a modified, harmless adenovirus type 9 to deliver CAG-CBE directly to brain cells of a mouse model of Huntington’s. After nearly six months, CAA interruptions were detected in more than 70% of HTT copies in brain cells, which showed also a significant reduction in CAG repeat length.
Similar findings were observed when using a similar, but distinct, base-editing approach in a mouse model of Friedreich’s ataxia, a neurological condition caused by excessive GAA repeats in a gene called FXN.
“Base editors introduced … interruptions at CAG and GAA repeats, mimicking stable, [non-disease-causing gene copies] that naturally occur in people,” the researchers wrote.
“Not only does this study show for the first time that inducing interruptions has a profound stabilizing effect on repeats, but that the base-editing approach we’ve used can also be applied to study any of over a dozen repeat [expansion] disorders,” Mandana Arbab, PhD, one of the study’s co-first authors, said. Arbab is now an assistant professor at Boston Children’s Hospital.
The researchers are also working on a new approach that could replace disease-causing excessive repeat tracts with a shorter, stable number of repeats all at once.
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