Developing resources to alleviate muscle atrophy in FSHD by genome engineering

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DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited form of muscular dystrophy after Duchenne and Myotonic dystrophy, affecting 1 in 20,000 live births. FSHD is primarily characterized by progressive weakness and atrophy of skeletal muscle of the face, shoulders and upper arms that typically manifest in a patients second or third decade of life. Currently there is no cure or effective treatment for this disease. FSHD is an autosomal dominant disorder and in almost all cases the genetic basis for disease involves a contraction in the size of the macrosatellite repeat D4Z4 in the subtelomeric region of chromosome 4q. D4Z4 is composed of a tandem array of a 3.3kb sequence with between 10-150 repeat units defining a single allele in healthy individuals. Intriguingly, whereas reduction in the size of D4Z4 to fewer than 10 repeat units is associated with FSHD, contraction alone is not sufficient to cause disease. Indeed, D4Z4 alleles comparable in size to those observed in FSHD patients have been detected in unaffected individuals, whereas in others complete loss of the macrosatellite has been reported in the absence of FSHD symptoms. Instead, FSHD is exclusively associated with contraction of D4Z4 on specific variants of the subtelomeric region of human chromosome 4q. Recent studies have demonstrated that permissive chromosomes share a canonical polyadenylation signal that upon a contracted allele stabilizes transcripts originating from the most distal D4Z4 repeat unit resulting in a toxic gain of function in muscle. The objective of this proposal is to generate the resources necessary to nullify pathogenic D4Z4 alleles. Recent technological advances have made possible genome engineering in human cells through custom-built zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN). Both approaches involve generation of sequence specific DNA binding proteins tethered to nuclease enzymes that induce double strand breaks at a desired sequence. Introduction of ZFNs and TALENs into cells along with a modified homologous sequence to the target site results in the introduction of desired changes into the endogenous locus as the supplied template is used to repair the damage. In Aim 1, we will develop ZFNs and TALENs directed to sequences proximal to the D4Z4 array. These will be used in combination with a repair template that introduces a telomere seeding sequence to remove the macrosatellite, resulting in a truncated chromosome lacking D4Z4 similar to that observed in unaffected individuals. In Aim 2, we will develop ZFNs and TALENs designed to sequences immediately distal to D4Z4 to convert the polyadenylation signal to a sequence found on non-permissive chromosomes. Generation of such resources will permit exploration of a novel therapy for individuals afflicted with this debilitating progressive disorder.