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. 2023 Feb 21;4(1):011307. doi: 10.1063/5.0131452

TABLE I.

Summary of in vitro and in vivo CRISPR-based preclinical work for DMD and their prominent findings. N/A: not tested.

CRISPR in vitro experiment in DMD
Subject Cas enzyme type DMD target therapy Method Immune response Off-target activity/mutagenesis Notable finding Reference
Injection mdx zygote SpCas9 mRNA Exon 23 HDR-mediated exon correction N/A No off-target mutations detected Dystrophin expression is restored in skeletal and heart muscle in corrected progeny mice. 61
Cas12a mRNA Exon 23 Exon frameshifting, HDR-mediated correction N/A No off-target mutations detected Dystrophin expression is restored in skeletal muscle, heart muscle, and brain in corrected progeny mice. 201
Electroporation DMD patient-derived iPSCs myoblast SpCas9 plasmid Exon 44 and 45 Splice site mutation, Exon frameshifting Exon insertion N/A No off-target mutations detected Dystrophin expression is restored using all methods. 23
SaCas9 plasmid IMTR of UTRN 3′ UTR Transcriptional modulation N/A No off-target mutations detected Upregulation of utrophin at up-to twofold and restoration of DGC expression. 82
DMD patient-derived myoblast SpCas9 plasmid Exon 45–55 Exon deletion N/A Minimal off-target mutations detected Dystrophin expression is restored using multi-exon deletion in treated cells and in myofiber after in vivo transplantation in immunodeficient mice. 57
dCas9-VP160 plasmid UTRN A or B promoter Transcriptional modulation N/A No off-target mutations detected Upregulation of utrophin at up to three and sevenfold for UTRN A and B, respectively. 80
Nucleofection DMD patient-derived iPSCs myoblast SpCas9 plasmid Exon 45–55 Exon deletion N/A No off-target mutations detected Dystrophin expression is restored in reframed exon 58
DMD patient-derived fibroblast SpCas9 protein Exon 44 Exon frameshifting N/A No off-target mutations detected Dystrophin expression is restored in myotubes from fibroblast trans-differentiation. 261
DMD patient-derived iPSCs cardiomyocytes nCas9 ABE, PE plasmid Exon 52 Splice site mutation, Exon frameshifting N/A No off-target mutations detected Dystrophin expression is restored in cardiomyocytes after base editing and prime editing. PE edited cells can normalize contractile abnormality. 73
LNP DMD patient-derived myoblast SpCas9 Exon 50, Exon 54 Exon deletion N/A N/A Dystrophin expression is restored in corrected myotubes 55
mdx muscle-derived fibroblast SpCas9 Exon 23 HDR-mediated exon correction N/A N/A HDR correction of DMD mutations was performed on 1% of all cells using ssODN 137
DMD patient-derived iPSCs cardiomyocytes SaCas9-TAM plasmid Exon 50 Splice site mutation N/A No off-target mutations detected Dystrophin expression is restored in cardiomyocytes with 95% of the reads acquired the intended mutation. 74
GNP mdx primary muscle cells SpCas9 protein Exon 23 HDR-mediated exon correction N/A Minimal off-target mutations detected Dystrophin expression is restored in corrected myotubes. 165
VLP DMD patient-derived iPSCs myoblast SpCas9 protein Exon 45 Splice site mutation N/A Minimal off-target mutations detected Dystrophin expression is restored in treated cells. 175
AdV DMD patient-derived myoblast SpCas9 protein Exon 48–50, Exon 45–52 Exon deletion N/A N/A Dystrophin expression is restored in corrected myoblast. 56
Primary human myoblast SpCas9 plasmid Exon 51 Exon deletion N/A N/A Exon deletion is detected in dystrophin gene. 54
DMD patient-derived myoblast eSpCas9 plasmid Exon 48–50 Exon deletion N/A N/A Dystrophin expression is restored in targeted removal of mutational hotspots. 133
DMD patient-derived myoblast eSpCas9 plasmid Exon 51, Exon 53 Exon frameshifting N/A Additional NLS might reduce off-target activities Dystrophin expression is restored in corrected myotubes. 262
LV DMD patient-derived myoblast SpCas9 plasmid Intron 2 Duplicated exon deletion N/A No off-target mutations detected Dystrophin expression is restored about 7%–11% in western blot (WB) analysis. 59
CRISPR in vivo experiment in DMD
Subject Cas enzyme type DMD target therapy Method Immune/toxicity response Off-target activity/mutagenesis Length of study Systemic/local Notable finding Reference
Electro-poration mdx mice SpCas9 plasmid Exon 50, Exon 54 Exon deletion N/A N/A 7 days Local A large deletion of mutated exon in DMD gene (160 kb) leading to restoration of reading frame in skeletal muscle is achieved. 55
LNP hEx45KI-mdx44 SpCas9 mRNA Exon 44 Exon deletion Repeated IM administration did not elicit immune response and showed no toxicity signs No off-target mutations detected 14 days to 12 months Local Dystrophin expression in skeletal muscle is stable for a year and repeated injections result in cumulative benefits. 154
ΔE × 44 mice SpCas9 protein Exon 44 Exon deletion N/A No off-target mutations detected 6 weeks Local Dystrophin expression in skeletal muscle is restored about 4.2% in WB analysis. 155
Golden retriever muscular dystrophy (GRMD) SpCas9 plasmid Intron 6, Exon 7 Exon insertion N/A No off-target mutations detected 3 months Local Dystrophin expression in skeletal muscle is minimally restored in HDR-mediated treatment. 156
GNP mdx mice SpCas9 protein Exon 23 HDR-mediated exon correction No elevation in plasma cytokine is detected up to two weeks post-injection and after multiple injections Minimal off-target mutations detected 2 weeks Local An improvement in skeletal muscle function is seen with 5.4% restoration of dystrophin expression in WB analysis 165
VLP ΔE × 52 mice SpCas9 protein Exon 53 Exon frameshifting N/A N/A 1 week Local Dystrophin restoration is confirmed by IF. 174
AAV9 ΔE × 50 dog SpCas9 plasmid Exon 51 Exon frameshifting N/A (while high dose immune suppression was administered) No off-target mutations detected 3 weeks Local Dystrophin restoration up to 90% in immunohistochemical staining (IHC) is detected in skeletal and heart muscles. 263
mdx mice SpCas9 plasmid Exon 23 Exon deletion N/A No off-target mutations detected 3–12 weeks Local and systemic Dystrophin expression in skeletal and heart muscle is restored within range of 5%–25% based on IHC. 45
Exon frameshifting N/A Minimal change in off-target mutations detected after 18 months of treatment. 18 months Systemic Dystrophin expression is restored 20% in heart and 2% in skeletal muscle based on WB. 264
SpCas9, SaCas9 plasmid Exon 23 Exon deletion N/A Minimal off-target mutations detected 4 weeks Local and systemic Dystrophin expression is restored six in skeletal muscle and 1% in heart muscle after systemic treatment based on WB. 47
CjCas9 plasmid Exon 23 Exon frameshifting N/A No off-target mutations detected 8 weeks Local Dystrophin restoration up to 40% based on IHC and improvement in muscle function are observed. 265
SaCas9 plasmid Exon 23 Exon deletion N/A N/A 6 weeks Local and systemic AAV can transduce satellite cells population with a lesser degree of gene editing. 116
ΔE × 20 mice nCas9-ABE plasmid Exon 20 Base editor-mediated correction N/A No off-target mutations detected 8 weeks Local Dystrophin expression is restored 17% in skeletal muscle based on IF. 75
Dup 18–30 mice SaCas9 plasmid Exon 18–30 Duplicated exon removal N/A No off-target mutations detected 7 weeks Systemic Dystrophin restoration ranging from 12% to 68% in skeletal muscle and 22%–36% in heart based on IF. 266
Patient derived xenograft DMD mouse Cas9, Cas12a plasmids Exon 46–54 Exon deletion N/A Minimal off-target mutations detected in both Cas proteins. Cas12a have fewer off-target sites with similar efficiency 30 days Local Dystrophin expression is restored more than 10% in skeletal muscle based on IF in both Cas9 and Cas12. 200
ΔE × 44 mice SpCas9 plasmid Exon 42–46 Exon deletion N/A No off-target mutations detected 4 weeks Local, Systemic Dystrophin expression is restored more than 90% in skeletal heart muscle based on IF with muscle improvement. 267
Exon 45 Splice site mutation, Exon frameshifting N/A N/A 4 weeks Systemic Dystrophin expression is restored ranging from 50% to 70% in skeletal and heart muscle based on WB. 268
Exon 45 Exon frameshifting Upregulation of genes related to immune system are detected in treated mice. Minimal change in off-target mutations detected after 18 months of treatment. No off-target and minimal on-target AAV integration are observed. 18 months Systemic Dystrophin expression is restored more than 40% in skeletal muscle based on WB 269
ΔE × 50 mice SpCas9 plasmid Exon 51 Splice site mutation N/A No off-target mutations detected 4–8 weeks Local, systemic Dystrophin expression is restored up to 90% in skeletal muscle and heart based on IHC with improvement in muscle function. 63
SaCas9 plasmid Exon 51 Exon frameshifting N/A No off-target mutations detected 4 weeks Systemic Dystrophin expression is restored up to 40% in skeletal and heart muscle based on WB with muscle function improvement. 270
ΔE × 52 mice SaCas9 plasmid Exon 47, Exon 58 Exon deletion N/A No off-target mutations detected 6 weeks Systemic Dystrophin expression is confirmed in heart based on WB. 128
Exon 52, Exon 52–79 Exon insertion N/A Minimal off-target mutations detected 8 weeks Local, systemic Full-length dystrophin expression is observed by WB. 178
ΔE × 43 mice, ΔE × 45 mice, ΔE × 52 mice SpCas9 plasmid Exon 53, Exon 44 Exon deletion, Exon frameshifting N/A No off-target mutations detected 3 weeks Local Dystrophin expression is restored ranging from 14% to 50% based on WB in all type of mice. 271
ΔE × 52 pig SpCas9 plasmid Exon 51 Exon deletion N/A No off-target effects are detected at systemic dose 6 weeks Local, systemic Dystrophin expression and function are restored in skeletal muscle. Survival rate and cardiac improvement are observed after treatment. 272
AAV8 mdx mice SaCas9 plasmid Exon 23 Exon deletion N/A Minimal off-target mutations detected 8 weeks Local Dystrophin expression is restored up to 8% based on WB with muscle function improvement. 46
Immune responses against SaCas9 nuclease are detected in adult but not in newborn dystrophic mice Minimal change in off-target mutations after 1 year treatment. Unintended vector integrations are detected in target sites. 12 months Local, systemic A decrease in dystrophin staining after local administration and an increase after systemic treatment. 194
AAV6 mdx4cv mice SpCas9, SaCas9 plasmids Exon 52–53 HDR-mediated correction N/A Minimal off-target mutations detected 4 weeks Local Dystrophin expression is restored up to 23% in skeletal muscle based on WB. 62
AdV mdx mice SpCas9 plasmid Exon 21–23 Exon deletion N/A N/A 3 weeks Local Dystrophin expression is restored up to 50% in skeletal muscle based on WB. 136
Exon 23 HDR-mediate exon correction N/A N/A 4 weeks Local Up to 8% dystrophin restoration in skeletal muscle after transplantation of corrected muscle stem cells. 137