PROJECT SUMMARY/ABSTRACT. Telomerase is critical for health and longevity, but there is a fundamental lack of understanding of how it is regulated in human cells. This knowledge gap impedes the ability to develop therapies for a growing spectrum of disorders in which telomerase dysfunction is implicated. The long-term goal of this project is to be able to manipulate telomerase in human cells for therapeutic benefit, with a key target being the hematopoietic system. The level of the noncoding telomerase RNA component TERC is a critical determinant of telomerase function in cells. Low TERC levels resulting from genetic mutations cause a wide spectrum of degenerative disorders, including dyskeratosis congenita (DC), aplastic anemia, MDS/leukemia, solid tumors, pulmonary fibrosis, and cirrhosis. What is not known is how TERC levels are regulated in cells, which is an important barrier to developing therapeutic strategies to manipulate telomerase. The overall objective of this proposal is to understand how TERC is regulated in human stem cells including hematopoietic stem cells (HSCs). The central hypothesis is that cis-acting elements at the TERC locus and other factors that function post-transcriptionally act in concert to determine the steady-state level of TERC in cells. The rationale for this work is that deciphering how patient mutations impact TERC biogenesis will yield a new understanding of how TERC levels are normally regulated, which will in turn reveal novel approaches to manipulate telomerase activity. The central hypothesis will be tested by pursuing two Specific Aims: (1) Identify transcriptional mechanisms regulating human TERC expression, and (2) Identify post-transcriptional mechanisms regulating human TERC levels. Under the first aim, ?scarless? genome-editing in induced pluripotent stem (iPS) cells from DC patients will be used to define the function of newly identified cis-acting elements in the human TERC locus, and to reveal trans-acting factors that modulate TERC transcription. Tools and techniques that have been developed and demonstrated to be feasible in the applicants' hands will be used. Under the second aim, the role of an RNA processing factor recently found to be disrupted in DC patients who have low TERC levels will be investigated. Using patient iPS cells, which have been created by the applicant and carry mutations in this gene, the post-transcriptional processing and maturation of TERC will be investigated. The approach is innovative because the new tools and insights now available allow a critical shift in focus from the regulation of the catalytic component of telomerase (TERT), which has been the subject of intense investigation, to the equally important but less well studied RNA component of telomerase, TERC. The proposed research is significant, because it is expected to yield new strategies to manipulate telomerase activity in a growing number of hematopoietic and degenerative disorders including aplastic anemia, MDS/leukemia, pulmonary and cardiovascular disease, in which telomerase dysfunction is implicated but for which there are few if any curative therapies.