Finding the Right CRISPR Targets for DM

Published on Mon, 11/13/2017

Cutting Through the Hype

As if we needed actual data to understand that CRISPR technology is a hot topic—a PubMed search on “CRISPR” yields over 7,000 hits, while the NIH RePORTER database search returns 2,316 active research grants with reference to the topic. Potentially transformative therapeutic strategies have always been hyped, but the explosion in the mass media coverage makes it difficult to believe that anyone remains who has not heard the term CRISPR and the unfortunate implication that marketed therapies are “imminent.”

Our task in the scientific, pharma/biotech and advocacy communities is to counter the therapeutic misconception that CRISPR will be a panacea for any inherited disease, while taking pragmatic steps to address the not inconsequential barriers of rigorous efficacy studies, scale-up production, delivery/exposure, efficiency, and even ethics to optimize and evaluate the potential of this important new tool.

Finding the Optimal CRISPR Targets

Initial efforts to apply CRISPR/Cas9 approaches to DM focused on reducing/removing expanded CTG tracts from the DMPK gene. An additional challenge for gene editing in expanded repeat disorders is the need to remove large DNA tracts. Indeed, Dr. Bé Wieringa and colleagues found that removal of expanded CTG repeats was feasible, but required cutting from both sides of the repeat to avoid increasing the genomic instability that drives DM (see DNA targeting may ultimately prove to be the optimal approach for DM, but more developmental and mechanistic work is required to facilitate in situ removal of large expansions safely and efficiently.

Within the CRISPR field, the approach of targeting RNA has attracted considerable interest. Two recent papers from Dr. Feng Cheng’s lab (Broad Institute of MIT and Harvard) have demonstrated RNA targeting using CRISPR/Cas13.  In the first (Cox et al., 2017), they evaluated programmable editing of RNA transcripts to alter coding and thereby correct mutations at the level of the transcript (a system they designate as RNA Editing for Programmable A to I Replacement or REPAIR). This system is designed to address single base mutations and thus may have limited to no applicability in repeat expansion disorders, although continued attention to the associated technological advances is warranted. The second publication (Abudayyeh et al., 2017) demonstrates that CRISPR/Cas13a has the potential for targeted knockdown of RNA, with similar efficiency to and better specificity than RNAi. Such an approach is comparable to knockdown of DMPK transcripts with antisense oligonucleotides that recruit RNaseH mechanisms.

To Target RNA or DNA in DM?

Previously, Dr. Gene Yeo’s group showed that RNA-targeted Cas9 in an in vitro model degrades toxic DMPK transcripts, increases in the MBNL protein, and reduces or eliminates the gene splicing defects that characterize DM (see

In a recent paper in Molecular Cell (Pinto et al., 2017), Dr. Eric Wang and colleagues (University of Florida) reported that deactivated Cas9 (dCas9) specifically binds expanded repeat tracts at the DMPK locus and impedes their transcription. They evaluated multiple CTG repeat lengths and showed that dCas9 coupled to a guide RNA achieved knockdown in a repeat length-dependent manner, with binding efficiency and potency of knockdown increasing with the number of repeats. Moreover, the group showed that the dCas9-gRNA strategy reduces nuclear foci, restores normative splicing, and blocks RAN translation in both HeLa cell models and DM1 patient myoblasts and in skeletal muscles of systemically injected HSA-LR mice.

Next Steps for DM

Efforts to target the expanded repeat DMPK RNA or DNA have shown potential as a therapeutic strategy, but it must be recognized that these efforts are at a preclinical proof of concept stage. Both approaches face questions of delivery, specificity of targeting and reagent efficiency once on target that are likely to become more complex when CRISPR as a candidate therapeutic transitions to clinical testing. For example, if AAV is used for CRISPR reagent delivery, the tissue targeting specificity inherent to many AAV serotypes must be overcome if the multi-systemic consequences of DM are to be addressed.

Hype is a poor substitute for pragmatic scientific progress. As we go forward, all parties are cautioned to avoid language that feeds therapeutic misconception—mouse studies are purely experiments in a so-so disease model—the mice are neither treated nor cured; clinical trials also are experiments, not treatments, until the agent receives regulatory approval. Finally, recruitment of substantive funding and scientific expertise to further optimize and test the RNA and DNA targeting strategies for DM is essential.



RNA editing with CRISPR-Cas13.
Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F.
Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180. [Epub ahead of print]

RNA targeting with CRISPR-Cas13.
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F.
Nature. 2017 Oct 12;550(7675):280-284. doi: 10.1038/nature24049.

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 Oct 18. pii: S1097-2765(17)30711-6. doi: 10.1016/j.molcel.2017.09.033. [Epub ahead of print]