Project Summary/Abstract
Autosomal dominant Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular
dystrophies, affecting 1 in 7,500 to 1 in 20,000 individuals. FSHD was formally classified as a major form of
muscular dystrophy in 1954, but the pathogenic events leading to the disease have only recently started coming
into focus. Today, it is now recognized that FSHD pathogenesis involves aberrant expression of the DUX4 gene,
which encodes a myotoxic transcription factor. The emergence of DUX4 as a primary insult underlying FSHD
represented a momentum shift in the field as it provided an important target for model development and therapy
design. Indeed, as FSHD is currently untreatable, developing effective FSHD therapies is a critical need in the
field. We hypothesized that an FSHD treatment should center on inhibiting toxic DUX4 expression in skeletal
muscles. The objective of this proposal is to develop effective prospective FSHD therapies aimed at reducing or
eliminating toxic DUX4 expression. To do this, we will use new cutting edge RNA silencing and RNA editing
approaches for which technology emerged only within the last 2-3 years. These approaches only cut RNA and
not DNA, and therefore pose no risk of permanent genome modification in host cells. Because these strategies
are only newly emerged, they have never been tested in vivo using gene therapy approaches in an animal model
of disease. We will do that here, and thus our proposal will provide first proof-of-principle for in vivo efficacy use
of CRISPR-Cas13 and RNA editing technology. Specifically, in Aim 1 we will develop a new CRISPR/Cas13
gene therapy approach to cleave DUX4 mRNA before it can be made into protein. In Aim 2, we will develop RNA
editing strategies that are designed to change the DUX4 transcript and render it incapable of producing toxic full-
length forms of DUX4 protein. Finally, since these strategies are new and abundant off-target data are currently
lacking, in Aim 3, we will assess the precision of RNA cleavage and editing approaches in human FSHD and
control muscle cells. Upon completion of these Aims, we expect to produce pre-clinical data supporting the
translation of new AAV-based RNA-targeted therapies for FSHD that can be ultimately used for translation
toward our goal of clinical application. These data may also support the broader use of this technology for other
genetic disorders.
