Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells

Figure 1. Design and generation of AdVs encoding RNA-guided nuclease components. (a) Schematic representation of the genome structure of control and DMD-targeting AdVs. The genomes of dual RGN-encoding AdV.Cas9IN52.IN53 and AdV.Cas9IN43.IN54 are depicted in relation to those of single RGN-encoding AdV.Cas9EX51 and AdV.Cas9EX53. The AdV.gRNAS1 and AdV.Cas9, expressing exclusively an irrelevant gRNA and Cas9, respectively, served as controls. The AdV.Cas9EX51 and AdV.Cas9EX53 particles code for Cas9:gEX51 and Cas9:gEX53 complexes, respectively. The target sites of Cas9:gEX51 and Cas9:gEX53 complexes are located in exon 51 and exon 53 of the DMD locus, respectively. AdV.Cas9IN52.IN53 and AdV.Cas9IN43.IN54 deliver a pair of gRNA cassettes together with a Cas9 transcriptional unit. The gRNAs gIN52 and gIN53 address the Cas9 nuclease to DMD sequences in intron 52 and intron 53, respectively; the gRNAs gIN43 and gIN54 target the same nuclease to sequences located in intron 43 and intron 54, respectively. All vectors deployed in this study were isogenic in the sense that they were all assembled on the basis of a second-generation (i.e. E1- and E2A- deleted) AdV backbone coding for chimeric fibers…

Figure 2. “All-in-one” AdV transduction of paired RGNs for DMD repair via short-range intragenic deletions. (a) DMD repair through NHEJ-mediated exon 53 excisions in DMD.Δ45-52 muscle cells. Genomic deletions spanning exon 45 through exon 52 disrupt the DMD reading frame and yield out-of-frame mRNA transcripts carrying a premature stop codon within exon 53 (red box). RGN multiplexes consisting of Cas9 bound to intron-specific gIN52 and gIN53 gRNAs are designed to trigger the excision of a 1,123-bp DNA segment encompassing exon 53. This strategy is expected to induce the synthesis of a Becker-like dystrophin as splicing of exon 44 to exon 54 generate in-frame mRNA transcripts. gEX53, gRNA targeting Cas9 to a sequence within exon 53; SD, splice donor; SA, splice acceptor. Orange hexagons, CH1 and CH2 actin-binding domain 1; black hexagons, hinges; green boxes, spectrin-like repeats; orange oval, WW domain; blue pentagons, EFH1 and EFH2 hand-regions containing cysteine-rich motifs which, amongst other proteins, bind to β-dystroglycan; green pentagon, ZZ zinc finger domain; grey oval, C-terminal domain. Vertical arrowhead, protein junction formed by the DMD editing procedure. (b) qPCR analysis…

Figure 3. “All-in-one” AdV transduction of paired RGNs for DMD repair via long-range intragenic deletions. (a) DMD repair through NHEJ-mediated long-range, multi-exon, deletion. DMD-causing mutations located in the major DMD mutational hotspot (spanning exon 45 through exon 55) can, in principle, be tackled by a single multiplexing strategy based on the excision of a DNA segment encompassing exon 44 through exon 54. This multi-exon deletion strategy can build on the coordinated action of a pair of RGNs, consisting of Cas9 bound to the intron-specific gIN43 or gIN54 gRNAs. These RGNs are designed to repair the DMD reading frame as the joining of exon 43 to exon 55 is expected to yield in-frame mRNA species coding for an internally truncated Becker-like dystrophin. gEX51, gRNA targeting Cas9 to a sequence within exon 51. Vertical arrowhead, protein junction formed by the DMD editing procedure. For the naming of the dystrophin structural domains see legend of Fig. 2a. (b) qPCR analysis for establishing the frequencies of DMD multi-exon deletions. The qPCR assay was applied on genomic DNA from DMD.Δ48-50 and DMD.Δ45-52 myoblasts transduced with “all-in-one” vector AdV.Cas9IN43.IN54. The…

Figure 4. Dystrophin and β-dystroglycan immmunofluorescence microscopy on DMD-edited myotubes. Immunostainings for dystrophin and β-dystroglycan were carried out in myotubes differentiated from DMD.Δ48-50 and DMD.Δ45-52 myoblasts transduced with the AdV.Cas9IN43.IN54. Each numeral refers to multiplicities of infection (MOI). The same immunostainings were also done in myotubes differentiated from DMD.Δ48-50 myoblasts transduced with AdV.Cas9EX51 at the indicated MOI. Myotubes derived from DMD.Δ48-50 and DMD.Δ45-52 transduced with AdV.gRNAS1 at an MOI 12.5 IU/cell served as negative controls. Myotubes isolated from a healthy donor (WT) and transduced with AdV.gRNAS1 provided for positive controls.