Repeated hexanucleotide expansions in the C9ORF gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Scientists are increasingly turning to gene editing in their search for a cure for these diseases. Now researchers led by Christian Mueller, Sanofi, New Jersey, and Zane Zeier, University of Miami, report that CRISPR gene-editing machines carried by adeno-associated viruses can excise expansions and eliminate pathology in neurons cultured mice and in induced neurons. neurons of a person with ALS/FTD. As reported in an article published on bioRXiv on May 17, CRISPR also cut human C9ORF expansion in three mouse models.

  • In mice, CRISPR cut C9ORF expansions, reducing pathology.
  • Ditto for gene editing in patient-derived cells.

The treatment reduced the production of toxic poly-dipeptides and the formation of RNA inclusions. The scientists did not test whether the treatment increased lifespan. Yet this delivery system brings gene therapy for C9ORF expansion carriers one step closer to the clinic.

“CRISPR holds the promise of curing any genetic disease, especially toxic gain-of-function mutations such as those seen with C9ORF extensions,” Claire Clelland, University of California, San Francisco, told Alzforum. In a separate article uploaded to bioRXiv on May 21, Clelland and colleagues described a similar strategy, reducing excision that might lead to the best clinical outcome. They transfected the gene-editing machinery directly into patient-derived neurons rather than using AAV vectors.

In people with ALS/FTD, the C9ORF extensions contain hundreds and sometimes thousands of GGGGCC repeats inserted into the first intron. It wreaks havoc on cells in three ways: it stifles the production of normal C9ORF; it creates extended transcripts that aggregate with other RNAs or proteins; and, in an aberrant form of protein synthesis, ribosomes translate all six reading frames, generating chains of poly-dipeptides that also aggregate (February 2013 news; February 2013 news).

Researchers had previously restored normal C9ORF expression and reduced the amount of toxic RNA inclusions and poly-dipeptides by transfecting the CRISPR machinery into induced pluripotent stem cells from an ALS donor (Ababneh et al., 2020). More recently, scientists led by Yichang Jia of Tsinghua University in Beijing used guide RNAs provided by lentiviruses to excise the expansion in transgenic C9ORF mice that constitutively express Cas9, the molecular scissors that nick DNA to start the editing process. This approach ablated RNA aggregates (Piao et al., 2022). Could Cas9 do the same if delivered virally?

To find out, first author Katharina Meijboom, who worked with Mueller when she was in medical school at the University of Massachusetts at Worcester, created two adeno-associated viruses: one carrying the Cas9 gene and the two other CRISPR guide RNAs. The gRNAs target a segment that includes most of C9ORF intron 1, the hexanucleotide repeats, and part of intron 2. It also includes exon 1b, which would normally incorporate into the most abundant of the three alternatively spliced ​​isoforms. The researchers expected that the loss of this non-coding exon would not alter protein production

To test the vectors, Meijboom added them to the primary cortical neurons of two transgenic mouse models: C9-BAC500 mice, created in the laboratory of collaborator Robert Brown at UMass, express six to eight copies of exons 1 to 6 of the human C9ORF, each containing 500 hexanucleotide repeats; C9-500 mice harbor one copy of human C9ORF with 500 repeats (December 2015 news; May 2016 news). Meijboom tested both vectors in cultured neurons from C9-500 mice. She also crossed the C9-BAC500 animals with mice constitutively expressing Cas9 in neurons, harvested the neurons from the offspring, and then added the AAV gRNA.

In neurons of both models, CRISPR cut the expansions. Six days after the addition of AAVs, the amount of toxic RNA and poly-glycine-proline (poly-GP) inclusions decreased by half. C9ORF and normal C9ORF mRNA levels were unaffected. Clelland believes that without measuring the effectiveness of editing, it is difficult to interpret these results. “If you’re only editing 10% of the cells, you might not expect an overall change in C9ORF protein level, but the edited cells might have a huge protein difference,” she said.

Next, the scientists edited C9ORF in vivo. They injected gRNA-AAV into the striatum of 2-3 month old C9-BAC500/Cas9 mice and AAV containing gRNA and Cas9 into the brain of C9-500 and C9-BACexp mice of the same age. . The latter carry 16 to 20 copies of human C9ORF, each with 550 repeats. Two months after injection, the expansion had been excised from the brain cells in all three models.

Meijboom measured editing efficiency and pathology only in C9-500 mice. Hexanucleotide expansion was suppressed in approximately 60% of cells, RNA foci dropped by 66%, and poly-GP and poly-glycine-arginine were reduced by half (image below) . Because C9-500 mice appear normal until 4-5 months of age, around the time they were sacrificed in these experiments, scientists were unable to measure changes in behaviour.

Out of Focus. RNA inclusions (red) dot striated tissue (left) from C9-BAC500/Cas9 (top) and C9-500 (bottom) mice. CRISPR/Cas9 treatment virtually abolished these foci (right). [Courtesy of Meijboom et al., bioRXiv, 2022.]

Could this CRISPR strategy eliminate expansions and attenuate pathology in human cells? Zeier added Cas9 gRNA and AAVs to induced pluripotent stem cells derived from a person with ALS/FTD due to expansion in a C9ORF allele. The treated iPSCs produced almost no GP poly-dipeptides. Additionally, they made about 50% more C9ORF protein than untreated cells, suggesting that CRISPR treatment can at least partially reverse haploinsufficiency.

CRISPR/Cas9 AAVs also suppressed C9ORF extensions when scientists added the vectors to induced motor neurons and brain organoids derived from the iPSC lineage, hinting that gene editing is possible throughout the brain. Altogether, the results suggest that excision of C9ORF expansions corrects the three hallmark pathologies of C9ORF ALS/FTD and may be able to arrest or slow disease progression.

“Most other C9ORF approaches, such as antisense oligonucleotides, tackle RNA inclusions and poly-dipeptides, not haploinsufficiency,” Mueller said (see November 2015 conference news) .

As described in their paper, scientists led by Clelland and Bruce Conklin, also at UCSF, pitted three different CRISPR constructs against each other to find the best strategy for clinical application. Co-first authors Maria Sckaff, Kamaljot Gill, and Aradhana Sachdev introduced CRISPR/Cas9 into neurons harboring a C9ORF allele with a 200-hexanucleotide expansion. Guide RNAs targeted only the repeat expansion, the regulatory exon 1a which is upstream only, or a segment before exon 1a through exon 3, which included the expansion. The first two are smaller and the last larger than the segment targeted by Meijboom. Sckaff, Gill and Sachdev measured C9ORF mRNA and poly-dipeptide levels.

All three constructs excised the expected DNA sections while leaving the normal C9ORF intact. Deletion of expansion or exons 1a-3 also eliminated poly-GP and poly-glycine-alanine (poly-GA) dipeptides, but deletion of exon 1a only eliminated poly-GA, which is translated from the sense strand. This suggests that silencing sense strand transcription did not prevent poly-GP transcription and translation from the antisense strand.

The authors believe that the gRNA pair that deleted exons 1a-3 is best for clinical application because it decreased poly-dipeptide pathology and had higher editing efficiency than gRNAs targeting only the region repeated.—Chelsea Weidman Burke

News Quotes

  1. RNA twist: C9ORF72 intron expansion makes protein aggregation

  2. A second study sees the intron in the translated FTLD gene

  3. C9ORF72 mice A-OK despite toxic RNAs, peptides

  4. New C9ORF72 mice develop ALS/FTD-like symptoms

  5. Listen, Gene Silencing strikes a chord at the RNA meeting

Research Patterns Quotes

  1. C9-BAC500 (brown)

  2. C9-BACexp (Baloh/Lutz)

paper quotes

  1. .
    Correction of amyotrophic lateral sclerosis-related phenotypes in motor neurons derived from induced pluripotent stem cells carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair.
    Hum Mol Genet. 2020 Aug 3;29(13):2200-2217.
  2. .
    Dual gRNA approach with limited off-target effect fixes repeat expansion of C9ORF72 in vivo.
    Scientific representative. 2022 Apr 5;12(1):5672.


  1. .
    Repeated expansion of C9ORF72 renders motor neurons vulnerable to excitotoxicity mediated by Ca2+-permeable AMPA receptors.
    Nat Common. 2018 Jan 24;9(1):347.