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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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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.
View all comments by Bhuvaneish Thangaraj SelvarajTsinghua 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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