Status Report: Genome Editing in DM1

The New Frontier?

While at first glance genome editing strategies offer considerable excitement for treating monogenetic disorders, on closer examination there are considerable hurdles to be overcome. Appropriately, the initial clinical testing of CRISPR/Cas9 is focused upon ex vivo editing or delivery to the restricted environment of the eye, since in vivo genome editing presents order of magnitude greater challenges of delivery and safety. To explore the path forward for DM, MDF’s Genome Editing Workshop report carefully evaluated the opportunities and challenges of this novel technology.

A Current Status Report: CRISPR/Cas for DM1

As of mid-August 2019, there have been nine original research publications on a CRISPR/Cas strategy for DM1 (see listing below). While academic research findings require considerable expertise, time, and effort to translate into effective therapies, MDF strives to monitor these technological advances. A new article by Dr. Derick Wansink (Radboud University Medical Center) and colleagues reviews eight of these published efforts using CRISPR/Cas genome editing in DM1, compares these efforts with others in neurological disorders with microsatellite expansions, and discusses some of the translational questions to achieve a therapy for DM1.

The authors describe application of CRISPR/Cas to excise the expanded repeat at the DMPK locus as the most straightforward application of the technology—as this strategy would permanently halt the primary genetic disease mechanism. They note preclinical validation of the approach achieved in three peer-reviewed publications (Dastidar et al., 2018; Provenzano et al., 2017; Wang et al., 2018) and in one on a preprint server (Yanovsky-Dagan et al., 2019); although one report (van Agtmaal et al., 2017) showed detrimental destabilization of the repeat tract. Challenges in expanded repeat excision include the inability to distinguish short and long repeats and that its double strand breaks may produce uncontrolled deletion of large repeats, resulting in an inability to predict residual repeat length. Whether repeats can be excised in terminally differentiated cells is also unclear.

Based upon studies in other microsatellite expansion disorders, the authors raised the possibility of reducing expanded repeat length via homology-directed repair, but note that this has not yet been tried in DM and raise doubts about its value. Likewise, allele-specific editing is under investigation for Huntington’s, but the authors note that identification of SNPs required in this approach have not been achieved for the DMPK mutant locus.

There are CRISPR/Cas alternatives to DNA deletions. One study (Wang et al., 2018) has shown the utility of a homology-directed recombination strategy to insert a premature polyA signal, thereby interfering with transcription of repeat-expanded DMPK.

Inactivated Cas9 (dCas9) is being touted as a way to exploit many of the advantages of CRISPR/Cas technology while reducing off-target safety concerns. Dr. Wansink and colleagues point to the work of Pinto et al. (2017), who achieved transcriptional block targeted to the mutant DMPK allele. If DMPK knockdown proves to not be deleterious (thus far it hasn’t), such an allele-specific strategy would have additional advantages.

Finally, the review’s authors discuss the RNA-targeted CRISPR/dCas9 work of Batra et al. (2017), findings of which have moved toward clinical development with the biotech company Locana. This approach, also potentially safer than DNA excision, is based upon the efficient targeting of RNA by dCas9 and subsequent blocking/displacement of MBNL from expanded repeat RNA hairpin structures.

Opportunities and Challenges for CRISPR/Cas in DM

Dr. Wansink and colleagues provided a nice summary of the path forward for genome editing in DM. A synopsis of their presentation, as well as MDF's observations, are presented here.

Opportunities facilitating translation of genome editing in DM include:

  • Availability of splicing event biomarkers (and, potentially, methylation status in CDM) capable of rapid readout of molecular target engagement and modulation;
  • Leveraging lessons from ongoing work in other repeat expansion disorders, including fragile X syndrome, Friedreich’s ataxia, and Huntington’s disease;
  • With the regulatory approval of Zolgensma and advances for other diseases, some of the hurdles for AAV delivered genetic therapies may be mitigated; and
  • The expanding interest and commitments to exploring genome editing for DM helps ensure that the field can take advantage of new technological advancements in editing reagents, delivery, and safety that are already on the horizon.

Challenges facing genome editing in DM included:

  • Safety issues with off-target editing that must be resolved for any candidate therapy where CRISPR/Cas reagents are delivered in vivo. These issues increase if the in vivo expressed reagents are not under control of an on/off regulator;
  • Delivery represents a major challenge for CRISPR/Cas9 utility in DM, as noted in MDF’s Genome Editing Workshop report, Unless alternative delivery using nanoparticles, antibodies, or other means proves feasible, the field will be looking at the additive challenges of AAV delivery for genome editing strategies;
  • Studies suggest that pre-existing immunity to bacterial Cas proteins affects a large percentage of the population, thus effective strategies will be needed to mitigate;
  • The review’s authors point toward the potential of ex vivo editing and cell therapy. While considerably safer, this approach poses considerable delivery issues and it’s difficult to see how multi-system consequences of DM1 could be addressed by this strategy; and
  • If CRISPR/Cas strategies mature sufficiently to move into clinical evaluation, careful choices of DM1 patient cohort, organ systems to be targeted and evaluated, and efficacy and safety endpoints will need to be made.


CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.
Raaijmakers RHL, Ripken L, Ausems CRM, Wansink DG.
Int J Mol Sci. 2019 Jul 27;20(15). pii: E3689. doi: 10.3390/ijms20153689. Review.

Original CRISPR/Cas Research Articles on DM1(PubMed August 2019):

CRISPR/Cas9-Induced (CTGCAG)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.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.
Batra R, Nelles DA, Pirie E, Blue SM, Marina RJ, Wang H, Chaim IA, Thomas JD, Zhang N, Nguyen V, Aigner S, Markmiller S, Xia G, Corbett KD, Swanson MS, Yeo GW.
Cell. 2017 Aug 24;170(5):899-912.e10. doi: 10.1016/j.cell.2017.07.010. Epub 2017 Aug 10.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.
Pinto BS, Saxena T, Oliveira R, Méndez-Gómez HR, Cleary JD, Denes LT, McConnell O, Arboleda J, Xia G, Swanson MS, Wang ET.
Mol Cell. 2017 Nov 2;68(3):479-490.e5. doi: 10.1016/j.molcel.2017.09.033. Epub 2017 Oct 19.

CRISPR/Cas9-Mediated Deletion of CTG Expansions Recovers Normal Phenotype in Myogenic Cells Derived from Myotonic Dystrophy 1 Patients.
Provenzano C, Cappella M, Valaperta R, Cardani R, Meola G, Martelli F, Cardinali B, Falcone G.
Mol Ther Nucleic Acids. 2017 Dec 15;9:337-348. doi: 10.1016/j.omtn.2017.10.006. Epub 2017 Oct 14.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophypatient-derived iPS and myogenic cells.
Dastidar S, Ardui S, Singh K, Majumdar D, Nair N, Fu Y, Reyon D, Samara E, Gerli MFM, Klein AF, De Schrijver W, Tipanee J, Seneca S, Tulalamba W, Wang H, Chai YC, In't Veld P, Furling D, Tedesco FS, Vermeesch JR, Joung JK, Chuah MK, VandenDriessche T.
Nucleic Acids Res. 2018 Sep 19;46(16):8275-8298. doi: 10.1093/nar/gky548.

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 Nov 7;26(11):2617-2630. doi: 10.1016/j.ymthe.2018.09.003. Epub 2018 Sep 11.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.
Angelbello AJ, Rzuczek SG, Mckee KK, Chen JL, Olafson H, Cameron MD, Moss WN, Wang ET, Disney MD.
Proc Natl Acad Sci U S A. 2019 Apr 16;116(16):7799-7804. doi: 10.1073/pnas.1901484116. Epub 2019 Mar 29.

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.
Lo Scrudato M, Poulard K, Sourd C, Tomé S, Klein AF, Corre G, Huguet A, Furling D, Gourdon G, Buj-Bello A.
Mol Ther. 2019 Aug 7;27(8):1372-1388. doi: 10.1016/j.ymthe.2019.05.021. Epub 2019 Jun 5.

Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses DMPK Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts
Yanovsky-Dagan S, Bnaya E, Diab MA, Handal T, Zahdeh F, van den Broek,WJAA, Epsztejn-Litman S, Wansink DG, Eiges R.
bioRxiv. 2019, 631457.


© Myotonic Dystrophy Foundation. All rights reserved.