Transduction of hTR into Hematopoietic Stem Cells Increases Telomerase Activity

Background: Telomerase, an enzyme complex that adds telomere repeats to the ends of linear chromosomal DNA, is an important regulator of cellular senescence and replication. The human telomerase complex is composed of several factors including an RNA component (hTR) that acts as a template, a reverse transcriptase (hTERT), and dyskerin, a stabilizing nuclear protein. Telomerase activity is tightly regulated by hTERT and hTR levels. Several reports have shown that overexpression of hTERT by gene transfer can upregulate telomerase activity in somatic cells, though the effects of hTR gene transfer on telomerase activity in primary cells are unclear. Importantly, mutations in the major components of the telomerase complex cause dyskeratosis congenita (DC), a premature aging syndrome associated with defective hematopoiesis. Of note, our group has recently demonstrated correction of an abnormal phenotype in autosomal dominant (AD) DC fibroblasts by gene transfer of hTR. Additionally, hematopoietic stem cells (HSC) from several AD DC subjects were collected which could be used for future gene therapy studies. In anticipation of these studies, we hypothesized that a lentiviral vector-based gene transfer of hTR into normal hematopoietic stem cells (HSCs) might result in an upregulation of telomerase activity and a corresponding increase in replicative capacity.

Methods: CD34 positively selected HSCs isolated from umbilical cord blood were expanded in cytokinesupplemented Stem Span media and counted periodically over a 14-day time course to determine proliferative capacity. Over this time course, cells were assessed for telomerase activity using the TRAP (telomeric repeat amplification protocol) assay. Additionally, cells were plated in a methylcellulose-based media in order to assess colony forming unit (CFU) formation. Transduction of HSCs was accomplished by infecting with a lentiviral (HIV) vector expressing GFP alone or GFP and hTR together. Transduced cells were assessed for GFP expression and then subjected to the same assessments as described above and comparisons were made to non-transduced cells.

Results: HSCs cultured in Stem Span showed a 100 fold expansion of total nucleated cells over a 14-day time course. Two days post infection, transduction efficiency of 60% was achieved for CD34+ HSCs transduced with GFP alone, and 10% for hTR/GFP. At nine days post infection there was a similar percentage of GFP-expressing cells, suggesting retention of the construct in differentiated cells. Comparing transduced and non-transduced cells, minimal differences were observed in cell proliferation and colony formation, indicating that conditions used for viral gene transfer did not have a deleterious effect on HSCs. Telomerase activity was markedly increased in hTR-transduced cells at day 2 and 4 post-infection, compared to non-transduced and GFP-transduced cells (18 to 33 fold estimated increase). At later time points post-infection (6 to 10 days), telomerase activity in hTR-transduced cells decreased to levels similar to those of non-transduced cells. Additionally, an accelerated loss of CD34 expression was noted in hTR transduced cells.

Conclusions: Lentiviral gene transfer of the hTR component of telomerase into normal HSCs results in an upregulation of telomerase activity without having any noticeable effect on cell proliferation or CFU formation but a possible alteration in differentiation. These studies indicate that transduction of hTR into HSCs may provide a means to upregulate telomerase and potentially correct the proliferative defects observed in bone marrow failure syndromes such as AD DC that are caused by prematurely shortened telomeres.