Homology-Independent Targeted Integration-mediated Gene Segment Replacement for Correction of DMD Mutations
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Date
2020-05
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Publisher
The Ohio State University
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder which causes muscle degeneration leading to loss of ambulation, cardiac dysfunction, respiratory complications, and premature death. The DMD gene is the largest gene within the human genome, and thus has a diverse mutational profile which prevents many corrective therapies from reaching a large patient cohort. My goal was to determine the feasibility of a new CRISPR/Cas9-based gene editing technique known as Homology Independent Targeted Integration (HITI) in replacing segments of the DMD gene. By replacing large segments of the DMD gene, a larger patient cohort could potentially be encompassed by a single therapy. As a proof of principle, I tested whether small (~1 kb) and medium sized (~175 kb) replacements were feasible at the 5สน end of the DMD gene using a HITI-mediated replacement with previously validated Cas9 guide RNAs (gRNAs). Genomic DNA PCR was used to detect the expected knock-in while sequencing confirmed that the integration was seamless with no insertion or deletions (indels). I then optimized this system by altering the molecular ratios of the two HITI gene editing components (CRISPR/Cas9 and HITI donor DNA plasmid) and found that a 1:1 ratio was optimal. Based on these studies, small and medium HITI replacements are feasible within the DMD gene, thus a large HITI replacement (~715 kb) of DMD exons 41-55 (which would potentially correct ~37% of patients) was designed. This consisted of two plasmids encoding i) Cas9 with a GFP reporter and ii) a HITI donor DNA with the coding sequence (CDS) of DMD exons 41-55, two gRNAs, and an RFP reporter. Knock in of the CDS of DMD exons 41-55 was detected and analyzed by genomic DNA PCR and Sanger sequencing. As with the proof-of-concept studies, the DMD exon 41-55 CDS was seamlessly integrated in place of the natural DMD exon 41-55 locus. These studies lay the groundwork for development of a HITI-mediated gene correction therapy that could potentially restore full-length dystrophin in a large patient cohort.
Description
Distinction at the Nationwide Children's Hospital Research Retreat Poster Presentation
Keywords
Gene Therapy, Gene Editing, CRISPR/Cas9, Duchenne Muscular Dystrophy