A Novel Genome Editing Strategy for DM1

Published on Tue, 12/11/2018

Focusing on Genome Editing for DM1

MDF has reported out on a recently held workshop that focused on the opportunities and challenges of genome editing for DM1. The feedback received from that workshop went into the design of a Request for Applications, where MDF challenged academic investigators for innovative genome editing approaches to DM1. Applications from the RFA are now under review and award announcements are expected in early 2019. MDF continues to monitor new developments in genome editing technology in general, as well as the development of targeted strategies that are potentially applicable for DM1.

Disrupting Expanded CUG Repeats in DMPK Transcripts

There have now been several published studies that followed the strategy of removing the expanded CTG tract from DMPK. One concern that has emerged is the potential for increasing the repeat instability already inherent in pathological length CTG expansions (van Agtmaal et al., 2017). These authors did demonstrate that dual CRISPR/Cas9-cleavage at either side of the expanded repeat tract could achieve efficient editing and correct biomarkers of DM in DM1 patient myoblasts and in a mouse model. But, dual cuts can also produce a low rate of inversions at the targeted genomic locus. These earlier findings suggest that translation of a dual cleavage approach to DM1 patients would have to address both genome editing reagent delivery and inversion rate issues.

Dr. Guangbin Xia (University of New Mexico) and colleagues recently compared two distinct genome editing strategies for feasibility in addressing DM1: (1) the “traditional” targeted deletion of the expanded CTG repeats in the DMPK gene and (2) a novel targeted insertion of a polyadenylation signal in the 3’ UTR to block formation of toxic CUG expanded repeats. This work was published in Molecular Therapy (Wang et al., 2018).

The research team tested the dual cut approach in DM1 neural stem cells derived from patient iPSCs, using S. pyogenes Cas9 and guide RNAs targeted up- and down-stream of the DMPK CTG repeat tract. This approach yielded a deletion frequency of < 10%, but also resulted in further instability/expansion of CTG repeats in the neural stem cell model. Higher editing efficiency (~53%) was seen with S. aureus Cas9, but further studies showed that this approach yielded inversions of the expanded CTG repeats at a rate as high as ~23%.

By contrast, insertion of a polyadenylation signal upstream of the DMPK repeat tract, using a S. pyogenes Cas9 nickase system, resulted in premature termination of transcription upstream of the repeats and elimination of nuclear foci in a subset of DM1 iPSC-derived neural stem cells. In additional studies, the research team showed that the modified DMPK transcript was stable, underwent post-transcriptional processing, and was normally exported to the cytoplasm in neural stem cells and cardiomyocytes. Similar results were obtained in studies of DM1 iPSC-derived skeletal myocytes, including loss of nuclear foci and retained ability to differentiate into myofibers in vitro.

Lessons for Therapeutic Development in DM1?

Advancement of an in vivo genome editing approach for any systemic disease faces a series of challenges, many of which were documented in the MDF workshop referenced above. This latest study shows that an S. pyogenes Cas9-directed dual editing approach in DM1 iPSC-derived neural cells and cardiomyocytes may suffer from several drawbacks, including low editing efficiency and inability to be packaged into AAV delivery vectors (due to size constraints). S. aureus Cas9 does not have the same AAV packaging constraints and was more efficient in deletion of repeat tracts, but, in the hands of this team, produced inversions leading to DMPK transcripts with expanded CAG repeats; RAN translation of these CAG tracts was not tested in the study but represents a concern due to known protein product toxicity. These findings suggest that the specific genome editing strategies tested here lack potential to move forward as a candidate therapy for DM1.

Alternatively, the study team demonstrated the potential of altering transcription of DMPK CTG repeat tracts via upstream insertion of a polyadenylation signal. This strategy results in generation of a DMPK pre-mRNA from mutant genes that is lacking the expanded repeat tract and appears to be processed normally, thereby avoiding any potential (although not yet discerned) consequences of DMPK haploinsufficiency.


CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.
van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel IDG, van den Broek WJAA, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, Wieringa B.
Mol Ther. 2017 Jan 4;25(1):24-43. doi: 10.1016/j.ymthe.2016.10.014. Epub 2017 Jan 4.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9.
Wang Y, Hao L, Wang H, Santostefano K, Thapa A, Cleary J, Li H, Guo X, Terada N, Ashizawa T, Xia G.
Mol Ther. 2018 Sep 11. pii: S1525-0016(18)30444-1. doi: 10.1016/j.ymthe.2018.09.003. [Epub ahead of print]