A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation

Figures

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  Figure 1. Strategies for CRISPR KKH SaCas9-mediated gene editing of human DMD exon 51 (A) An out-of-frame deletion of human DMD exons 48 to 50 (ΔEx48–50) results in splicing of exons 47 to 51, generating a premature termination codon in exon 51. A single-cut editing strategy was designed to enable CRISPR-KKH SaCas9 DNA cutting to restore the open reading frame of the DMD gene. Small insertions and deletions (indels) with two nucleotide deletions (3n-2) can reframe exon 51. Large indels disrupting the 5′-AG-3′ splice acceptor sequence cause exon 51 skipping, resulting in splicing of exon 47 to exon 52. (B) Illustration of the sgRNA targeting human DMD exon 51. This sgRNA recognizes a 5′-AACAGT-3′ PAM in exon 51 and generates a DSB 4 bp upstream of the 5′-TGA-3′ premature termination sequence (indicated in red). The 5′-AG-3′ splice acceptor sequence is indicated in yellow. (C) Illustration of a plasmid-encoding KKH SaCas9 with 2A-GFP, driven by a hybrid form of cytomegalovirus and chicken beta-actin promoter (CBh). The plasmid also encodes a sgRNA driven by the U6 promoter. Cells transfected with this plasmid express GFP, allowing for selection of KKH SaCas9-expressing cells by FACS. (D)…

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  Figure 2. Restoration of dystrophin expression in DMD ΔEx48–50 cardiomyocytes after CRISPR-KKH SaCas9-mediated single-cut gene editing (A) DMD ΔEx48–50 iPSCs were edited by KKH SaCas9 (corrected DMD iPSCs) and then differentiated into corrected cardiomyocytes (CMs) for downstream analysis. (B) Immunocytochemistry shows dystrophin restoration in mixtures of DMD ΔEx48–50 CMs following KKH SaCas9-mediated single-cut gene editing. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm. (C) Western blot shows dystrophin restoration in mixtures of DMD ΔEx48–50 CMs following KKH SaCas9-mediated single-cut gene editing. Dilutions of protein extract from healthy control CMs were used to standardize dystrophin protein expression. Vinculin was used as the loading control. (D) Representative traces of spontaneous calcium activity of iPSC-derived CMs cultured with calcium indicator Fluo-4AM. Traces show change in fluorescence intensity (F) in relationship to resting fluorescence intensity (Fo). (E) Quantification of calcium release phase of contraction, as measured by time to peak, in iPSC-derived CMs. Data are represented as mean ± SEM. One-way ANOVA was performed with post hoc…

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  Figure 3. Systemic delivery of All-In-One AAV-packaged KKH SaCas9 restores dystrophin expression in ΔEx50 mice (A) Illustration of the All-In-One AAV vector used to deliver KKH SaCas9 gene-editing components. KKH SaCas9 expression is driven by a muscle-specific CK8 promoter. Two copies of the same sgRNA targeting mouse Dmd exon 51 are driven by two RNA polymerase III promoters, 7SK and U6. (B) Illustration of systemic delivery of All-In-One AAV vectors in ΔEx50 mice. Postnatal day 4 ΔEx50 mice were injected intraperitoneally with 2 × 1014 or 4 × 1014 vg/kg of All-In-One AAV vectors. Four weeks after systemic delivery, ΔEx50 mice and WT littermates were dissected for analysis. (C) Immunohistochemistry shows restoration of dystrophin in the tibialis anterior (TA), triceps, diaphragm, and heart of ΔEx50 mice 4 weeks after systemic delivery of AAV-packaged KKH SaCas9 and sgRNA. Dystrophin is shown in green. n = 6 for each muscle group. Scale bars, 100 μm.

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  Figure 4. Western blot and genomic analysis of skeletal muscles and heart of ΔEx50 mice receiving systemic All-In-One AAV delivery of KKH SaCas9 gene-editing components (A) Western blot analysis shows restoration of dystrophin expression in the TA, triceps, diaphragm, and heart of ΔEx50 mice 4 weeks after systemic delivery of All-In-One AAV-packaged KKH SaCas9 and sgRNA. The dose of the AAV vector is indicated. Dilutions of protein extract from WT mice were used to standardize dystrophin protein expression. Vinculin was used as the loading control (n = 3 for each dose). (B) Quantification of dystrophin expression in the TA, triceps, diaphragm, and heart. Relative dystrophin intensity was calibrated with vinculin internal control before normalizing to the WT control. (C) Genomic indel quantification by deep-sequencing analysis of the TA, triceps, diaphragm, and heart of ΔEx50 mice 4 weeks after systemic delivery of All-In-One AAV-packaged KKH SaCas9 and sgRNA. Data are represented as mean ± SEM.

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  Figure 5. Systemic delivery of All-In-One AAV-packaged CRISPR-KKH SaCas9 improves muscle function in ΔEx50 mice (A and B) Specific force (mN/mm2) of the soleus (A) and extensor digitorum longus (EDL) (B) in WT, ΔEx50 mice untreated, and ΔEx50 mice treated with All-In-One AAV-packaged KKH SaCas9. Data are represented as mean ± SEM. Brown-Forsythe and Welch ANOVA test was performed. ∗p < 0.05, ∗∗p < 0.005 (n = 6). (C and D) Maximal tetanic force of the soleus (C) and EDL (D) in WT, ΔEx50 mice untreated, and ΔEx50 mice treated with All-In-One AAV-packaged KKH SaCas9. (E and F) Fatigue resistance analysis of soleus (E) and EDL (F) in WT, ΔEx50 mice untreated, and ΔEx50 mice treated with All-In-One AAV-packaged KKH SaCas9. Data are represented as mean ± SEM. Nonlinear regression with extra sum-of-squares F test was performed (n = 6).