Advanced iPSC-based modeling of LMNA-related congenital muscular dystrophy enables development of genetic therapies for muscle laminopathies

Figures

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  Figure 1. CRISPR-Cas9-mediated disruption of Let-7c BS and EBS upregulates UTRN in human DMD myoblasts (A) Scheme of UTRN gene with blow-up of guide RNA (gRNA) design to target ERF binding site (EBS); Let-7c binding site (BS); miR-196b BS; and the overlapping region of miR-150, miR-133b, and miR-296-5p BS. For each target, gRNA sequences are indicated by a colored line, PAM sites are framed, and SpCas9 cutting sites are indicated by a dotted line. (B) DNA editing efficiency at the indicated target loci after treatment with corresponding Cas9-gRNA complex in human DMD myoblasts bearing a deletion of exon 52 (hDMD-Δ52). InDel, insertions and deletions. n = 4–13 per group. (C) DNA indel pattern in edited myoblasts. “+1 InDel pattern” indicates the frequency of each inserted nucleotide. n = 6–13 per group. (D) UTRN mRNA expression normalized to peptidylprolyl isomerase A (PPIA) and represented as fold change to control. n = 4–11 per group. (E) UTRN protein expression. Left: representative western blot assay of control, Let-7c, or EBS edited myoblasts. Right: western blot quantification of UTRN, normalized to β-tubulin and represented as fold change to control. n = 7–8 per group. Each dot…

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  Figure 2. Let-7c gRNA efficiently disrupts its target without major off-target effects (A) Left: schematic representation of the dual reporter plasmid. Gaussia luciferase (GLuc) is under the control of the strong ubiquitous SV40 promoter and upstream of the 3′ UTR sequence of UTRN. The secreted embryonic alkaline phosphatase (SEAP) gene is included in the plasmid to normalize for transfection efficiency. From this full-length 3′ UTR construct, 11 additional sequences were generated, containing a full deletion of the Let-7c BS (ΔLet-7c BS), an insertion of one nucleotide (Let-7c +1 nt), a deletion of eight nucleotides (Let-7c -8 nt), or missing AREs and miR clusters. Deleted regions are indicated as Δstarting-ending nucleotide. Right: GLuc expression normalized to SEAP and represented as fold change to the full-length condition 48 h post transfection of the reporter systems. n = 3–4 per group. (B) Off-target analysis by Guide-seq in hDMD-Δ52 myoblasts treated with gRNA Let-7c. The top 10 genomic sequences identified are listed. On the left, alignments of the gRNA to the genomic DNA sequences are shown. Perfect matches are represented by black dots and mismatches are represented by colored…

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  Figure 3. Let-7c BS disruption upregulates UTRN in human 3D engineered muscles and ameliorates calcium dysregulation and contraction (A) Workflow of 3D muscle generation from edited immortalized hDMD-Δ52 myoblasts. (B) DNA editing efficiency at the Let-7c BS in 3D muscles made from edited myoblasts. n = 7 per group. (C) UTRN mRNA expression normalized to PPIA and represented as fold change to unedited 3D muscles. n = 3 per group. (D) UTRN protein expression. Top: western blot assay of WT, DMD, or DMD-Let-7c BS edited 3D muscles. Bottom: western blot quantification of UTRN, normalized to GAPDH and represented as fold change to control. n = 3 per group. (E) Representative immunofluorescence staining of UTRN and myosin heavy chain (MyHC) on control DMD or DMD-Let-7c BS edited 3D muscle transverse sections. White arrows indicate UTRN signal concentration at the proximity of the sarcolemma. Scale bar: 50 μm. (F) Calcium transients assay performed on 3D muscles. Each dot represents the mean amplitude of peak signals obtained upon stimulation. n = 4 per group. (G) Mean contraction force measured in micro-Newtons (μN) during 12-V twitch stimulation. n = 4 per group. Each dot represents one 3D…

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  Figure 4. miR-Let-7c BS disruption induces a 2-fold increase in UTRN expression in C2C12 mouse myoblasts (A) DNA editing efficiency at the Let-7c BS after treatment with corresponding Cas9-gRNA complex in murine C2C12 myoblasts. n = 3 per group. (B) DNA indel pattern in gRNA mLet-7c-2 (left) and gRNA mLet-7c-4 (right) edited myoblasts. n = 3 per group. (C) UTRN mRNA expression normalized to PPIA and represented as fold change to control. n = 3 per group. (D) UTRN protein expression. Left: western blot assay of mLet-7c-2 or mLet-7c-4 treated myoblasts. Right: western blot quantification of UTRN, normalized to vinculin and represented as fold change to control. n = 2 per group. ns, non-significant. (E) Murine Let-7c miRNA (mmu-Let-7c) expression was measured by qPCR in the tibialis anterior (TA), diaphragm (DIA), and heart (HR) of WT and mdx mice, normalized to sno202 expression, and represented as fold change to the expression in the TA of WT mice. n = 4 mice per group. Each dot represents one biological replicate. n indicates the number of biological replicates per group. Bars represent the mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s (C and D) or…

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  Figure 5. Intramuscular AAV9 delivery of Cas9 and Let-7c BS gRNA results in upregulation of UTRN in mdx mice (A) Workflow of the experiment. 4-week-old mdx mice were injected with a total of 3e11 vector genomes (vg) of AAV9-Cas9 and AAV9-gRNA-mLet-7c-4 intramuscularly in one tibialis anterior (TA) with a 1:5 Cas9:gRNA ratio. WT and mdx groups treated with PBS were used as controls. After 4 weeks of treatment, TAs were collected for analysis. (B) Quantification of total AAV9 vector copy number in the TA of PBS or gRNA-mLet-7c-4-treated mice. (C) Native GFP signal in TA transverse sections acquired with the AxioScan device. Zoom: 10×. Scale bars are indicated. (D) DNA editing efficiency at the Let-7c BS measured by NGS in gRNA-mLet-7c-4-treated mice. (E) UTRN mRNA expression normalized to PPIA and represented as fold change to mdx group. (F) UTRN protein expression. Left: western blot assay of PBS or gRNA-mLet-7c-4-treated mdx mice TAs. Right: western blot quantification of UTRN, normalized to vinculin and represented as fold change to mdx. Each dot represents one TA. n = 3–4 TAs per group. Bars represent the mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s…

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  Figure 6. Intramuscular AAV9 delivery of Cas9 and Let-7c BS gRNA tends to ameliorate DMD histological and functional parameters in mdx mice (A) Representative images of Sirius red (SR; top) and hematoxylin and eosin (HE; bottom) stainings of TA transverse sections. Regions of necrosis and infiltration are indicated by black stars. Examples of centrally nucleated fibers are indicated by a black arrow. Scale bars: 200 μm. (B) Quantification of SR-positive area (collagen deposition). (C) Quantification of centrally nucleated fibers. (D) TA mass measured in milligrams (mg). (E) Tetanic force measured in Newtons (N) during TA in situ contraction assay and normalized by TA weight (N/mg). Each dot represents one TA. n = 3–4 TAs per group. Bars represent the mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test.

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  Figure 7. Systemic AAV9 delivery of Cas9 and Let-7c BS gRNA results in upregulation of UTRN in mdx mice (A) Workflow of the experiment. 4-week-old mdx mice were injected with a total of 1e13 vg of AAV9-Cas9 and AAV9-gRNA-mLet-7c-4 in the tail vein with a 1:5 Cas9:gRNA ratio. A group treated with a ROSA-targeting gRNA was used as control. After 5 weeks of treatment, tibialis anterior (TA), diaphragm (DIA), and heart (HR) were collected for analysis. (B) Quantification of total AAV9 vector copy number in the TA, DIA, and HR of gRNA-ROSA or gRNA-mLet-7c-4-treated mice. (C) DNA editing efficiency at the Let-7c BS measured by NGS in the TA, DIA, and HR of gRNA-mLet-7c-4-treated mice. (D) UTRN protein expression. Left: western blot assay of gRNA-ROSA or gRNA-mLet-7c-4-treated mdx mice TA, DIA, and HR. Right: western blot quantification of UTRN, normalized to β-tubulin and represented as fold change to ROSA-treated mice. (E) Immunofluorescence staining of UTRN in TA, DIA, and HR of control or treated mice indicates sarcolemma localization of the upregulated protein. Scale bars: 100µm. Each dot represents one mouse. n = 3 mice per group. Bars represent the mean ± SD. Statistical analysis was…