Gene Therapy Lowers Toxic RNA Due to C9ORF72 Mutations in ALS

Margarida Maia, PhD avatar

by Margarida Maia, PhD |

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C9ORF72 mutation | ALS News Today | research into gene therapy for mutation-driven ALS | image of cells in lab dish

One-time delivery of a CRISPR-based gene therapy significantly reduced the buildup of toxic RNA molecules resulting from C9ORF72 mutations in cells and mouse models of amyotrophic lateral sclerosis (ALS), the therapy’s developer, Locanabio, reported.

The approach was designed using the company’s CORRECTx platform, which uses CRISPR technology to modify RNA molecules in multiple ways, including editing, destroying, or replacing harmful pieces of RNA that often contribute to disease. RNA, the intermediate molecule that is first produced when a gene is “read,” is used as a template for protein production.

Notably, treatment did not affect levels of healthy C9ORF72 RNA, the company reported, meaning that cells could still produce the C9orf72 protein important for the function of motor neurons — the nerve cells that control voluntary movement and are lost in ALS patients.

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“These new data demonstrate the potential advantages of our CORRECTx technology, which can be designed to selectively target [disease-causing] RNA transcripts in a single construct while sparing expression of the wild-type [healthy] C9ORF72 allele which may be important for cellular function,” John Leonard, PhD, chief scientific officer of Locanabio, said in a press release.

Findings were in the presentation “AAV9-mediated delivery of RNA targeting systems eliminate hexanucleotide repeat expansions in C9ORF72 ALS/FTD models,” given at the 2022 American Society of Gene and Cell Therapy (ASGCT) Annual Meeting in Washington, D.C.

Mutations in the C9ORF72 gene are the most common cause of ALS, found in about 40%–50% of cases of familial disease and about 5%–10% of sporadic ALS cases. These mutations are also frequent in frontotemporal dementia (FTD), a related condition.

The most common C9ORF72 mutation is a hexanucleotide repeat, in which six nucleotides — specifically those of G and C, two “letters” of the genetic code — are repeated an excessive number of times. This causes two types of RNA molecules, called G4C2 (for GGGGCC) and C4G2 (for CCCCGG), to accumulate in the nucleus and serve as templates for small proteins that form clumps and are damaging to cells.

Locanabio developed a CRISPR/Cas13d-based system to target these toxic RNA molecules for destruction. The approach consists of two pieces of “guide RNA” that recognize the toxic RNA sequences, G4C2 or C4G2, and guide the Cas13d enzyme to these molecules to cut them into pieces. The RNA molecules and the enzyme are delivered to cells via a harmless virus called an adeno-associated virus 9 (AAV9).

To understand whether this gene therapy could be used to lower the levels of the repeat expansions, the researchers wrapped an AAV9 package containing a guide molecule that led the way to G4C2. The package was delivered to neurons, or nerve cells, in mice whose C9ORF72 gene contained 500 hexanucleotide repeats. These mice show the hallmarks of ALS and FTD, and can serve as a model for the diseases, the team noted.

In an initial experiment, the researchers found that the CRISPR-based therapy reduced levels of the hexanucleotide repeat expansions by almost half in the lab-grown neurons about 10 weeks after injection.

They then delivered the package into mice via an injection beneath the pia mater, which is the membrane enveloping the brain and the spinal cord. A one-time delivery resulted in “no overt safety concerns” at 10 weeks after delivery, the scientists reported.

This CRISPR-based gene therapy also reduced the levels of the hexanucleotide repeat expansions in the spine’s lumbar region, without affecting RNA levels of the C9ORF72 gene.

A second package, which the researchers called a dual array, contained two guide molecules leading the way to both G4C2 and C4G2. When delivered into lab-grown cells, it lowered the levels of both G4C2 and C4G2.

“We show a novel approach that can effectively target both sense and antisense [hexanucleotide repeat expansions] in c9ALS/FTD with a single product,” the researchers wrote in the study’s abstract.

“These studies support further development of our CRISPR-Cas13d multi-guide RNA-targeting system as a potential therapeutic approach for ALS and other neurologic disorders,” Leonard added.

As the packaged therapy can be designed to target specific RNA sequences, the approach has the potential to be used across many genetic diseases, the company noted.

“We are pleased with the progress of our ALS program and are very encouraged by these data presented today, which supports our plans to leverage the multi-targeting and mutation specific capabilities of the CORRECTx platform to expand our pipeline and treat other serious diseases with limited or no treatment options,” said James Burns, PhD, CEO of Locanabio.