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Review
. 2022 May 25;12(6):865.
doi: 10.3390/jpm12060865.

FSHD Therapeutic Strategies: What Will It Take to Get to Clinic?

Charis L Himeda  1 , Peter L Jones  1
Affiliations
Review

FSHD Therapeutic Strategies: What Will It Take to Get to Clinic?

Charis L Himeda et al. J Pers Med. .

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is arguably one of the most challenging genetic diseases to understand and treat. The disease is caused by epigenetic dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, pathogenic misexpression of DUX4 in skeletal muscle. The complex nature of the locus and the fact that FSHD is a toxic, gain-of-function disease present unique challenges for the design of therapeutic strategies. There are three major DUX4-targeting avenues of therapy for FSHD: small molecules, oligonucleotide therapeutics, and CRISPR-based approaches. Here, we evaluate the preclinical progress of each avenue, and discuss efforts being made to overcome major hurdles to translation.

Keywords: AAV; CRISPR; DUX4; FSHD; antisense; facioscapulohumeral muscular dystrophy; gene therapy; muscular dystrophy; skeletal muscle; therapeutics.

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Conflict of interest statement

C.L.H. and P.L.J. are co-founders of EpiSwitch Rx, Inc., a company focused on bringing therapeutics for FSHD to the clinic. They are inventors on two U.S. patent applications pertaining to the use of CRISPR inhibition for FSHD and one pertaining to small molecule development for FSHD. P.L.J. is an inventor on a patent application identifying therapeutic targets for FSHD and on a patent for epigenetic diagnosis of FSHD.

Figures

Figure 1
Figure 1
Incomplete silencing of the FSHD locus leads to misexpression of the pathogenic DUX4-fl transcript in skeletal muscles. DUX4-FL subsequently activates a program of embryonic gene expression, which triggers many aberrant downstream events. While some pathways downstream of DUX4 are amenable to targeting (orange), it is not clear which individual pathway, if any, is causal for pathology. Thus, serious efforts to develop specific FSHD therapeutics (green) have focused on restoring silencing to the locus or preventing expression of DUX4-fl mRNA or protein. Purple indicates DUX4-targeting strategies that are inherently promiscuous; asterisk indicates small molecules targeting DUX4 epigenetic regulation or transcription. Blocking DUX4-FL activity (blue) is a theoretically viable avenue, although still in the very early stages of development.
Figure 2
Figure 2
Therapeutic targets within the FSHD locus. Two D4Z4 repeats (grey) of a disease-permissive allele are depicted, along with distal sequences. Exons 1 and 2 are located within the repeat, and exon 3 is located in the distal sequence. Full-length DUX4 transcripts (DUX4-fl) are expressed in cleavage-stage embryos and stabilized by a polyadenylation signal (PAS) in exon 7. Following this stage, expression of DUX4-fl is normally silenced in most somatic tissues, although the locus can produce unstable, nonpathogenic, short DUX4 isoforms (not shown). In FSHD myocytes, incomplete somatic silencing at the locus mediates a switch from the production of these nonpathogenic isoforms to pathogenic DUX4-fl transcripts, which are stabilized by an exon 3 PAS within permissive haplotypes. Regions for therapeutic targeting are indicated by colored bars: antisense oligonucleotides (pink and red bars; the latter are reported sequences), microRNAs (yellow bars), CRISPR inhibition/epigenetic modulation (purple bars), CRISPR editing (green bar), and small interfering RNAs recruiting the Dicer/Argonaute silencing system (blue bar). The orange asterisks indicate DUX4-FL stop codon. Additional abbreviation: DAE, distal auxiliary element.
Figure 3
Figure 3
CRISPR gene modification approaches to FSHD. (A) The Cas9 nuclease cuts DNA at sites targeted by single-guide RNAs (sgRNAs). DNA repair by nonhomologous end joining (NHEJ) mediates the disruption of genomic sequences by insertions/deletions (indels), whereas homology-directed repair (HDR) mediates precise editing in the presence of a donor template. CRISPR editing of the D4Z4 repeat (*) necessitates cutting the genome in hundreds of unintended places, as these repeats exist in many copies on the noncontracted 4q chromosome and both 10q chromosomes, in addition to polymorphic D4Z4s at other loci. Thus, groups attempting CRISPR editing have focused on the polyadenylation signal (PAS) in DUX4 exon 3, which provides a safer target. (B) RNA-programmable base editing utilizes a wild-type tRNA adenosine deaminase (TadA) and an engineered version of TadA (TadA*) fused as a dimer to a nicking version of Cas9 (nCas9). This enables adenine-to-guanine substitution without introducing double-stranded DNA breaks. (C,D) As an alternative strategy, enzymatically dead Cas9 (dCas9) is fused to the KRAB domain for transcription inhibition (panel C) or to chromatin-modifying proteins, which can act in a broader fashion across the locus (panel D). Fusing minimized versions of these regulators to dCas9 from Staphylococcus aureus allows packaging into rAAV vectors to silence pathogenic DUX4 expression in FSHD muscle. Additional abbreviation: PAM, protospacer-adjacent motif.
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