Facioscapulohumeral Muscular Dystrophy (FSHD) is a dominant degenerative muscle disease with no cure or treatment. The genetic cause of FSHD reduction of copy number of subtelomeric D4Z4 repeats at 4q35 encoding DUX4 homeobox protein, which is a potent transcription factor that is toxic to the cell. Contractions lead to loss of repeat-induced silencing, allowing for transcription of DUX4. Interestingly, the complete absence of D4Z4 repeats mimics the WT allele in that there is no pathologic consequence. Since D4Z4 is the last element on chr4 before the telomere, deleting the end of chr4 effectively corrects the mutant allele. In this study, I propose to use a genome engineering approach mediated by site-specific nucleases to delete the 4q35 telomeric region containing D4Z4 repeats, thus specifically eliminating DUX4. Targeted integration of an artificial telomere to generate terminal chromosomal deletions has never been demonstrated before, and has a unique application in FSHD, a dominant disease whose gene is the terminal gene on chromosome 4. A common diagnostic method for FSHD shows demethylation of D4Z4 using gDNA of blood cells, and our lab has shown that D4Z4 is demethylated in FSHD iPS cells, meaning that any cell type in an FSHD patient could be subject to deregulation by DUX4. We have previously shown that DUX4 recruits p300 to induce global changes in histone acetylation, massively disrupting gene expression, thus pathology is not obviously muscle-specific. Why the disease is muscle-specific and which cells in muscle may be deregulated by DUX4 are unknowns. Because there is no evidence that DUX4 expression is myoblast or myofiber-specific, I hypothesize that DUX4 might be misexpressed in non-myogenenic cells that are involved in myogenic support, which would lead to FSHD pathogenic progression. These cell types have been largely overlooked in the field. I will therefore generate a panel of isogenic pairs of corrected/diseased iPS lines, and study the presence and consequence of DUX4 expression in myogenic as well as supportive cell types (obtained by differentiating the iPS cells).
Aim 1 will focus on the myogenic lineage and make use of xenograft models pioneered by our collaborators, the Perlingeiro Lab.
Aim 2 will focus on the supportive cell types and evaluate cell non-autonomous effects on myogenesis. These studies will develop a unique and innovative method of genome engineering and shed light on the pathophysiological mechanism of muscle degeneration in FSHD.
(relevance to public health) Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy, affecting the muscles of the head and the upper body, leading to severe loss of motor function and understanding FSHD pathogenesis is essential for development of novel therapies and treatments. The proposed experiments use a novel telomere-editing technique which deletes the FSHD permissive allele in FSHD induced pluripotent stem cells, thus this strategy provides a potential gene editing therapy as well as a method of generating genetically identical disease-normal pairs of various cell types, an approach that eliminates the noise of genetic background differences that have plagued the study of FSHD. By revealing the effect of eliminating DUX4 in different cell types, the work proposed in this study will provide valuable insights into FSHD pathogenesis, as well as determining the usefulness of this gene editing technique for potential future iPSC-based gene therapies.
