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Wong M.S.,Southwestern Medical Center | Chen L.,Southwestern Medical Center | Foster C.,Sierra science | Kainthla R.,Southwestern Medical Center | And 2 more authors.
Cell Reports | Year: 2013

Telomerase is present in human cancer cells but absent in most somatic tissues. The messenger RNA of human telomerase (hTERT) is alternatively spliced into mostly nonfunctional products. We sought to understand splicing so that we could decrease functional splice isoforms to reduce telomerase activity in order to complement direct enzyme inhibition. Unexpectedly, minigenes containing hTERT exons 5-10 flanked by 150-300 bp intronic sequences did not produce alternative splicing. A 1.1 kb region of 38 bp repeats ∼2 kb from the exon 6/intron junction restored the exclusion of exons 7 and 8. An element within intron 8, also >1 kb from intron/exon junctions, modulated this effect. Transducing an oligonucleotide complementary to this second element increased nonfunctional hTERT messenger RNA from endogenous telomerase. These results demonstrate the potential of manipulating hTERT splicing for both chemotherapy and regenerative medicine and provide specific sequences deep within introns that regulate alternative splicing in mammalian cells by mechanisms other than the introduction of cryptic splice sites. © 2013 The Authors.


Harley C.B.,Geron | Harley C.B.,Telome Health Inc. | Liu W.,TA science | Blasco M.,Spanish National Cancer Center | And 4 more authors.
Rejuvenation Research | Year: 2011

Most human cells lack sufficient telomerase to maintain telomeres, hence these genetic elements shorten with time and stress, contributing to aging and disease. In January, 2007, a commercial health maintenance program, PattonProtocol-1, was launched that included a natural product-derived telomerase activator (TA-65 ®, 10-50mg daily), a comprehensive dietary supplement pack, and physician counseling/laboratory tests at baseline and every 3-6 months thereafter. We report here analysis of the first year of data focusing on the immune system. Low nanomolar levels of TA-65 ® moderately activated telomerase in human keratinocytes, fibroblasts, and immune cells in culture; similar plasma levels of TA-65 ® were achieved in pilot human pharmacokinetic studies with single 10- to 50-mg doses. The most striking in vivo effects were declines in the percent senescent cytotoxic (CD8 +/CD28 -) T cells (1.5, 4.4, 8.6, and 7.5% at 3, 6, 9, and 12 months, respectively; p=not significant [N.S.], 0.018, 0.0024, 0.0062) and natural killer cells at 6 and 12 months (p=0.028 and 0.00013, respectively). Most of these decreases were seen in cytomegalovirus (CMV) seropositive subjects. In a subset of subjects, the distribution of telomere lengths in leukocytes at baseline and 12 months was measured. Although mean telomere length did not increase, there was a significant reduction in the percent short (<4 kbp) telomeres (p=0.037). No adverse events were attributed to PattonProtocol-1. We conclude that the protocol lengthens critically short telomeres and remodels the relative proportions of circulating leukocytes of CMV + subjects toward the more "youthful" profile of CMV - subjects. Controlled randomized trials are planned to assess TA-65 ®-specific effects in humans. © 2011 Mary Ann Liebert, Inc.


Vaziri H.,Ontario Cancer Institute | Chapman K.B.,Biotime, Inc. | Guigova A.,Ontario Cancer Institute | Teichroeb J.,Ontario Cancer Institute | And 13 more authors.
Regenerative Medicine | Year: 2010

Aim: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. Materials & methods: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. Results: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. Conclusion: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine. © 2010 Future Medicine Ltd.

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