Schematic of chromosome 4, D4Z4, and DUX4-expressing AAV vectors.

<p>A: A representation of the telomeric region of the chromosome 4 long arm (4q35). Drawing is not to scale. The 4q35 subtelomere harbors polymorphic, 3.3 kb D4Z4 repeat arrays, as well as other genes, some of which are indicated. This region is normally embedded in repressive heterochromatin. Contraction of the D4Z4 repeat array (in FSHD1) or mutations in SMCHD1 (in FSHD2) leads to epigenetic changes in the 4q35 region, and subsequently permits transcription of the DUX4 gene. An “FSHD permissive” haplotype creates a polyA signal in the pLAM region located downstream of the array. DUX4 transcripts initiated in the last D4Z4 unit extend to this signal and are stabilized by a polyA tail, thereby allowing the mRNA to be translated into the toxic, pro-apoptotic DUX4 protein. B: Two different AAV vectors were engineered to express DUX4. The first generation vector utilized a CMV promoter and SV40 polyA signal. The DUX4 ORF was tagged at the 3’ end with sequences encoding a V5 tag, thereby producing a full-length DUX4 protein containing a carboxy-terminal V5 epitope fusion. ITR, AAV2 inverted terminal repeats. The second generation AAV.D4Z4.V5 vector essentially recapitulates the terminal D4Z4 repeat and pLAM sequences isolated from an FSHD patient, but engineered to express DUX4 with a carboxy-terminal V5 epitope fusion.