315. Highly Efficient Targeted Addition of Large XIST Transgenes in a Population of Somatic Cells

Abstract

Recent advances in genome editing technologies enable precise and corrective genome modification to treat genetic diseases in a targeted way. High efficiency of genome editing can be achieved through error-prone NHEJ pathway, however, the absolute rate of precise genome editing through homology-directed repair (HDR) pathway remains relatively low, especially for targeted addition of large DNA sequences. Although it is sufficient for the generation of cell lines derived from single targeted clones when coupled with drug selection, poor rates of homologous recombination greatly limit the practical application of targeted gene integration in somatic tissue. XIST encodes a large long non-coding RNA (19 kb) that plays an essential role in X chromosome inactivation in mammalian females. Recently, we have shown that XIST can be inserted to chromosome 21 and silence the extra copy of entire chromosome in Down syndrome stem cells derived from a single-targeted clones. Successful trisomy silencing in vitro holds promise for potential development of ‘chromosome therapy” in patients with Down syndrome. Here, using Zinc finger nuclease (ZFN) technology, we tested the feasibility of targeted addition of large XIST transgenes into different loci of human genome in a population of transformed cells, as well as primary fibroblasts. Our results demonstrate ZFNs enable highly efficient targeted addition of several large XIST transgenes (up to 21 kb) into multiple selected loci of human genome, which are the largest sequences for targeted addition to date. Surprisingly, the frequency of targeted integration is independent of the size of transgenes but possibly depends on ZFN cleavage activity. Highly efficient targeted addition of large XIST transgenes allows to directly compare silencing of XIST transgenes that contain different functional domains in a pool of many independent integrants. This study provides a powerful tool to test functional mini-XIST transgenes that can be packaged in a viral vector, and has particularly important applications in potential development of in vivo delivery of XIST transgenes into an organ of patients with trisomic chromosomal abnormalities.