A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene

Abstract

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR–dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

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Fig. 1: SadCas9–2×VP64-mediated upregulation of Lama1 in vitro.
Fig. 2: Upregulation of Lama1 in tibialis anterior muscles of dy2j/dy2j mice improves muscle fibrosis.
Fig. 3: Upregulation of Lama1 in older dy2j/dy2j mice halts disease progression.

Data availability

The authors declare that the main data supporting the findings of this study are available within the paper and its Supplementary Information.

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Acknowledgements

We thank members of the Cohn laboratory for technical support and input in this study; P. Yurchenco for providing reagents used in this study, C. Rand, I. Vukobradovic and R. Smith for assistance with functional and behavioural studies, D. Holmyard for transmission electron microscopy analysis and S. Pereira for genomic data acquisition. This work was supported by AFM-Telethon, Cure CMD, Muscular Dystrophy Association (to D.U.K.), Rare Disease Foundation Microgrant (to P.S.B. and S.E.), SickKids Restracomp (to S.E.), Crafoord Foundation (to K.I.G.), CIHR Summer Studentship (to R.M.M.), Swedish Research Council, Anna and Edwin Berger Foundation, Greta and Johan Kock Foundation, Olle Engkvist Byggmastare Foundation and Osterlund Foundation (to M.D.), Natural Sciences and Engineering Research Council of Canada, Canadian Institute for Health Research, SickKids Foundation and R. S. McLaughlin Foundation Chair in Pediatrics (to R.D.C.).

Reviewer information

Nature thanks James M. Ervasti, Reid D. Landes, Daniel Michele and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

D.U.K., E.A.I. and R.D.C. conceived and supervised the study. P.S.B., K.L., R.K. and K.M.P. cloned and screened guides in vitro. D.U.K., P.S.B., K.L. and E.H. performed in vivo injections and contractile assays. D.U.K., P.S.B., K.L., K.M.P. and R.M.M. analysed muscle histology. K.I.G. and M.D. performed extended muscle imaging and provided reagents. D.U.K. performed longitudinal open field assay and vector genome quantification. D.U.K. and P.S.B. performed nerve imaging with the help from K.M.P. and R.M.M. D.U.K. and D.A.-B. performed nerve conduction velocity under supervision by S.A.P. S.E. analysed RNA-sequencing data and off-target prediction. D.U.K. wrote the manuscript with inputs from the other authors. All authors provided feedback and agreed on the final manuscript.

Correspondence to Ronald D. Cohn.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 SadCas9–2×VP64 enhances expression of minCMV-driven tdTomato in vitro.

a, b, HEK293T cells were transfected with a plasmid encoding minCMV-driven tdTomato only. c, d, HEK293T cells were transfected with a combination of a plasmid containing minCMV-driven tdTomato and one containing SadCas9–2×VP64 and a sgRNA targeting the minCMV promoter. a, c, Schematic. b, d, Cells were imaged for tdTomato expression by fluorescent microscopy. Bright-field and tdTomato images are shown. The experiment was repeated three times independently with similar results. Scale bars, 50 µm.

Extended Data Fig. 2 Early intervention to upregulate Lama1 prevents disease progression in dy2j/dy2j mice.

a, P2 neonatal dy2j/dy2j mice were injected with AAV9s carrying no guide (n = 6; 7.5 × 1011 viral genomes) or three guides (n = 4; split into two vectors; total dose of 2 × 7.5 × 1011 viral genomes) in the temporal vein and euthanized 7 weeks later. b, c, Immunofluorescence staining shows Lama1 expression in tibialis anterior (b) and gastrocnemius (c) muscles. Scale bars, 100 µm. d, e, General muscle histopathology was evaluated by haematoxylin and eosin staining on tibialis anterior (d) and gastrocnemius (e) muscles. Scale bars, 200 µm. All mice (n = 6, no guide and n = 4, three guides) were analysed for experiments shown in be, which were repeated three times independently with similar results. fi, Fibrosis (f, h) and minimum Feret diameter (g, i) were quantified for all mice from both treatment groups (n = 6, no guide and n = 4, three guides) by two independent experimenters with similar results. Data are mean ± s.d. **P = 0.0025 (f), *P = 0.0103 (g), *P = 0.0142 (h), P = 0.0711 (i); two-tailed Student’s t-test. Source data

Extended Data Fig. 3 Upregulation of Lama1 corresponds to improvement of muscle functions.

Three-week-old dy2j/dy2j mice were injected systemically in the tail vein with AAV9s carrying no guide (n = 9) at the dose of 3 × 1011 viral genomes (vg) per gram of mouse, or three guides at different doses of 7.5 × 1010 (n = 4), 1.5 × 1011 (n = 3) or 3 × 1011 (n = 8) viral genome per gram of mouse. Two AAVs were needed for the three-guide cohorts, therefore the total doses of virus injected were 1.5 × 1011, 3 × 1011 and 6 × 1011 viral genomes per gram of mouse. a, Tibialis anterior muscles isolated that were isolated four weeks later were stained for Lama1 expression. Asterisks indicate Lama1-positive fibres in the low-dose cohort. The experiment was repeated three times independently with similar results. Scale bars, 100 µm. b, c, In vivo contractile force assays (b) and open field tests (c) were performed at the end of the treatment regimen. Data are mean ± s.d. b, P values from left to right are: P = 0.9849, P = 0.9082, **P = 0.0044; one-way ANOVA with Dunnett’s multiple comparisons test. c, P values from left to right are: P = 0.9640, P = 0.9646, **P = 0.0085; one-way ANOVA with Dunnett’s multiple comparisons test. Source data

Extended Data Fig. 4 Representative images of Lama1-positive muscle sections.

Three-week-old mice were injected systemically with three guides (n = 5) at the dose of 3 × 1011 viral genomes per gram of mouse (split into two vectors, thus total dose was 2 × 3× 1011 viral genomes per gram of mouse) in the tail vein, and euthanized at the age of 11–12 weeks old. Muscles were stained for Lama1 expression (red). Scale bars, 500 µm. The experiment was repeated three times independently with similar results.

Extended Data Fig. 5 Expression of laminin subunits in tibialis anterior muscles.

Expression of laminin (LM) subunits in tibialis anterior muscles of wild-type, dy2j/dy2j (intravenously injected with AAVs carrying no guide at 3 × 1011 viral genomes per gram of mouse or three guides at a total dose of 2 × 3× 1011 viral genomes per gram of mouse) and dy2j/dy2j LMα1 (with transgenic overexpression of LMα1) mice (n = 3 for each group). Expression of the LMα1 chain in the tibialis anterior muscle is comparable between dy2j/dy2j mice treated with three guides and transgenic mice. No major differences in expression of LMα2 and LMγ1 were detected between the groups. The LMα4 chain is upregulated in muscles of dy2j/dy2j mice treated with the no-guide AAV and the expression of the LMα4 chain is partially normalized in muscle of dy2j/dy2j mice treated with the three-guide AAV. Scale bar, 50 μm. The experiment was repeated three times independently with similar results.

Extended Data Fig. 6 Expression of laminin subunits in gastrocnemius muscles.

Expression of laminin subunits in gastrocnemius muscles from wild-type, dy2j/dy2j (intravenously injected with AAVs carrying no guide at 3 × 1011 viral genomes per gram of mouse or three guides at a total dose of 2 × 3 × 1011 viral genomes per gram of mouse) and dy2j/dy2j LMα1 (with transgenic overexpression of LMα1) mice (n = 3 for each group). Expression of the LMα1 chain in the gastrocnemius muscles is comparable between dy2j/dy2j mice treated with three guides and transgenic mice. No major differences in expression of LMα2 and LMγ1 were detected between the groups. The LMα4 chain is upregulated in muscle of dy2j/dy2j mice treated with the no-guide AAV and the expression of the LMα4 chain is partially normalized in muscle of dy2j/dy2j AAV-mice treated with three guides. Scale bar, 50 μm. The experiment was repeated three times independently with similar results. Source data

Extended Data Fig. 7 Myelination of sciatic nerves.

Transmission electron microscopy images of sciatic nerves isolated from dy2j/dy2j mice injected with AAV9s carrying no guide (3 × 1011 viral genomes per gram of mouse) or three guides (split into two vectors; total dose of 2 × 3 × 1011 viral genomes per gram of mouse). Three mice were analysed per group. Myelin thickness was measured as the difference between the distance of the myelin and axon (m+a, blue solid line) and axon only (a; red dotted line), quantified using ImageJ and presented as mean ± s.d. Statistical analysis was performed using a two-tailed t-test. *P = 0.0231. Scale bar, 5 μm. The experiment was repeated three times independently with similar results. Source data

Extended Data Fig. 8 Quantitative evaluation of AAV genome distribution.

Genomic DNA isolated from the tibialis anterior muscle, sciatic nerve and liver of dy2j/dy2j mice (n = 8) injected with AAVs carrying three guides at the dose of 3 × 1011 viral genomes per gram of mouse (split into two vectors; total dose of 2 × 3 × 1011 viral genomes per gram of mouse) was amplified to analyse the presence of viral genomes by qPCR. Data are mean ± s.e.m. The experiment was repeated three times independently with similar results. Source data

Extended Data Fig. 9 Genome-wide analysis of gene expression of SadCas9-2×VP64-treated dy2j/dy2j mice.

ac, Differential expression analysis derived from RNA-sequencing results from the quadriceps of treated and untreated mice (n = 3 per group). a, b, No guide: dy2j/dy2j mice injected with AAV9 carrying only the SadCas9–2×VP64 at the dose of 3 × 1011 viral genomes per gram of mouse. a, c, Three guides: dy2j/dy2j mice injected with AAV9 carrying the SadCas9–2×VP64 and three guides at the dose of 3 × 1011 viral genomes per gram of mouse (total dose of 2 × 3 × 1011 viral genomes per gram of mouse). b, c, Untreated: dy2j/dy2j mice that had not been injected with AAV9. False discovery and multiple testing were controlled for and an adjusted P value was computed using the Benjamini–Hochberg method. Differentially expressed genes were defined as genes with a more than twofold change and adjusted P < 0.05. For each comparison, significantly differentially expressed genes (false-discovery rate-adjusted P < 0.05) are coloured. Red data points indicate a log-transformed fold change in expression greater than one; blue data points indicate a log-transformed fold change in expression less than one. d, Hierarchical clustering was performed on the normalized counts-per-million expression data.

Extended Data Fig. 10 Top 500 differentially expressed genes.

Heat map illustrating the log-normalized counts per million for the top 500 genes that were differentially expressed in quadriceps isolated from wild-type mice, or dy2j/dy2j mice injected with AAV9s carrying SadCas9–2×VP64 only (no guide, n = 3) or with three guides (three guides, n = 3), and compared to age-matched, untreated dy2j/dy2j mice. Red indicates higher expression; blue indicates lower expression.

Supplementary information

Supplemental Figure 1

Uncropped scans with size marker indications Boxes indicate area presented in the corresponding manuscript figures.

Reporting Summary

Supplemental Method 1

Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines Detailed reporting of design, experimental procedures, and experimental animals in this study.

Supplementary Tables

This file contains Supplementary Tables 1-6 (legends included at the top of each sheet).

Supplementary Video 1

Phenotype of control dy2j/dy2j mouse following early intervention. The dy2j/dy2j mouse was injected with AAV9 containing SadCas9-2xVP64 (no guide) at P2 (pre-symptomatic stage) via temporal vein and video was taken at the age of 7-week old. Hind limb paralysis, contracture and kyphosis resulting from lack of functional Lama2 and compensatory Lama1 are apparent.

Supplementary Video 2

Phenotype of treated dy2j/dy2j mouse following early intervention. The dy2j/dy2j mouse was injected with AAV9 containing SadCas9-2xVP64 and sgRNAs targeting Lama1 proximal promoter (three guides) at P2 (pre-symptomatic stage) via temporal vein and video was taken at the age of 7-week old. Upregulation of compensatory Lama1 expression ameliorates the hind limb paralysis, contracture and kyphosis.

Supplementary Video 3

Phenotype of control dy2j/dy2j mouse following intervention at symptomatic stage. The dy2j/dy2j mouse was injected with AAV9 containing SadCas9-2xVP64 (no guide) at 3-week old (pre-symptomatic stage) via tail vein. Video was taken at the age of 7-week old. Hind limb paralysis, contracture and kyphosis resulting from lack of functional Lama2 and compensatory Lama1 are apparent.

Supplementary Video 4

Phenotype of treated dy2j/dy2j mouse following intervention at symptomatic stage. The dy2j/dy2j mouse was injected with AAV9 containing SadCas9-2xVP64 and sgRNAs targeting Lama1 proximal promoter (three guides) at 3-week old (pre-symptomatic stage) via tail vein. Video was taken at the age of 7-week old. Dystrophic features and disease progression were significantly improved and partially reversed following upregulation of Lama1.

Source data

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