Therapeutic Development

Peter and Takako Jones Lab

We are beginning to identify and understand the pathogenic mechanisms underlying FSHD (summarized in Figure 7), however, currently there are no validated therapies for the disease. The FSHD field as a whole is designing a wide range of approaches to attack many of these pathways in the hope of alleviating or preventing pathology. However, FSHD patients display a wide range of clinical presentation and disease progression can be highly variable. Thus, different patients may require or respond to different therapeutic approaches; potentially combinatorial approaches would be best. The good news is that now that we understand a molecular basis for disease many groups are moving forward into therapeutic development and preclinical testing.

Figure 7: Therapeutic targets for FSHD Chart

The key event causing FSHD is the epigenetic disruption of the chromosome 4q35 D4Z4 macrosatellite array either by physical deletion of regulatory heterochromatin (FSHD1) or genetic mutations in epigenetic regulatory proteins (FSHD2). The primary mediator of FSHD is the aberrant expression and splicing of the DUX4 gene encoded within the distal-most D4Z4 repeat unit. Therapeutic strategies are designed to target (1) upstream of DUX4 focusing on the epigenetic and myogenic regulation of DUX4 expression, (2) the pathogenic DUX4-fl mRNA and (3) certain tractable downstream effects of DUX4 expression. Our group is using small molecules, antisense, small RNA, and CRISPR-based approaches to block DUX4-fl expression and function. These proof-of-principle studies are being performed using both in vitro and in vivo models of FSHD.

Figure 8: CRISPR Based aproaches to treatment chartImportant notes on CRISPR
(Figure 8)

CRISPR/Cas9 is a gene-editing technology that involves two key components: 1) the Cas9 protein from bacteria and 2) a guide RNA that targets Cas9 to a specific sequence within the genome. Normal Cas9 (the version most commonly used) is an enzyme that cuts DNA at the site targeted by the guide RNA. The cut DNA is then repaired by either nonhomologous end joining (NHEJ), which is imprecise and often results in a small insertion or deletion (InDel) that disrupts the targeted sequence, or homology directed DNA repair, which allows for the insertion of a changed or new DNA sequence into the genome at a specific location. Both of these result in site-specific permanent changes in the genomic sequence (thus the phrase "gene editing technology" associated with typical CRISPR/Cas9 approaches). The ethical controversy surrounding CRISPR the potential for this gene editing technology and to be used in the human germ line (sperm or eggs) to produce humans with specifically altered genomes. Its use in somatic cells for gene therapy type approaches is not controversial, just very difficult using the current technology. A variation to the standard CRISPR/Cas system uses the specific gene targeting capacity of they system but does not use the genome editing. Instead, a non-cutting version of Cas9 ("dead Cas9" or dCas9) is fused to a protein domain of desired function, normally a transcriptional activator (such as VP16 or VP64) or repressor (such as KRAB) to turn on or turn off the expression of a desired gene, respectively.

CRISPR in the context of FSHD vs other diseases:

Most diseases are caused by mutation(s) in a single protein-encoding gene, which results in the production of a dysfunctional protein or no protein at all. Thus, researchers studying these "typical" diseases are using Cas9 to cut out the mutated part of the disease gene in order to replace it with the correct DNA sequence. For FSHD, we don't need to replace a missing/dysfunctional protein; rather, we need to stop expression from the pathogenic DUX4 gene. Hence, CRISPR inhibition (CRISPRi) of DUX4 expression with dCas9-KRAB (as opposed to editing with Cas9) may be uniquely suited to FSHD. We showed that is in fact possible to accomplish in our recent paper published in Molecular Therapy. An alternative way to silence expression of the DUX4 gene via CRISPR/Cas9 would be to alter the sequence of the polyadenylation signal (PAS) required for producing DUX4-fl mRNA. In this scenario, Cas9 with a functional nuclease activity would be targeted to the 4q35 DUX4 exon 3 PAS and NHEJ repair would result in the loss of the PAS creating a chromosome 4q that is non-permissive for FSHD. It should be noted that while all of these approaches are viable tools in the lab, their transition to clinical practice would require several technical advances for delivery to muscle in a person and dealing with potential adverse immune system reactions. Therefore, any potential clinical trials for FSHD using CRISPR/Cas are many years away.