Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3′ UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3′ UTR is a viable approach to develop therapeutic genome editing for DM1.

Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses DMPK Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts

“…Indeed, the removal of the CTG repeat from DMPK using CRISPR/Cas9 has been reported in myoblasts from DM1 patients (27, 28) and trans-differentiated patient fibroblasts (29), DM1 transgenic mice (27), patient-derived induced pluripotent stem cells (iPSCs) and their myogenic cell derivatives (24, 28, 29). These efforts generally restored myogenic capacity and reversed nuclear foci formation, splicing alteration and RNA-binding protein distribution in the corrected cells, providing new therapeutic opportunities for ameliorating disease pathology.…”

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond

“…One strategy for applying gene editing to these types of repeat expansion diseases is to remove the repeat region in the genomic DNA through NHEJ. Multiple groups have demonstrated effective deletion using gRNAs that flank the repeat region in DM1 patient cells (3,32,105,131). However, this deletion was not always precise, since different types of events occurred, including partial deletions, inversions, or a single cut inducing loss of the entire repeat.…”

“…Here, a polyadenylation (polyA) signal template is integrated before the repeat expansion. This termination signal prevented the repeat from being expressed in the RNA, thus inhibiting its toxic effects (131).…”

“…Using two vectors for one platform may effectively halve the effective dose. SaCas9 is an alternative, smaller nuclease that can be packaged along with a gRNA into a single AAV and has been successfully applied in some models of NMDs (15,36,59,60,97,98,104,110,122,131). However, a higher percentage of the population is estimated to have pre-existing immunity to SaCas9 compared with SpCas9 (23,119).…”

Targeting Toxic Nuclear RNA Foci with CRISPR-Cas13 to Treat Myotonic Dystrophy

“…Genome editing approaches using CRISPR-Cas9 DNA endonucleases have been employed to directly edit the DMPK gene locus. However, CRISPR-Cas9 editing at either the 5' or 3' ends of CTG genomic repeats can induce large and uncontrolled sequence deletions, and the use of double guide-RNAs flanking the repeat expansion can lead to frequent sequence inversions, which remain toxic 20,21 . Since CRISPR-Cas9 approaches for manipulating DNA remain inefficient and controversial due to the risk of germline editing, an alternative approach targeting the repeat RNAs using deactivated Cas9 (dCas9) fused to an active ribonuclease has recently been shown to be effective in cells 22 .…”