Characterization and restoration of a telomere and telomerase deficiency in the disease Dyskeratosis congenita
Telomeres consist of hexameric tandem repeats (TTAGGG)n of DNA located at the chromosome ends and are found in species that maintain their chromosomes in a linear form to protect chromosome integrity, function, and replication. Telomere attrition is a natural process that takes place as cells divide and age. This is due to what has been termed “the end replication problem” which simply means that DNA polymerase cannot completely replicate the 5’ end of newly synthesized linear DNA strands because it requires a 3’ primer. This natural attrition has been speculated to have “tumor suppressor” effects by preventing uncontrolled cellular proliferation. In accordance with this tumor suppression, telomere attrition may also result in senescence, cellular dysfunction, and aging. At minimum, the telomerase enzyme consists of an RNA component called hTR that acts as a template for a reverse transcriptase component, hTERT, to catalyze the addition of telomere repeats to the telomere ends. Telomerase is active in human germline cells and most cancers. Most normal human somatic cells such as fibroblasts express hTR but have low to undetectable levels of hTERT and thus low levels of telomerase. Some highly proliferative cells such as epidermal skin cells and hematopoietic cells also have active telomerase, although in these cells it is thought to be tightly regulated by differentiation.
Different lines of evidence pointed to the possibility that certain human diseases could be related to aberrant telomere length or telomerase function. Dyskeratosis congenita (DC), inherited in both an X-linked and an autosomal dominant (AD) manner, is a bone marrow failure disorder associated with a premature aging syndrome and characterized by the triad of leukoplakia, abnormal skin pigmentation, and nail dystrophy. A variety of other somatic abnormalities normally seen in aged individuals have also been reported in DC, including hair loss, gray hair, osteoporosis, cancer, and pulmonary and hepatic fibrosis. Causes of early mortality include bone marrow failure, pulmonary failure and malignancies. DC symptoms arrive earlier in subsequent generations due to an anticipatory effect seen in this disease where younger generations express DC traits earlier in life because of telomere restoration failure. The autosomal dominant form of DC (AD DC), a rarer and less severe form of DC, is generally caused by haploinsufficiency mutations in TR itself, although recent papers have found mutations implicating hTERT and the telomerase associated protein NOP10. Recently, mutations in hTR and hTERT have been documented in some patients with aplastic anemia and pulmonary fibrosis without a DC phenotype, suggesting that defects in telomerase or telomere length maintenance may have organ specific effects including potential roles in pulmonary disease.
Previous work by our group has attempted therapeutic efforts in skin fibroblasts and have characterized a proliferation defect as well as telomere and telomerase deficiencies in a multi-generation AD DC family. Upon infecting patients' fibroblasts with retroviral vectors of hTR, hTERT or a combination of the two, we were able to successfully restore telomere length, telomerase activity and proliferative capacity, in effect rejuvenating the AD DC cells. A drawback to expressing hTERT, a gene associated with 90% of cancers, is that some of our clonal populations bypassed replication senescence and may be immortal indicating potential transformation.
Future research includes development of HIV vectors tailored to study hematopoietic stem cells and further characterization of cellular defects in these patients' cells.
