Table 2.
Summary of studies that have used CRISPR/Cas9 approaches for the treatment of DMD.
| Cas Enzyme | Strategy | Target Gene Region(s) | Model(s) | Delivery | Study Highlights | Reference |
|---|---|---|---|---|---|---|
| SpCas9 | NHEJ reframing, HDR exon correction | Dmd exon 23 | mdx mice | 1-cell embryo injection | Dystrophin restoration observed by IHC (up to 100%) and WB; 17% Dmd HDR correction resulted in 47–60% dystrophin-positive fibers in skeletal muscles and the heart | 2014 Long et al. [43] |
| SpCas9 | NHEJ reframing, exon skipping, HDR exon knock-in | DMD intron 44/exon 45 | DMD hiPSCs, hiPSC-derived skeletal muscle cells (ex44 del.) | Electroporation | Dystrophin restoration in derived skeletal muscle cells observed by WB and IHC for all strategies; CRISPR was as effective as using TALEN | 2015 Li et al. [41] |
| SpCas9 | NHEJ reframing, single/multiple exon deletion | DMD exons 45–55 (for reframing each exon), introns 50 and 51 (ex51 del.), introns 44 and 55 (ex45–55 del.) | immortalized DMD patient muscle cells (ex48–50 del.), immunodeficient NSG mice | Electroporation | Generated targeted deletions of exon/s in vitro, particularly of the large exon 45–55 region which led to dystrophin rescue by WB; mice transplanted with treated myoblasts (exon 51-deleted) showed dystrophin-positive fibers by IHC | 2015 Ousterout et al. [21] |
| dSpCas9-VP16 | Utrophin upregulation | UTRN A/B promoter | immortalized DMD patient muscle cells (ex45–52 del.) | Electroporation | 1.7–6.9-fold upregulation of utrophin achieved; restored β-dystroglycan expression observed by WB with as little as 1.7-fold upregulation | 2016 Wojtal et al. [44] |
| SpCas9 | Duplicated exons removal | DMD intron 27 | primary DMD patient fibroblasts (ex18–30 dup.) | LV transduction, with Adeno-MyoD | 4.42% full-length dystrophin production achieved post-treatment, accompanied with α-dystroglycan restoration | 2016 Wojtal et al. [44] |
| SpCas9 | Single exon deletion | Dmd exon 23, introns 22 and 23 (ex23 del.) | mdx mice | AAV9 delivery (i.m., i.p., i.v.) | All modes of injection led to appearance of dystrophin-positive fibers as evaluated by IHC: ~25.5% 6 wks post-i.m., ~4.6% and ~9.6% in skeletal and cardiac muscles respectively 12 wks post-i.v., ~1.8% and ~3.2% in skeletal and cardiac muscles respectively 8 wks post-i.p. | 2016 Long et al. [45] |
| SaCas9 | Single exon deletion | Dmd introns 22 and 23 (ex23 del.) | mdx mice | AAV8 delivery (i.m., i.p., i.v.) | Intramuscular injections led to ~59% of transcripts with exon 23 deleted, which restored about 8% dystrophin of healthy levels by WB, proper relocalization of DGC proteins, and muscle function improvement; systemic injections restored dystrophin production in the heart and skeletal muscles | 2016 Nelson et al. [46] |
| SpCas9, SaCas9 | Single exon deletion | Dmd introns 22 and 23 (ex23 del.) | mdx mice, mdx satellite cells | AAV9 delivery (i.m., i.p., i.v.) | Dual-vector (Cas9 and gRNAs on separate constructs) had higher cutting efficiency than a single-vector system (Cas9 and gRNAs on the same construct) in vitro; dystrophin restoration >10% observed in the heart and skeletal muscles upon systemic treatment; correction also possible in satellite cells | 2016 Tabebordbar et al. [47] |
| SpCas9 | Hybrid exon formation via internal exon deletion | DMD exons 50 and 54 | immortalized DMD patient muscle cells (ex51–53 del.), hDMD/mdx mice | Lipotransfection (in vitro)/ electroporation (in vivo) | Dystrophin restoration successful in vitro by WB, not shown in vivo; hybrid exon formation thought to preserve dystrophin rod domain structure better | 2016 Iyombe-Engembe et al. [48] |
| SpCas9 | NHEJ reframing, single/multiple exon deletion | DMD exons 51, 53, introns 52 and 53 (ex53 del.), 43 and 54 (ex44–54 del.) | immortalized DMD patient muscle cells (ex48–50, or 45–52 del.) | Sequential LV then AdV transduction/AdV transduction | Study showed the possibility of combining both TALEN and CRISPR approaches in one gene editing strategy; also, comparable editing was obtained with Cas9 and gRNA delivered either together or separately in AdV | 2016 Maggio et al. [49] |
| SpCas9 | Multiple exon deletion | Dmd introns 20 and 23 (ex21–23 del.) | mdx mice | Electroporation/AdV transduction | Treatment restored proper calcium dynamics in muscle (electroporation), and restored dystrophin to 50% of wild-type levels, as well as dystrophin-associated complex sarcolemmal localization and muscle membrane integrity (transduction) | 2016 Xu et al. [50] |
| SpCas9 | Multiple exon deletion | DMD introns 44 and 55 (ex45–55 del.) | DMD hiPSCs, hiPSC-derived skeletal and cardiac muscle cells (ex46–51 or 46–47 del., ex50 dup.), immunodeficient NSG-mdx mice | Nucleofection | CRISPR-mediated deletion of the large exon 45–55 region achieved, restored membrane function and dystrophin, β-dystroglycan expression by WB and IHC; mice transplanted with hiPSC-derived skeletal muscle cells showed dystrophin-positive fibers by IHC | 2016 Young et al. [42] |
| SpCas9 | NHEJ reframing, single/multiple exon deletion | DMD exons 51, 53 (for reframing) introns 52 and 53 (ex53 del.), introns 43 and 54 (ex44–54 del.) | immortalized DMD patient muscle cells (ex48–50, or 45–52 del.) | AdV transduction | AdV with 2gRNA-SpCas9 constructs work as good as those with 1gRNA-SpCas9 constructs in terms of corrective ability and dystrophin restoration | 2016 Maggio et al. [51] |
| SpCas9, SaCas9 | Multiple exon deletion, HDR exon correction | Dmd exon 53, introns 51 and 53 (ex52–53 del.) | mdx4cv mice (nonsense ex53 mutation) | AAV6 delivery (i.m., i.v.) | Dual vector approach (SpCas9 and gRNA separate) yielded higher correction efficiency than single vector approach (SaCas9 and gRNA together); systemic treatment restored dystrophin expression in the heart (~34% dystrophin-positive fibers) and skeletal muscles (~10–50% dystrophin-positive fibers) | 2017 Bengtsson et al. [52] |
| LbCpf1, AsCpf1 | NHEJ reframing, single exon skipping, HDR exon correction | DMD exon 51, intron 50 | DMD hiPSCs, hiPSC-derived cardiac muscle cells (ex48–50 del.), mdx mice | Nucleofection (in vitro)/ 1-cell embryo injection (in vivo) | Cpf1 editing successfully restored dystrophin expression and improved mitochondrial function in cardiomyocytes; 5/24 pups (injected at the embryo stage) showed HDR correction and had ameliorated dystrophic phenotypes | 2017 Zhang et al. [53] |
| SpCas9 | Duplicated exon removal | DMD exon 2, intron 2 | immortalized DMD patient muscle cells (ex2 dup.) | PEI transfection/LV transduction | Use of a single gRNA can delete a duplicated exon, resulting in slight dystrophin rescue by WB and IHC | 2017 Lattanzi et al. [54] |
| SpCas9 | HDR exon correction | Dmd exon 23 | mdx mice, mdx satellite cells | Lipotransfection (template, gRNA), AdV transduction (Cas9)/AdV transduction | Higher transduction efficiency obtained when AdVs were used for both Cas9 and gRNA-HDR template delivery; mice transplanted with corrected satellite cells showed dystrophin-positive fibers by IHC | 2017 Zhu et al. [55] |
| SpCas9 | Multiple exon deletion | DMD introns 44 and 55 (ex45–55 del.) | humanized mdx mice with DMD exon 45 del. | Electroporation | Exon 45–55 deletion by CRISPR possible in vivo; first use of the humanized DMD mouse model with exon 45 del. for CRISPR studies | 2017 Young et al. [56] |
| SpCas9 | Multiple exon deletion | DMD introns 2 and 7 (ex3–9 del.), introns 5 and 7 (ex6–7 del.), introns 6 and 11 (ex7–11 del.) | DMD hiPSCs, hiPSC-derived cardiac muscle cells (ex8–9 or ex3–7 del.) | Nucleofection | Dystrophin with ex7–11 del. showed the least functionality, while those with ex3–9 del. had the highest functionality in terms of assessing iPSC-derived cardiomyocyte calcium cycling | 2017 Kyrychenko et al. [57] |
| SpCas9 | HDR correction | Dmd exon 23 | mdx primary muscle cells, mdx mice | CRISPR-Gold nanoparticles (i.m.) | 5.4% HDR correction of the Dmd mutation in mdx was observed after CRISPR treatment and cardiotoxin injection, dystrophin-positive fibers found by IHC; 0.8% HDR correction observed without cardiotoxin co-injection, which led to significantly improved hanging test performance | 2017 Lee et al. [58] |
| SpCas9 | NHEJ reframing, single exon skipping | Dmd exon 51 | mice with Dmd exon 50 del. | AAV9 delivery (i.m., i.p.) | Successful dystrophin restoration in the heart and skeletal muscles; systemic injections led to improved muscle function; first application of CRISPR in the ex50 del. mouse model | 2017 Amoasii et al. [59] |
| SpCas9 | Single exon deletion | Dmd introns 50 and 51 (ex51 del.) | primary human skeletal muscle cells | HCAdV delivery | Up to 93.3% exon 51 deletion observed in vitro upon delivery of CRISPR agents by HCAdV | 2017 Ehrke-Schulz et al. [60] |
| SpCas9 | NHEJ reframing, exon skipping | DMD exon 51, introns 47, 50, 54 | DMD hiPSCs, hiPSC-derived cardiac muscle cells (ex48–50 del., pseudo-ex47, ex55–59 dup.) | Nucleofection | All strategies corrected the respective patient mutations and restored dystrophin production in iPSC-derived cardiomyocytes; 3D-engineered heart muscle produced from treated iPSC-derived cardiomyocytes showed improved contractile force | 2018 Long et al. [61] |
| CjCas9 | NHEJ reframing | Dmd exon 23 | mice with deletions in Dmd exon 23 | AAV9 delivery (i.m.) | CjCas9 displayed higher targeting specificity than SpCas9; use of CjCas9-based CRISPR can lead to successful dystrophin restoration and improvement in muscle function as well | 2018 Koo et al. [62] |
| SaCas9 | Hybrid exon formation via multiple exon deletion | DMD exons 47 and 58 | DMD skeletal muscle cells (ex51–53 del., ex49–50 del., ex51–56 del., ex50–52 del.), humanized mdx mice with DMD ex52 del. | LV transduction (in vitro)/AAV9 delivery (in vivo; i.v.) | gRNAs designed to produce exon deletions that best preserved dystrophin protein structure were able to show dystrophin restoration in vitro and in vivo (slight rescue in the heart) | 2018 Duchêne et al. [63] |
| SpCas9 | NHEJ reframing, exon skipping | Dystrophin exon 51 | deltaE50-MD canine model (ex50 del.) | AAV9 delivery (i.m., i.v.) | First published study on dystrophin gene correction in a dog model; ~3–70% dystrophin restoration of healthy levels in skeletal muscles and ~92% in the heart found by WB | 2018 Amoasii et al. [64] |
| nSpCas9-ABE7.10 | Base editing to correct a nonsense mutation | Dmd exon 20 | mice with a nonsense mutation in Dmd exon 20 | trans-splicing AAV2/9 delivery (i.m.) | ~3.3% base editing frequency achieved 8 weeks post-treatment with no detectable off-target effects; ~17% dystrophin-positive fibers and restored localization of nNOS observed by IHC | 2018 Ryu et al. [65] |
| dSa/SpCas9-TAM | Base editing to induce exon skipping | DMD intron 50 5′ splice site | DMD hiPSCs, hiPSC-derived cardiac muscle cells (ex51 del.) | Lipotransfection | ~100% base editing efficiency achieved; corrected iPSC-derived cardiomyocytes had restored dystrophin protein, low CK and miR-31 levels, and restoration of β-dystroglycan expression | 2018 Yuan et al. [66] |
Abbreviations: NHEJ, non-homologous end joining; HDR, homology-directed repair; ex, exon; NSG, NOD scid IL2R gamma; hiPSC, human induced pluripotent stem cells; LV, lentivirus; AAV, adeno-associated virus; AdV, adenovirus; HCAdV, high-capacity adenoviral vector; PEI, polyethyleneimine; i.m., intramuscular; i.p., intraperitoneal; i.v., intravenous; WB, Western blot; IHC, immunohistochemistry; TALEN, transcription activator-like effector nuclease; nNOS, neuronal nitric oxide synthase; CK, creatine kinase; gRNA, guide RNA.