Project Summary/Abstract
Facioscapulohumeral dystrophy (FSHD) is among the most commonly inherited muscular dystrophies, affecting
up to 870,000 people worldwide. There are currently no approved treatments for FSHD and therapy
development remains an unmet need. Historically, FSHD has been subdivided into two forms, a common type,
called FSHD1 (95% of cases) and rare type, called FSHD2 (5% of cases). These forms are clinically
indistinguishable, and both FSHD1 and FSHD2 are ultimately caused by aberrant de-repression in muscle of a
wild-type gene, called double homeobox 4 (DUX4), which encodes a transcription factor (DUX4) that is toxic to
muscle. DUX4 de-repression in FSHD is caused by chromatin changes at the DUX4 locus. Specifically, in
healthy muscle, DUX4 DNA is normally embedded in heterochromatin and repressed; in FSHD muscle, genetic
factors associated with FSHD change the epigenetic status of the DUX4 locus, making it more euchromatin-like
and allowing toxic DUX4 expression. FSHD1 and FSHD2 are distinguished by the genetic mechanisms that
give rise to the FSHD epigenetic lesion, including mutation in chromatin modifier genes that normally promote
heterochromatin deposition at the DUX4 DNA locus. Mutations in one such gene, called structural maintenance
of chromosomes hinge domain 1 (SMCHD1), lead to DUX4 DNA hypomethylation and enables DUX4
expression. Recent in vitro data in FSHD patient myoblasts from FSHD1 and FSHD patients suggested that
SMCHD1 over-expression can rescue the FSHD-associated epigenetic lesion, regardless of underlying cause.
Thus, we hypothesize that the DUX4 locus in muscles can be repressed by SMCHD1 over-expression, thereby
offering a novel therapy for FSHD via chromatin remodeling. Our goal is to test this hypothesis in vivo using a
novel gene therapy strategy in two complementary humanized FSHD mouse models. Our gene therapy
approach involves using adeno-associated viral vectors (AAV) to deliver SMCHD1 to muscle, but AAV has a
limited packaging capacity, and the full-length SMCHD1 open reading frame (ORF) is too large to fit into a single
AAV vector. To circumvent this size problem, we created an AAV.SMCHD1 split-vector system, where one vector
contains a promoter and the 5’ half of the SMCHD1 gene, and a second vector contains the 3’ half of SMCHD1
and a poly A signal. The two vectors share several hundred base pairs of SMCHD1 sequence to allow
homologous recombination in vivo. Our preliminary data support the efficiency of this system to recombine in
mouse muscle. Here we synergize expertise of two labs to test the functional impacts of SMCHD1 split-vector
gene therapy to correct the epigenetic lesions and gene expression defects associated with FSHD in two different
humanized mouse models, a transgenic line expressing a human FSHD-permissive DNA fragment (D4Z4-2.5)
and a human FSHD muscle xenograft model. Successful completion of our Specific Aims will provide a
foundation for translating AAV.SMCHD1 split vector gene therapy as a prospective new treatment for FSHD.
