CRISPR Prevents Pathology in Cell and Mouse Models of ALS/FTD
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Hexanucleotide repeat expansions within the gene for C9ORF 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 treatment for these diseases. Now, researchers led by Christian Mueller, Sanofi, New Jersey, and Zane Zeier, University of Miami, report that CRISPR gene-editing machinery ferried in by adeno-associated viruses can excise the expansions and eliminate pathology in cultured neurons from mice and in induced neurons from a person who had ALS/FTD. As reported in a paper posted to bioRxiv on May 17, CRISPR also clipped the expansion from human C9ORF in three mouse models.
- In mice, CRISPR clipped C9ORF expansions, reduced 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 if the treatment increased lifespan. Still, this delivery system brings gene therapy for C9ORF expansion carriers one step closer to the clinic.
“CRISPR holds the promise to cure any genetic disease, especially toxic gain-of-function mutations such as those seen with C9ORF expansions,” Claire Clelland, University of California, San Francisco, told Alzforum. In a separate paper uploaded to bioRxiv on May 21, Clelland and colleagues described a similar strategy, narrowing down the 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, C9ORF expansions contain hundreds and sometimes thousands of GGGGCC repeats stuffed into the first intron. This wreaks havoc in cells three ways: it squelches production of normal C9ORF; it creates expanded transcripts that aggregate with other RNAs or proteins; and, in an aberrant form of protein synthesis, ribosomes translate all six reading frames, generating strings of poly-dipeptides that also aggregate (Feb 2013 news; Feb 2013 news).
Researchers had previously restored normal C9ORF expression and reduced the amount of toxic RNA inclusions and poly-dipeptides by transfecting CRISPR machinery into induced pluripotent stem cells from an ALS donor (Ababneh et al., 2020). More recently, scientists led by Yichang Jia, Tsinghua University, Beijing, used lentivirus-delivered guide RNAs 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 the RNA aggregates (Piao et al., 2022). Could Cas9 do the same if virally delivered?
To find out, first author Katharina Meijboom, who worked with Mueller when she was at the University of Massachusetts Medical School, Worcester, created two adeno-associated viruses: one carrying the Cas9 gene and the other two CRISPR guide RNAs. The gRNAs target a segment that includes most of C9ORF intron 1, the hexanucleotide repeats, and some of intron 2. It also includes exon 1b, which normally would incorporate into the most abundant of three alternatively spliced isoforms. The researchers expected that loss of this non-coding exon would not change protein production
To test the vectors, Meijboom added them to primary cortical neurons from two transgenic mouse models: C9-BAC500 mice, created in the lab of collaborator Robert Brown at UMass, express six to eight copies of exons 1-6 of human C9ORF, each containing 500 hexanucleotide repeats; C9-500 mice harbor one copy of human C9ORF with 500 repeats (Dec 2015 news; May 2016 news). Meijboom tested both vectors in neurons cultured from the C9-500 mice. She also crossed the C9-BAC500 animals with mice constitutively expressing Cas9 in neurons, harvested neurons from the offspring, then added the gRNA AAV.
In neurons from both models, CRISPR snipped the expansions. Six days after adding the AAVs, the amount of toxic RNA inclusions and poly-glycine-proline (poly-GP) fell by half. Levels of C9ORF mRNA and normal C9ORF were unaffected. Clelland thinks that without measuring editing efficiency, it is hard to interpret these findings. “If you edited only 10 percent of cells, you might not expect to have any change in C9ORF protein level overall, but the edited cells could have a huge difference in protein,” she said.
Next, the scientists edited C9ORF in vivo. They injected the gRNA-AAV into the striatum of 2- to 3-month-old C9-BAC500/Cas9 mice and both gRNA and Cas9-containing AAVs into the brains of C9-500 and C9-BACexp mice of the same age. The latter carry 16-20 copies of human C9ORF, each with 550 repeats. Two months after injection, the expansion had been excised from brain cells in all three models.
Meijboom measured editing efficiency and pathology only in C9-500 mice. The hexanucleotide expansion was removed in about 60 percent of cells, RNA foci had fallen by 66 percent, and poly-GP and poly-glycine-arginine were halved (image below). Since C9-500 mice appear normal until they are 4 to 5 months old, around the time they were sacrificed in these experiments, the scientists were unable to measure behavioral changes.
Out of Foci. RNA inclusions (red) dot striatal tissue (left) from C9-BAC500/Cas9 (top) and C9-500 (bottom) mice. CRISPR/Cas9 treatment all but abolished these foci (right). [Courtesy of Meijboom et al., bioRXiv, 2022.]
Could this CRISPR strategy remove expansions and attenuate pathology in human cells? Zeier added the gRNA and Cas9 AAVs to induced pluripotent stem cells derived from a person who had ALS/FTD due to an expansion in one C9ORF allele. The treated iPSCs produced almost no GP poly-dipeptides. What’s more, they made about 50 percent more C9ORF protein than did untreated cells, suggesting that CRISPR treatment may at least partially reverse haploinsufficiency.
The CRISPR/Cas9 AAVs also clipped out C9ORF expansions when the scientists added the vectors to induced motor neurons and brain organoids derived from the iPSC line, hinting that the gene editing is possible in the whole brain. All told, the findings suggest that excising C9ORF expansions corrects the three hallmark pathologies of C9ORF ALS/FTD and may be able to stop or slow disease progression.
“Most other C9ORF approaches, such as antisense oligonucleotides, go after RNA inclusions and poly-dipeptides, not haploinsufficiency,” Mueller said (see Nov 2015 conference news).
As described in their paper, scientists led by Clelland and Bruce Conklin, also at UCSF, pitted three different CRISPR constructs against one another 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 the repeat expansion only, the regulatory exon 1a that lies upstream only, or a segment from before exon 1a through exon 3, which included the expansion. The first two are smaller, and the last bigger than the segment Meijboom targeted. Sckaff, Gill, and Sachdev measured C9ORF mRNA and poly-dipeptide levels.
All three constructs excised the expected sections of DNA yet left normal C9ORF untouched. Removing the expansion or exons 1a-3 also cleared poly-GP and poly-glycine-alanine (poly-GA) dipeptides, but removing just exon 1a only eliminated poly-GA, which is translated from the sense strand. This suggests that silencing transcription of the sense strand did not prevent transcription and translation of poly-GP from the antisense strand
The authors think that the gRNA pair that removed exons 1a-3 is the best for clinical application because it decreased poly-dipeptide pathology and had higher editing efficiency than the gRNAs targeting just the repeat region.—Chelsea Weidman Burke
References
News Citations
- RNA Twist: C9ORF72 Intron Expansion Makes Aggregating Protein
- Second Study Sees Intron in FTLD Gene Translated
- C9ORF72 Mice A-OK Despite Toxic RNAs, Peptides
- New C9ORF72 Mice Develop Symptoms Resembling ALS/FTD
- Listen Up, Gene Silencing Strikes a Chord at RNA Meeting
Research Models Citations
Paper Citations
- Ababneh NA, Scaber J, Flynn R, Douglas A, Barbagallo P, Candalija A, Turner MR, Sims D, Dafinca R, Cowley SA, Talbot K. Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons 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. PubMed.
- Piao X, Meng D, Zhang X, Song Q, Lv H, Jia Y. Dual-gRNA approach with limited off-target effect corrects C9ORF72 repeat expansion in vivo. Sci Rep. 2022 Apr 5;12(1):5672. PubMed.
Further Reading
Papers
- Selvaraj BT, Livesey MR, Zhao C, Gregory JM, James OT, Cleary EM, Chouhan AK, Gane AB, Perkins EM, Dando O, Lillico SG, Lee YB, Nishimura AL, Poreci U, Thankamony S, Pray M, Vasistha NA, Magnani D, Borooah S, Burr K, Story D, McCampbell A, Shaw CE, Kind PC, Aitman TJ, Whitelaw CB, Wilmut I, Smith C, Miles GB, Hardingham GE, Wyllie DJ, Chandran S. C9ORF72 repeat expansion causes vulnerability of motor neurons to Ca2+ -permeable AMPA receptor-mediated excitotoxicity. Nat Commun. 2018 Jan 24;9(1):347. PubMed.
Primary Papers
- Meijboom KE, Abdallah A, Fordham NP, Nagase H, Rodriguez T, Kraus C, Gendron TF, Krishnan G, Esanov R, Andrade NS, Rybin MJ, Ramic M, Stephens ZD, Edraki A, Blackwood MT, Kahriman A, Henninger N, Kocher JPA, Benatar M, Brodsky MH, Petrucelli L, Gao FB, Sontheimer EJ, Brown RH, Zeier Z, Mueller C. CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. bioRxiv. May 17, 2022 bioRxiv
- Sckaff M, Gill K, Sachdev A, Birk AM, Aladesuyi Arogundade O, Watry HL, Keough KC, Tsai YC, Ziegle J, Conklin BR, Clelland CD. Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by a C9orf72 repeat expansion mutation in patient derived cells. bioRxiv. May 21, 2022 bioRxiv
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Comments
University of Edinburgh
This paper comprehensively determines, using a variety of experimental models including in-vivo studies, that deleting hexanucleotide repeat expansion mutations in the C9ORF72 locus reduces pathological hallmarks of C9ORF72 ALS/FTD. This opens up an exciting opportunity for CRISPR/Cas9-mediated gene therapy for C9ORF72 ALS/FTD. More work is needed to demonstrate if this approach will slow or stop ALS/FTD disease progression, which is the acid test.
Tsinghua University
Given that C9ORF72 GGGGCC repeat expansion is the most common genetic cause for both ALS and FTD, development of therapeutic approaches to treat the repeat expansion-mediated pathologies in vivo is urgently needed.
The expansion acts at DNA, RNA, and protein levels to contribute to the disease pathogenesis. At the DNA level, the repeat expansion forms abnormal nucleotide structures, which causes haploinsufficiency of C9ORF72 (Belzil et al., 2016; Xi et al., 2015; Xi et al., 2013; Zhang et al., 2019). At the RNA level, the expanded GGGGCC repeats are bidirectionally transcribed into repeat RNAs, which form sense and antisense RNA foci to sequester RNA binding proteins and disturb their normal functions (Gitler and Tsuiji, 2016; Balendra and Isaacs, 2018; DeJesus-Hernandez et al., 2011; Cooper-Knock et al., 2014; Lee et al., 2013; Sareen et al., 2013; Donnelly et al., 2013; Conlon et al., 2016; Mori et al., 2013). At the protein level, toxic dipeptide repeat (DPR) proteins are generated from repeat-associated non-ATG (RAN) translation (Gitler and Tsuiji, 2016; Balendra and Isaacs, 2018; Ash et al., 2013; Mori et al., 2013; Mori et al., 2013; Gendron et al., 2013; Zu et al., 2013; Freibaum et al., 2015; Zhang et al., 2015; Lee et al., 2016; Zhang et al., 2018; Zhang et al., 2016).
The location of C9ORF72 repeat expansion is in the intron, the sequence nonencoding protein, which makes it suitable for DNA fragment removal by CRISPR/Cas9 in a "cutting-deletion-fusion" manner without affecting C9ORF72 protein coding.
In April 2022, my group at Tsinghua University designed a dual-gRNA approach with limited off-target effect and achieved high removal rate in a mouse modeling expressing 100-1000 repeat expansion (Piao et al., 2022).
The manuscript posted in bioRxiv (Meijboom et al., 2022) on May 17 employed a similar approach to what we had described to remove the repeat DNA expansion in vitro and in vivo, especially in patient-derived iPS cells and organoids. These authors further demonstrated that the removal of repeat expansion by dual gRNAs can even recover the haploinsufficiency of C9ORF72 in the patient-derived cells.
Current in vivo therapeutic approaches target C9ORF72 transcripts, including ASO-mediated (Akabas et al., 1992) and microRNA-mediated target RNA silencing (Akabas et al., 1992), however, both sense and antisense repeat RNAs can generate toxic DPR proteins. Therefore, these in vivo approaches have not achieved both sense and antisense RNA silencing with a single shot. The CRISPR/Cas9-based DNA editing approaches published by my group and now posted in bioRxiv will provide a one-time treatment solution to correct expansion-mediated toxicities at DNA, RNA, and protein levels at a time.
Although the approach is promising, we still have obstacles to overcome. The first one is the off-target effect of CRISPR/Cas9 system. So far, we are unable to 100 percent rule out off-target effects of any given gRNA, but can limit it to a certain level. However, scientists have designed means to estimate the off-target effect in silico and to examine it by experiments. To avoid unwanted cutting that may damage other gene functions, we took the in silico predictor and in vivo detector and lowered our gRNA off-target effects to an undetectable level (Piao et al., 2022).
The second obstacle is the long-lasting expression of CRISPR/Cas9 in the AAV-infected neurons. Because neurons in the brain are non-dividing cells and AAV-mediated gene expression in neuron is long-lasting, it may increase a p53-mediated type of DNA damage (Haapaniemi et al., 2018), which, in turn, can increase the chance of neurodegeneration. Therefore, we urgently need a system for transient expression of CRISPR/Cas9 in the targeted neurons. I am enthusiastic about the new approach, and methods to be developed to treat these devastating diseases in the near future.
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