Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Ohio State University Wexner Medical Center

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD. 

CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.

Anoctamin 5 (ANO5) is a member of a conserved gene family (TMEM16), which codes for proteins predicted to have eight transmembrane domains and putative Ca(2+)-activated chloride channel (CaCC) activity. It was recently reported that mutations in this gene result in the development of limb girdle muscular dystrophy type 2L (LGMD2L), Miyoshi myopathy type 3 (MMD3), or gnathodiaphyseal dysplasia 1 (GDD1). Currently, there is a lack of animal models for the study of the physiological function of Ano5 and the disease pathology in its absence.

Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy.

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in  mice after a single systemic delivery of recombinant adeno-associated virus.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.

Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb-girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti-Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N- and C-terminus, respectively. The monoclonal antibody (mAb) N421A/85 was found to specifically detect human Ano5 by immunoblotting and immunofluorescence staining. The antigen epitope was mapped to a region of 28 residues within the N-terminus. Immunofluorescence staining of muscle cryosections from healthy control subjects showed that Ano5 is localized at the sarcoplasmic reticulum. The muscle biopsy from a LGMD2L patient homozygous for the c.191dupA mutation showed no Ano5 signal, confirming the specificity of the N421A/85 antibody. Surprisingly, strong Ano5 signal was detected in a patient with compound heterozygous mutations (c.191dupA and a novel splice donor site variant c.363 + 4A > G at the exon 6-intron 6 junction). Interestingly, insertion of the mutant intron 6, but not the wild-type intron 6, into human ANO5 cDNA resulted in a major transcript that carried the first 158-bp of intron 6. Transfection of the construct encoding the first 121 amino acids into C2C12 cells resulted in protein aggregate formation, suggesting that aggregate-forming Ano5 peptide may contribute to the pathogenesis of muscular dystrophy.

A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation-prone Ano5 peptide.

Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5 (ANO5), which belongs to the anoctamin protein family. Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes; instead, one of these mice was observed to present with some abnormality in sperm motility. In contrast, a third line of ANO5-knockout (KO) mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology. Many of the ANO5-related patients carry point mutations or small insertions/deletions (indels) in the ANO5 gene. To more closely mimic the human ANO5 mutations, we engineered mutant ANO5 rabbits via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.

Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births.  mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The  knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies.This article has an associated First Person interview with the first author of the paper.

Yandi Gao

Department of Surgery,
Davis Heart and Lung Research Institute,
Biomedical Sciences Graduate Program,
Biophysics Graduate Program,
Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program

Ohio State University Wexner Medical Center

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice