Description: Normal diploid cells can only divide a finite number of times in vitro and in vivo before they senesce, however cancer cells overcome this replicative barrier and can divide indefinitely. Part of the oncogenic processes is the maintenance of chromosome ends, or telomeres. Most cancer cells use an enzyme called telomerase to maintain telomeres and thus telomerase inhibition has been attempted as a cancer specific therapy. However current telomerase inhibition strategies are either toxic to normal cells or have met difficulties in clinical trials. Thus, new ways to target telomerase positive cancer cells to generate stable, long term cancer remissions are being pursued. To do this, an in depth understanding of telomerase regulatory mechanisms is essential. Dogma in the field has caused a focus on transcriptional regulation of TERT, however recent evidence indicates that alternative splicing of TERT may be equally or more important than transcriptional regulation of TERT. An integral aspect of telomerase regulation is the alternative splicing of the protein component, telomerase reverse transcriptase (TERT). In human cells TERT is a 16-exon gene and is spliced into different isoforms, most of which are either degraded or do not form proteins that can maintain telomeres. Full-length TERT containing all 16 exons is the only known isoform (full length TERT) that can form ribonucleoprotein complexes that can maintain telomeres. Thus, understanding the cis and trans-acting factors that regulate splicing choice of TERT pre-mRNAs is critical to finding a means switch TERT splicing from full length to other isoforms. By switching the splicing to non-telomerase activity generating isoforms, we would be able to shorten telomeres and interrupt cellular immortality of the cancer cells. To this end we have performed an siRNA screen which generated a list of 94 unique genes that potentially directly regulate TERT and telomerase in human cancer cells. So far, we have elucidated three genes, NOVA1, PTBP1, and SF3B4, all bind to a region in TERT intron 8, and act as splicing enhancers and promoting the formation of full length TERT. Reduction of any of these three proteins results in telomere shortening. Deletion of where these oroteins bind in TERT intron 8 via CRISPR/Cas9, also shortens telomeres. We also utilize long read RNA sequencing to identify the catalog of TERT alternative splicing isoforms/variants. Using this information, we are attempting to develop innovative therapies to treat cancers. To this end our laboratory is pursuing antisense oligonucleotide therapies to shift the splicing of TERT towards degraded or non-functional TERT isoforms as potential cancer therapies.