Translating dosage compensation to Trisomy 21: a novel approach to Down syndrome. J. B. Lawrence1, J. Jiang1, Y. Jing1, C. J. Cost2, J. Chiang1, H. J. Kolpa1, A. M. Cotton3, D. M. Carone1, B. R. Carone1, D. A. Shivak2, M. Byron1, P. D. Gregory2, C. J. Brown3, F. D. Urnov2, L. L. Hall1 1) Department of Cell and Developmental Biology, University Massachusetts Medical School, Worcester, MA; 2) Sangamo BioSciences, Richmond, CA; 3) Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.

   DS researchers have sought to define the more DS critical genes on Chr21, but this has proven difficult due to high genetic complexity and phenotypic variability of DS, confounded by normal variation between any individuals. There remains a critical need for better ways to understand the underlying cell and developmental pathology of human DS, key to design of therapeutics. Despite advances in strategies to correct single-gene defects of rare monogenic disorders in vitro and in some cases in vivo, genetic correction of chromosomal imbalance in living trisomic cells has been outside the realm of possibility, in any context. Several years ago we began work on a high-risk project to test the idea that functional correction of trisomy may be feasible by inserting a single gene that can epigenetically silence a whole chromosome. Nature has evolved a mechanism to dosage compensate X-chromosome dosage differences between mammalian males and females: the X-linked XIST silences one X-chromosome in female cells. We previously showed that XIST produces a large non-coding RNA that coats the whole chromosome in nuclei. The RNA induces a host of heterochromatin modifications that transcriptionally silence one X-chr in cis. While XIST has been intensely studied, its potential translational relevance for chromosome pathology has not been pursued.
   Therefore, we first demonstrated that the targeted addition of a very large 21 kb XIST transgene could be efficiently achieved in human cells, using genome editing with zinc finger nucleases. With 99% accuracy, we inserted XIST into an intron of the DYRK1A locus on Chr21, thereby generating an inducible system to express XIST RNA on one Chr21 in DS patient-derived pluripotent stem cells. Remarkably, the RNA localizes across and comprehensively silences one of the three Chr 21s, as shown by eight different methods, including molecular, cytological, and genomic. Reversal of specific cellular phenotypes was demonstrated. Results show the clear promise of this new strategy as a novel approach to identify DS cellular pathologies and genome-wide pathways most directly perturbed by trisomy 21, distinct from pervasive genetic and epigenetic variation between cell isolates and subclones. Silencing of trisomy 21 by manipulation of a single gene in living cells in vitro surmounts the first major obstacle to development of potential chromosome therapy.