A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells

Abstract

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Figure 1. CDMD hiPSCs are pluripotent and genetically stable

A. CDMD hiPSCs were generated from DMD fibroblasts. Brightfield images depict fibroblasts before and after reprogramming to hiPSCs. Immunocytochemical staining reveals that cells express pluripotency markers NANOG (green) and SOX2 (red). Scale bar 100μm. B. Karyotyping of all lines is shown. C. CDMD hiPSCs were injected into mice to test teratoma formation in vivo. Representative hematoxylin and eosin stainings of the three germ layers (endoderm, mesoderm, and ectoderm) are shown. D. Patient mutations for each CDMD hiPSC line are shown. In addition, the number of exons and the approximate distance necessary for successful NHEJ is indicated, based on comparative genomic hybridization data for the patient’s underlying mutation size.

Figure 2. Generation of stable, pluripotent CDMD hiPSC lines with an exon 45–55 deletion

A. Shown is a cartoon (not to scale) of the region of DMD targeted for CRISPR/Cas9-mediated deletion using gRNAs specific to introns 44 and 55 (lightning bolts). Successful NHEJ deletes exons 45–55 and restores the reading frame for mutations within this region. Different deletion sizes are required depending on the patient’s underlying mutation (black arrow heads). B. PCR genotyping of 117 and 109 single cell clones from parental lines CDMD 1006 and 1003, respectively, was carried out on cells nucleofected with gRNAs 44C4 and 55C3. One clone from CDMD 1006 (CDMD 1006-1) and three from CDMD 1003 (CDMD 1003-49, 1003-57, 1003-81) were identified as stably deleted. Deletion PCR genotyping results for 6 hiPSC clonal lines is shown. One pair of primers (red arrows in A) was located internal to the deletion and only produced a 1201bp band in the undeleted clones CDMD 1003-13 and 1003-51. Another primer set (purple arrows in A) flanked the deletion region and produced a 788bp band only when the deletion and NHEJ occurred successfully, as in the reframed clones CDMD 1006-1, 1003-49, 1003-57, 1003-81. C. Each clonal line maintained normal morphology (brightfield) and expressed NANOG (green) and SOX2 (red) by immunocytochemistry. Scale bar 100μm. Shown to the right is the sequence of the gDNA at the rejoining site between introns 44 (I44) and 55 (I55). Sequencing revealed a 16bp deletion in CDMD 1006-1, a 2bp insertion in CDMD 1003-49, and 1bp insertions in CDMD 1003-57 and CDMD 1003-81. See also Figure S1, S2, S3, and S4 A–B.

Figure 3. Reframed CDMD hiPSC-derived skeletal muscle and cardiomyocytes restore dystrophin expression

A. Immunocytochemical staining of human myosin heavy chain (MyHC, red) and dystrophin (green) of wild type (CDMD 1002), out-of-frame (CDMD 1003, 1006) or reframed (CDMD 1003-49, 1006-1) cardiomyocytes derived from hiPSCs by directed differentiation. Inset depicts zoomed in region defined by the white box. Scale bar 50μm. B. Immunocytochemical staining of MyHC (red) and dystrophin (green) of wild type (CDMD 1002), out-of-frame (CDMD 1006) or reframed (CDMD 1006-1, 1003-49) skeletal muscle myotubes derived from hiPSCs. Myotubes were fused after MyoD overexpression (OE) or from sorted NCAM⁺ cells after an adapted directed differentiation 50-day protocol. Inset depicts zoomed in region defined by the white box. Scale bar 100μm. C. Western blots of cell extracts probed with anti-dystrophin. Extracts were from out-of-frame and reframed cardiomyocytes (left) and skeletal muscle myotubes (right), derived from CDMD hiPSCs. Wild type (wt) hiPSCs (CDMD 1002) or human skeletal muscle myotubes (HSMM) were used as a control for dystrophin. The molecular weight shift caused by the exon 45–55 deletion (1779bp, ~66kDa) is evident in reframed vs. wild type dystrophin (arrows). A non-specific band around 220kDa was seen in some samples. Samples were also probed with anti-MyHC as a loading control (bottom panels). See also Figure S4C–D.

Figure 4. Reframed hiPSC-derived cardiomyocytes and skeletal muscle cells demonstrate restored function in vitro and in vivo

A. Representative graphs of CK release assays from cells exposed to hypoosmotic conditions. Cardiomyocytes and skeletal muscle myotubes derived from hiPSCs were subjected to a range of osmolarities below 240mosmol and CK release to the supernatant was measured as an indication of membrane fragility. Data are presented as average ± standard error. B. Fold change in expression of miR31 measured by ddPCR in myotubes derived from out-of-frame or reframed hiPSCs by MyoD OE, normalized to wild type (CDMD 1002). Data are presented as average ± standard deviation. C. Western blots of cell extracts probed with anti-β-dystroglycan. Extracts were from out-of-frame and reframed skeletal muscle myotubes derived by MyoD OE. HSMM was used as a positive control. Samples were also probed with anti-MyHC as a loading control (bottom panel). D. Immunocytochemical staining of MyHC (red) and β-dystroglycan (green), a component of the DGC, in wild type (CDMD 1002), out-of-frame (CDMD 1006) or reframed (CDMD 1006-1) skeletal muscle myotubes. Inset depicts zoomed in region defined by the white box. Scale bar 50μm. E. Assessment of human dystrophin restoration in wild type (CDMD 1002), out-of-frame (CDMD 1003), and reframed (CDMD 1003-49) MyoD OE cells engrafted into the TA of NSG-mdx mice. Engrafted human cells were identified by co-immunostaining for human spectrin and lamin A/C (shown in red). Positive staining for human dystrophin is shown in green and all fibers are shown using laminin (grey). All sections were stained with DAPI (blue) to identify nuclei. Scale bar 100μm. F. Assessment of β-dystroglycan restoration in human fibers from wild type (CDMD 1002), out-of-frame (CDMD 1003), and reframed (CDMD 1003-49) MyoD OE cells engrafted into the TA of NSG-mdx mice. Engrafted human cells were identified by co-immunostaining for human spectrin and lamin A/C (shown in red). Positive staining for dystrophin is shown in grey and β-dystroglycan is shown in green. All sections were stained with DAPI (blue) to identify nuclei. Cell order is same as noted in E. Scale bar 20μm. See also Figure S4E–F.