Experimental Gene Therapy Successfully Silences Key ALS Gene C9orf72, Preclinical Studies Show

A gene therapy candidate targeting a key amyotrophic lateral sclerosis (ALS) mutation in the C9orf72 gene is able to lower the accumulation of toxic RNA clumps and reduce the activity of this mutated gene, in cells collected from a patient with frontotemporal dementia (FTD) and in a mouse model of ALS, according to two preclinical studies.

uniQure’s miQURE is designed to silence disease-causing genes while not causing off-target damage. It is intended to induce silencing of the entire target organ by using tiny, cell-derived vesicles called exosomes. The gene therapy candidates using miQURE incorporate micro-RNAs (miRNAs) — RNA molecules that regulate gene expression — that can be delivered with a type of harmless viral vector called an adeno-associated virus (AAV) to provide sustained activity.

The most common genetic cause of familial and sporadic ALS and FTD — found in a substantial number of ALS patients and caused by progressive degeneration of the brain’s frontal and temporal lobes — is an expanded GGGGCC repeat, a sequence of four guanine (G) nucleotides followed by two cytosine (C) nucleotides in the first intron of C9orf72. While nucleotides are the building blocks of DNA, introns are DNA bits normally spliced out in protein production.

In humans, loss-of-function mutations in C9orf72 — leading to reduced or complete lack of protein function — have not been associated with disease, suggesting that a reduction in this gene’s expression (protein production) is likely tolerable.

Most studies linking C9orf72 to disease showed an accumulation of so-called RNA foci in the cell nucleus and deposition of specific proteins in the cytoplasm (within the cell but excluding the nucleus). RNA foci are toxic clumps of mutated C9orf72 RNA that sequester RNA-binding proteins, leading to cellular dysfunction and death.

The two studies were conducted by scientists at uniQure and used the company’s miQURE technology, a next-generation gene-silencing platform. Both were published in the journal Molecular Therapy – Nucleic Acids.

In the first, “Artificial microRNAs targeting C9orf72 can reduce accumulation of the intra-nuclear transcripts in ALS and FTD patients,” the researchers showed that miQURE’s miRNAs lead to lower levels of the C9orf72 messenger RNA — generated from DNA to produce the protein — in both the cell nucleus and the cytoplasm.

Findings also revealed an approximately 50% reduction in RNA foci in cells expressing the mutant form of C9orf72.

Then, two different miRNA candidates — miC-101 and miC-451 — were incorporated into the AAV5 subtype of the viral vector. This enabled silencing of C9orf72 in vitro, including in neurons derived from induced pluripotent stem cells (iPSCs) from a patient with FTD. iPSCs are developed by reprogramming cells so that they revert to an embryonic-like state to grow into all cell types.

The second study, “Targeting RNA-mediated toxicity in C9orf72 ALS/FTD by RNAi based gene therapy,” further showed that AAV5 carrying miRNAs that target C9orf72 are able to reduce the accumulation of C9orf72 RNA, as well as induce silencing of the target gene in both the nucleus and cytoplasm.

The previously shown reductions in C9orf72 RNA and RNA foci were then validated in a mouse model of ALS that carries the human form of the mutant gene. Reduction in C9orf72 RNA were found in the brain’s striatum, a key brain area in motor control, upon delivery of the potential gene therapy specifically into this region.

“Our data provide evidence on the efficacy of artificial miRNAs against C9orf72 as a promising AAV-based gene therapy for ALS and FTD,” the scientists wrote.

Sander van Deventer, MD, PhD, uniQure’s chief scientific officer, said in a press release that the ability to silence C9orf72 in the nucleus “may prove to be critical for therapeutic efficacy of gene therapies for these diseases.”

These results support the continuation of uniQure’s gene therapy program in ALS and FTD, according to van Deventer, who also said, “We are very pleased to have these data published in a highly relevant journal for the field and look forward to further exploring this opportunity.”

miQURE has also been incorporated into AMT-130, a treatment candidate for Huntington’s disease, and is expected to be used in AMT-150 for spinocerebellar ataxia type 3.