Time-controlled and muscle-specific CRISPR/Cas9-mediated deletion of CTG-repeat expansion in the DMPK gene

Abstract

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3′ untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo .

Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple disease loci

“…Unexpectedly, this did not lead to higher levels of editing compared to a two-vector system 17,18 . Therefore, the size of the Cas9 enzyme and its delivery in the brain remain important challenges.…”

“…This is an issue because greater quantities of viral vectors must be injected to ensure that both AAVs infect the same cells. One way around this is to use smaller Cas9 orthologues, for example the Cas9 from S. aureus 59 , as has been done in vivo for the DMPK locus 17,18 . Unexpectedly, this did not lead to higher levels of editing compared to a two-vector system 17,18 .…”

“…One way around this is to use smaller Cas9 orthologues, for example the Cas9 from S. aureus 59 , as has been done in vivo for the DMPK locus 17,18 . Unexpectedly, this did not lead to higher levels of editing compared to a two-vector system 17,18 . Therefore, the size of the Cas9 enzyme and its delivery in the brain remain important challenges.…”

The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease

“…At the DNA level, another approach could be a CRISPR/Cas9-based therapy for genetic engineering to restore the wild-type repeat number of the polymorphic regions, a method that is already developed for other repeat-associated diseases like Huntington’s disease ( 225 – 227 ), Duchenne muscular dystrophy ( 228 ), myotonic dystrophy type-1 ( 229 , 230 ), spinocerebellar ataxia type-3 ( 231 ), Friedreich’s ataxia ( 231 , 232 ) and amyotrophic lateral sclerosis ( 233 ). Off-target effects of this technology has been initially a challenge, but there are intensive efforts on reducing it, e.g., by dual CRISPR/Cas9 technology ( 234 ).…”

A Selective Alkylating Agent for CTG Repeats in Myotonic Dystrophy Type 1

“…DM1 therapeutic efforts have been focused largely on inhibition of the undesired interactions between r­(CUG) exp and MBNL1, because over 80% of DM1 spliceopathy is related to MBNL1 sequestration. , However, it has been suggested that this narrow targeting neglects the contribution of pathogenic pathways other than those involving MBNL1 . Therefore, new approaches have been developed to reduce the toxic load of r­(CUG) exp through its degradation using RNase H-mediated antisense oligonucleotide targeting or small molecules with RNase-like activity. − Another strategy is to use transcription inhibitors that selectively target d­(CTG) exp repeats thereby reducing the amount of toxic r­(CUG) exp generated. − With the rapid progress in CRISPR/Cas9-mediated gene editing, it has been possible to delete the expanded CTG repeats in DM1 patient cells; however, delivery issues and the introduction of unintended mutations remain a challenge prior to clinical development. − …”