Genome Editing using Custom Endonucleases and Dystrophin cDNA Insertions Targeting Intron 1 of the Duchenne Muscular Dystrophy Gene

Marc Moore

Research output: ThesisDoctoral Thesis

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Duchenne muscular dystrophy (DMD) is a hereditary X-linked neuromuscular disease resulting from mutations across the DMD gene. The subsequent absence of the encoded dystrophin protein prevents the correct formation of the dystrophin-associated protein complex (DAPC), a structural link between the sarcolemmal actin cytoskeleton and the myofibre extracellular matrix. Dystrophin deficiency compromises myofibre stability giving rise to progressive muscle wasting, the prominent pathological feature of this disease. Over time, muscle deterioration results in loss of ambulation and upper body movement, decline in respiratory and cardiac function and ultimately premature death in the third decade of life. Current treatment is limited to corticosteroid use, which delays clinical manifestations but does not address the underlying aetiology of the disease. In addition, many therapies currently under development address specific mutation types or are reliant upon repeated administration; this serves to reduce their applicability to patients.
Genome editing refers to modification of genetic material at a precise genomic locus using special enzymes called customisable endonucleases. Recently, interest in genome editing approaches has increased with the advent of the clustered regularly interspaced palindromic repeats - Cas9 system (CRISPR/Cas9), in which an RNA guide can be used to direct the Cas9 nuclease to cleave the genome at a specific sequence.
In the present study a genome editing strategy was designed and evaluated to restore functional dystrophin expression from the endogenous DMD gene. The approach involved modifying intron 1 of the locus to insert dystrophin cDNAs downstream of the endogenous full-length dystrophin promoter and exon 1 elements, and was developed and verified in HEK293T cell and human DMD patient myoblast cultures. The approach was developed to permit full-length, mini-dystrophin and microdystrophin cDNA insertions to be examined. Specific CRISPR/Cas9 and guide RNAs (gRNAs) were designed and assessed for their ability to introduce double strand breaks (DSBs) at the DMD intron 1 locus. An exogenous DNA repair template construct, targeted to the DSB region in intron 1 was designed and further modified to contain a microdystrophin coding cDNA from DMD exon 2 onwards
Co-delivery of CRISPR/Cas9, gRNA and exogenous repair template constituents in HEK293T cells, resulted in specific integration of the microdystrophin cDNA at the DMD intron 1 site. This was confirmed at genomic, transcriptomic and protein levels in polyclonal and zeocin-selected clonal cell populations. Application of the genome editing system in human DMD myoblasts also gave rise to evidence of specific microdystrophin cDNA integration, although further refinement of the methodology is required to ensure myoblast survival following positive selection with the zeocin antibiotic.
The research reported demonstrates the potential of genome editing the 5’ end of the DMD gene and offers a strategy towards permanent corrective gene therapy applicable to almost all DMD patient genotypes.
Original languageEnglish
Awarding Institution
  • Royal Holloway, University of London
  • Dickson, George, Supervisor
  • Popplewell, Linda, Supervisor
  • Yáñez-Muñoz, Rafael J., Supervisor
  • Chen, Philip, Advisor
Thesis sponsors
Award date1 Jul 2020
Publication statusUnpublished - 2018


  • CRISPR-Cas
  • Duchenne muscular dystrophy
  • DMD
  • Gene editing
  • Homology Directed Repair
  • Gene Therapy
  • Myoblasts
  • HEK293T

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