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Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats

Nature Biomedical Engineering volume 6, pages 207–220 (2022)Cite this article

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Abstract

Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals’ skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies.

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Fig. 1: MBNL1Δ corrects alternative-splicing defects and overall transcriptome of DM1 patient-derived muscle cells.
Fig. 2: MBNL1∆ binding to CUGexp displaces endogenous MBNL1 from RNA foci and reduces CUGexp-RNA levels in DM1 patient-derived muscle cells.
Fig. 3: MBNL1∆ forms less stable CUGexp-RNA complexes than MBNL1.
Fig. 4: Intramuscular expression of MBNL1Δ using AAV vectors has no deleterious effect in WT mice.
Fig. 5: Intramuscular injection of AAV-MBNL1∆ corrects splicing defects and myotonia in HSALR mice.
Fig. 6: Long-term correction of DM1-associated defects in MBNL1∆-treated HSALR mice.
Fig. 7: Systemic treatment with AAV-MBNL1Δ improves myotonia, splicing defects and decreases CUGexp-RNA levels in HSALR mice.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. NGS data are available at the GEO repository (GSE189516). The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from ANR (Agence National de la Recherche), AFM (Association Francaise contre les Myopathies) and Association Institut de Myologie. M.M. was supported by the DIM biotherapies, Paris Ile-de-France Region. We thank I. Holt and G. Morris (CIND, RJAH Orthopeadic Hospital, UK) as well as The Muscular Dystrophy Association Monoclonal Antibody Resource for the MBNL1 (MB1a) antibody; T. Cooper for the 960 CTG construct; C. Thornton for the MBNL1 polyclonal antibody and the HSALR mouse model; the iVector facility of the Institut du Cerveau; the human cell immortalization facility and the AAV facility of the Myology Institute, the Penn Vector Core -Gene Therapy Program- University of Pennsylvania (Philadelphia) for providing the pAAV2/9 plasmid (p5E18-VD29); and B. Cadot, S. Ziyyat-Benkhelifa, L. Julien, A. Jollet, C. Neuillet and V. Allamand for their help.

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  1. These authors contributed equally: Ludovic Arandel, Magdalena Matloka.

Authors and Affiliations

  1. Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, Paris, France

    Ludovic Arandel, Magdalena Matloka, Arnaud F. Klein, Frédérique Rau, Alain Sureau, Michel Ney, Aurélien Cordier, Maria Kondili, Micaela Polay-Espinoza, Naira Naouar, Arnaud Ferry, Mégane Lemaitre, Joëlle Marie & Denis Furling

  2. Sorbonne Paris Cité, Université Paris Descartes, Paris, France

    Arnaud Ferry

  3. Sorbonne Université, Inserm, Phénotypage du petit animal, Paris, France

    Mégane Lemaitre

  4. Université de Lille, Inserm, CHU Lille, Lille Neuroscience and Cognition, Lille, France

    Séverine Begard, Morvane Colin, Chloé Lamarre, Hélène Tran, Luc Buée & Nicolas Sergeant

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Contributions

L.A. and M.M. conducted most of the experiments. F.R. performed FRAP experiments, J.M and A.F.K. performed LLPS experiments, and M.N., A.F.K., A.S., J.M., A.C., H.T., C.L. and L.B. supported some experiments. M.C. and S.B. produced lentiviral vectors, A.F. and M.L. measured the muscle force, and M.P-E., M.K. and N.N. performed RNA-seq analysis. N.S. and D.F. supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Nicolas Sergeant or Denis Furling.

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Competing interests

The method described in this paper is the subject of a patent application (PCT/EP2015/058111). The authors declare no other competing financial interest.

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Nature Biomedical Engineering thanks Gene Yeo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Intramuscular injection of AAV-GFP-MBNL1 corrects splicing defects in HSALR mice but has deleterious effects in WT mice.

(a) Correction of Atp2a1 exon 22, Clcn1 exon 7a and Mbnl1 exon 5 alternative splicing assessed by RT-PCR in Gastrocnemius of HSALR mice after local intramuscular injection of AAV9-GFP-MBNL1 (1 × 1011 vg, n = 3) and compared to saline vehicle-injected contralateral muscle or muscle from WT mice (n = 4). Data analysed by one-way ANOVA followed by Tukey’s test (****p < 0.0001). (b) Hematoxylin and Eosin (HE) staining performed on GA muscle sections of WT mice injected with AAV9-GFP-MBNL1 vectors (1×1011 vg) or saline for 2 or 3 weeks. (c) Expression of Myog and Myh8 measured by RT-qPCR in FVB muscles injected with AAV9-GFP-MBNL1 or saline for 2 weeks.

Extended Data Fig. 2 Intramuscular injection of AAV-GFP has no effect on DM1 splicing events regulated by MBNL1.

(a) Representative Western blot and quantification of MBNL1 protein level in WT Gastrocnemius muscles five weeks after intramuscular injection of AAV9-GFP (1×1011 vg; n = 4) or saline. Data analyzed by unpaired Student’s t-test (ns: not significant). (b) Splicing profiles of Clcn1 exon 7a and Atp2a1 exon 22 assessed by RT-PCR in WT mice five weeks after intramuscular injection of AAV9-GFP (1×1011 vg) compared to saline vehicle-injected contralateral muscles or muscles form HSALR mice (n = 3-6). Data analyzed by one-way ANOVA followed by Tukey’s test (****p < 0.0001). (c) Modulation of 23 DM1-misspliced events regulated by MBNL1 in GA muscles of WT mice injected with AAV9-GFP-MBNL1∆ or AAV9-GFP compared to saline vehicle-injected contralateral muscles or muscles form HSALR mice (n = 3).

Extended Data Fig. 3 Local and systemic administration of AAV-V5-MBNL1∆ corrects splicing defects in muscles of HSALR mice.

(a) Correction of Atp2a1 exon 22, Clcn1 exon 7a and Mbnl1 exon 5 alternative splicing assessed by RT-PCR in Gastrocnemius of HSALR mice after local intramuscular injection of AAV9-V5-MBNL1∆ (n = 3-4, upper panel) or AAV9-MBNL1∆ (n = 3, lower panel) and compared to saline vehicle-injected contralateral muscle or muscle from WT mice (n = 4). Data analyzed by one-way ANOVA followed by Tukey’s test (****p < 0.0001). (b) Correction of Atp2a1 exon 22 and Mbnl1 exon 5 alternative splicing misregulation in Gastrocnemius (GA) and Quadriceps (QUA) muscles of HSALR mice following systemic MBNL1∆ treatment (n = 5) compared to saline-injected HSALR (n = 4) and WT mice (n = 3). Data analyzed by one-way ANOVA followed by Tukey’s test (****p < 0.0001). (c) Levels of MBNL1 proteins assessed by Western blot in GA muscles of MBNL1∆-treated HSALR mice (n = 4) and saline-injected HSALR or WT mice (n = 3). Data analyzed by one-way ANOVA followed by Tukey’s test (ns: not significant).

Extended Data Fig. 4 Analysis of DM1 splicing events in heart, liver and kidney of HSALR mice treated systemically with AAV-MBNL1∆.

(a) Splicing profiles of Scn5a exon 6b, Mbnl1 exon 5, Dmd exon 78 and Mbnl2 exon 5 in heart following systemic injection of AAV-V5-MBNLΔ or saline vehicle in HSALR mice (n = 5-6) and compared to WT mice injected with saline vehicle (n = 3) (b) Splicing profiles of Mbnl1 exon 5 and Mbnl2 exon 5 in kidney (K) and liver (L) assessed by RT-PCR following systemic injection of AAV-V5-MBNLΔ or saline vehicle in HSALR mice (n = 3-4), and compared to WT mice injected with saline vehicle (n = 2-3). Data analyzed by one-way ANOVA followed by Tukey’s test (ns = not significant).

Supplementary information

Supplementary Information

Supplementary figures, tables and video captions.

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Supplementary Table 1

Differential alternative-splicing events (|∆Psi| > 0.2; adjusted P < 0.05) inferred by analysis of RNA-seq data using rMTAS in WT vs DM1 cells and WT vs MBNL1Δ-treated DM1 cells.

Supplementary Table 2

Differential gene expression (|log2FC| > 1; adjusted P < 0.05) inferred by analysis of RNA-seq data using DESeq2 in WT vs DM1 cells and WT vs MBNL1Δ-treated DM1 cells.

Supplementary Table 3

Differential alternative-splicing events (|∆Psi| > 0.2; adjusted P < 0.05) inferred by analysis of RNA-seq data using rMTAS in saline vs AAV9-GFP-MBNL1Δ-treated WT gastrocnemius muscles and saline vs AAV9-GFP-treated WT gastrocnemius muscles.

Supplementary Table 4

List of primers and siRNA.

Supplementary Video 1

Representative video showing RNA droplets formed by fluorescently labelled (CUG)46 RNA repeats.

Supplementary Video 2

Representative video showing CUGexp-RNA/MBNL1 condensates.

Supplementary Video 3

Representative video showing CUGexp-RNA/MBNL1∆ condensates.

Source data

Source Data Extended Data Fig

Full gel/blot scans for the relevant main figures and Extended Data figures.

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Arandel, L., Matloka, M., Klein, A.F. et al. Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats. Nat Biomed Eng 6, 207–220 (2022). https://doi.org/10.1038/s41551-021-00838-2

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