Author + information
- Yanhui Li, MD, PhD,
- Gang Zhou, MD, PhD,
- Ivone G. Bruno, PhD and
- John P. Cooke, MD, PhD∗ ()
- ↵∗Houston Methodist Research Institute, Department of Cardiovascular Sciences, 6670 Bertner Avenue, Mail Stop: R10 South, Houston, Texas 77030
Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder of accelerated aging (1). Patients die in their teens of myocardial infarction and stroke (1). HGPS is caused by an autosomal 1824C to T mutation in the LMNA gene that encodes lamin A. The mutation results in a constitutively farnesylated lamin A called progerin, which severely disrupts nuclear architecture and cell functions (1). HGPS is associated with accelerated telomere erosion, which is a known determinant of senescence (2). The mechanism of telomere erosion is not known, although accumulation of progerin in the nuclear inner membrane may adversely affect the physical and functional interaction of the telomere with the nuclear lamina (3). The present study was driven by the hypothesis that transient expression of human telomerase might restore telomere length sufficiently to reverse the senescent phenotype of HGPS cells.
We assessed telomere length by MMqPCR (monochrome multiplex quantitative polymerase chain reaction) (4) in fibroblasts derived from HGPS patients (n = 17; age range 1 to 14 years; LMNA 1824 C>T; obtained from Progeria Research Foundation [Peabody, Massachusetts] and Coriell Cell Repositories [Camden, New Jersey]) and compared them with wild-type neonatal fibroblasts (BJ) and fibroblasts from normal adults (n = 3; age range 31 to 68 years). Cells were studied at a similar passage number (passage 5 to 7). As expected, the telomeres of HGPS patients were generally short for their chronological age. Within the HGPS group, there was an inverse relationship between age and telomere length (Figure 1A). However, there was heterogeneity in average telomere length among HGPS patients. Twelve of the 17 patients (71%; age: 1 to 14 years) had shortened telomeres, similar to the length of telomeres observed in cells from a healthy older (69 years) subject. In contrast, 5 patients (29%; age: 1 to 8 years) had normal telomere length (Figure 1A). This heterogeneity in telomere lengths in HGPS has not been reported previously, and could be due to differences in other factors, such as oxidative stress, which is known to cause telomere erosion.
We transfected HGPS fibroblasts with human telomerase (hTERT) mRNA, or catalytically inactive hTERT (CI) mRNA. CI TERT has a mutation at the catalytic site of the reverse transcriptase domain of TERT that abolishes catalytic activity so that it can bind to, but does not extend, the telomere (5). The mRNA was generated by in vitro transcription, modified with pseudouridine and 5-methylcytidine to increase stability, and purified by high-pressure liquid chromatography to remove impurities.
HGPS fibroblasts (AG1972, short telomere patient, passage 18) underwent 3 consecutive transfections with hTERT or CI hTERT mRNA in succession at 48-h intervals using Lipofectamine RNAiMax at 1 μg/ml for 4 h. Proliferation of HGPS cells was characteristically poor (Figure 1B) whether untreated or exposed to the CI hTERT. In contrast, treatment with hTERT mRNA dramatically restored HGPS cell proliferation, reduced cell loss, and extended overall cellular lifespan (Figure 1B). This enhanced proliferation effect after hTERT mRNA treatment was also observed in fibroblasts from other short telomere HGPS patients, but not fibroblasts from HGPS patients with normal telomere lengths (data not shown). The dramatic improvement in HGPS cell proliferation after hTERT mRNA treatment was associated with other evidence of cellular rejuvenation, which will be reported in detail elsewhere. These effects included an increase in telomerase activity and telomere length, a reduction in senescence-associated β-galactosidase staining, and a decreased secretion of inflammatory cytokines. Notably, hTERT mRNA treatment did not cause immortalization of the cells, because they showed normal growth kinetics, with a log phase of growth followed by a plateau phase (data not shown). None of these salutary effects were observed after treatment with the CI form of hTERT, supporting the notion that telomere extension played a critical role in the reversal of HGPS pathobiology.
These studies indicate that transient expression of telomerase mRNA might be a rapid and effective way to reverse senescence in HGPS cells. Although sustained telomerase expression would raise the safety concern of immortalization, our approach did not transform the cells. Of note, hTERT treatment did not provide the same benefit for HGPS cells with normal telomeres. Accordingly, assessment of telomere length might be an important diagnostic adjunct to therapy for HGPS. This therapeutic approach will be made more feasible by new developments in mRNA delivery that permit systemic delivery of mRNA, such as novel nanovectors.
Please note: This work was supported in part by grants to JPC from the Progeria Research Foundation (PRF), the Cullen Foundation, and the National Institutes of Health (U01 HL100397). The PRF and Coriell Cell Repositories provided cell lines and DNA samples. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- 2017 American College of Cardiology Foundation
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