Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes

Rashnonejad, Afrooz; Amini-Chermahini, Gholamhossein; Taylor, Noah K.; Wein, Nicolas; Harper, Scott Q.

doi:10.1016/j.omtn.2020.12.004

Cited by 24 publications

(21 citation statements)

References 61 publications

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“…However, the effect of these types of interference may be transient, which make them less ideal for long-term treatment of FSHD. When these types of targeting strategies are combined with a gene therapy approach, as reported by Rashnonejad et al [ 73 ].…”

Section: Discussionmentioning

confidence: 99%

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

Ashoti

Limone²,

Kranenburg³

et al. 2022

PLoS ONE

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Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.

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Section: Discussionmentioning

confidence: 99%

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

Ashoti

Limone²,

Kranenburg³

et al. 2022

PLoS ONE

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show abstract

“…Since the aberrant expression of the double homeobox 4 ( DUX4 ) gene has been suggested as a predominant cause of FSHD pathogenesis [ 12 , 13 , 14 ], targeting DUX4 has great potential to provide a cure for the disease. Therefore, we and others have developed several approaches for suppressing DUX4 expression or its downstream activity [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. Despite FSHD affecting multiple skeletal muscles, only a few studies describe the systemic effects of therapeutic strategies on FSHD-like animal models.…”

Section: Introductionmentioning

confidence: 99%

Long-Term Systemic Treatment of a Mouse Model Displaying Chronic FSHD-like Pathology with Antisense Therapeutics That Inhibit DUX4 Expression

et al. 2022

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Silencing the expression of the double homeobox 4 (DUX4) gene offers great potential for the treatment of facioscapulohumeral muscular dystrophy (FSHD). Several research groups have recently reported promising results using systemic antisense therapy in a transgenic small animal model of FSHD, the ACTA1-MCM/FLExDUX4 mouse model. However, the treatment was applied in non-DUX4-induced mice or shortly after DUX4 activation, which resulted in conditions that do not correctly represent the situation in a clinic. Here, we generated progressive FSHD-like pathology in ACTA1-MCM/FLExDUX4 mice and then treated the animals with vivoPMO-PACS4, an antisense compound that efficiently downregulates DUX4. To best mimic the translation of this treatment in clinical settings, the systemic antisense oligonucleotide administration was delayed to 3 weeks after the DUX4 activation so that the pathology was established at the time of the treatment. The chronic administration of vivoPMO-PACS4 for 8 weeks downregulated the DUX4 expression by 60%. Consequently, the treated mice showed an increase by 18% in body-wide muscle mass and 32% in muscle strength, and a reduction in both myofiber central nucleation and muscle fibrosis by up to 29% and 37%, respectively. Our results in a more suitable model of FSHD pathology confirm the efficacy of vivoPMO-PACS4 administration, and highlight the significant benefit provided by the long-term treatment of the disease.

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“…As potential therapies for FSHD, multiple groups are developing technologies to inhibit the function or expression of DUX4-FL (e.g. Bosnakovski et al, 2019 ; Bouwman et al, 2021 ; Das and Chadwick, 2021 ; Himeda et al, 2020 ; Lim et al, 2020 , 2021 ; Lu-Nguyen et al, 2021 ; Mariot et al, 2020 ; Mellion et al, 2021 ; Oliva et al, 2019 ; Rashnonejad et al, 2020 ; Šikrová et al, 2021 ). Direct targeting of DUX4 will likely be the primary therapeutic strategy for FSHD, because once begun, any technique that inhibits DUX4 expression or function should block multiple pathogenic changes that are dependent on DUX4 transcriptional activity ( Mitsuhashi et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

Downstream events initiated by expression of FSHD-associated DUX4: Studies of nucleocytoplasmic transport, γH2AX accumulation, and Bax/Bak-dependence

et al. 2022

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Abnormal expression in skeletal muscle of the double homeobox transcription factor DUX4 underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Though multiple changes are known to be initiated by aberrant DUX4 expression, the downstream events initiated by DUX4 remain incompletely understood. In this study, we examined plausible downstream events initiated by DUX4. First, we found that nucleocytoplasmic protein export appeared to be decreased upon DUX4 expression as indicated by nuclear accumulation of a shuttle-GFP reporter. Second, building on studies from other labs, we showed that phospho(Ser139)-H2AX (γH2AX), an indicator of double-strand DNA breaks, accumulated both in human FSHD1 myotube nuclei upon endogenous DUX4 expression and in Bax-/-;Bak-/- (double knockout), SV40-immortalized mouse embryonic fibroblasts upon exogenous DUX4 expression. In contrast, DUX4-induced caspase 3/7 activation was prevented in Bax-/-;Bak-/- double knockout SV40-MEFs, but not by single knockouts of Bax, Bak, or Bid. Thus, aberrant DUX4 expression appeared to alter nucleocytoplasmic protein transport and generate double-strand DNA breaks in FSHD1 myotube nuclei, and the Bax/Bak pathway is required for DUX4-induced caspase activation but not γH2AX accumulation. These results add to our knowledge of downstream events induced by aberrant DUX4 expression and suggest possibilities for further mechanistic investigation.

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Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes

Cited by 24 publications

References 61 publications

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen

Long-Term Systemic Treatment of a Mouse Model Displaying Chronic FSHD-like Pathology with Antisense Therapeutics That Inhibit DUX4 Expression

Downstream events initiated by expression of FSHD-associated DUX4: Studies of nucleocytoplasmic transport, γH2AX accumulation, and Bax/Bak-dependence

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