Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

Abstract

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx^4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

Figure 1. CRISPR/Cas9-mediated gene editing in mdx^4cv mice.

(a–e) Strategies for creating a dystrophin mRNA carrying an ORF by removing the mdx^4cv TAA premature stop codon (the mdx^4cv C to T point-mutation is depicted in red). (a) Strategy 1 (Δ5253) utilizes both dual- and single-vector approaches to target introns 51 and 53 (arrows=sgRNA target sites shown in a 5′-3′ direction based on target strand) to direct excision of exons 52 and 53 (b). (c) Strategy 2 (53*) utilizes a dual-vector approach to target exon 53 on either side of the stop codon, relying on HDR (utilizing a WT DNA template) or NHEJ to generate either full-length WT dystrophin (d) or a partial in-frame deletion of exon 53 (e).

Figure 2. In vivo gene editing introduces a functional ORF in mdx^4cv mouse muscles.

(a) Deep sequencing quantification on PCR amplicons generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n=4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. (b) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n=4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. (c) Subclone sequencing of the treatment-specific RT–PCR product (red box in b) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). (d) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n=5) and RT–PCR amplicons generated from pooled transcripts (right, n=4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. (e) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).

Figure 3. Dystrophin expression in treated muscles improves muscle morphology.

(a) TA muscles from treated mice were collected and analysed for expression of the mCherry reporter gene (top) or cryosectioned for immunostaining of dystrophin (bottom). Widespread dystrophin expression resulted from both strategies 1 and 2 (Scale bar, 1 mm). (b) Western analysis of muscles from treated and untreated mice (WT and mdx^4cv) showing dystrophin (Dys), SpCas9, SaCas9 and GAPDH expression. Dystrophin was detected using antisera raised against the C terminus (CT); the SaCas9 nuclease carried an HA epitope tag to enable detection with anti-HA antibodies. (c) Quantification of GAPDH-normalized dystrophin expression in treated TAs compared with WT muscles (n=4). (d) Immunostained cross-sections from treated and control mice were analysed for the percentage of all myofibers expressing dystrophin (n=5). (e) Shown is the cross-sectional area (CXA) size distribution of individual myofibers from treated and control muscles (n>12,500 total fibres per group). (f) The total myogenic cross-sectional area (CXA) that was dystrophin-positive is shown for treated and WT control muscles (n=5). (g) Individual myofiber size distribution for treated TAs relative to dystrophin expression. (h) The percentage of myofibers containing centrally located nuclei is shown for dystrophin-positive treated myofibers and for total myofibers of control TA muscles (n=5). Data are shown as mean±s.e.m. ***P<0.001, (One-way ANOVA multiple comparisons test with Turkey’s post hoc test).

Figure 4. CRISPR/Cas9-mediated dystrophin correction localizes nNOS to the sarcolemma and improves muscle function.

(a) Immunofluorescent staining for nNOS, laminin and dystrophin in IM-treated and control muscles (Scale bar, 100 μm). (b) Specific force generating levels of treated mdx^4cv mouse TA muscles 18 weeks post-IM transduction with 2.5 × 10¹⁰ v.g. of each vector (SaCas9Δ5253 (n=8), SpCas9/Δ5253 (n=6), SpCas9/53* (n=8) and of untreated age-matched WT (n=3) and mdx^4cv (n=6) muscles. Bars represent mean±s.e.m. (*P<0.05, ***P<0.001). (c) Protection against eccentric contraction-induced injury as demonstrated by measuring contractile performance immediately before increasing length changes during maximal force production in TA muscles of untreated (n=5) versus IM-treated mdx^4cv mice (SaCas9Δ5253 (n=8), SpCas9/Δ5253 (n=7), SpCas9/53* (n=8)). Values are represented as mean±s.e.m. Statistical significance was determined via multiple Student’s t-test comparisons, (**P<0.01, ****P<0.0001).

Figure 5. Systemic gene editing results in widespread dystrophin expression.

Immunofluorescence analysis of mdx^4cv mouse muscles at 4 weeks post systemic transduction with dual (sp5253) and single (sa5253) vector approaches in strategy 1. (a) Muscle cross-section showing widespread transduction of multiple muscle groups following high dose (1 × 10¹³/4 × 10¹² v.g. of nuclease/targeting vectors) dual-vector delivery based on mCherry reporter gene expression, Scale bar, 3 mm. Whole cardiac cross-sections showing dystrophin expression following dual-vector delivery at the high dose (b), low dose (c, 1 × 10¹²/1 × 10¹²) and following single vector delivery at the low dose (d, 1 × 10¹²), Scale bars, 1 mm. Insets depict magnified field of views. Widespread but variable dystrophin expression is observed in multiple muscle groups following high dose dual-vector delivery; including TA (e), diaphragm (f), soleus (g) and gastrocnemius (h), Scale bars, 100 μm. Western analysis of cardiac lysates demonstrates expression of near full-length dystrophin in low dose (LD) and high dose (HD) treatment groups, with increased dystrophin expression at higher vector doses (i).