Transient Expression of Modified TERT mRNA Extends Telomeres and Opens Door for Therapy
June 2015
Scientists may have uncovered a novel way to cure disease and slow aging through the transient expression of telomerase reverse transcriptase (TERT), a protein that elongates chromosomes.
Since replication machinery cannot copy to the very ends of DNA, chromosomes get shorter with each cellular division. Sections of repetitive DNA, called telomeres, have evolved to protect the ends of chromosomes. As these molecular caps shorten, the cell enters into replicative senescence which is often followed by cell death. While this “molecular clock” is necessary for maintaining healthy tissue and organs, this process is thought to play a role in aging and is disrupted in a variety of diseases.
TERT is the catalytic subunit of telomerase, the enzyme that extends telomeres. Increased expression of TERT disrupts the natural cell cycle and can lead to immortal cells and increase risk for cancer. Increased TERT levels are documented in 85-90% of all malignant cells and several strategies for targeting TERT have entered into clinical trials.
Despite this, researchers in Helen Blau’s lab at Stanford University are intentionally upregulating TERT levels in human myoblasts and fibroblasts. Their hope is to utilize TERT to increase the utility of various cell types for disease modeling, drug screening, and use in cell therapies for telomere-related diseases. Due to the negative implications of creating immortal cells, they are utilizing mRNA to rapidly and transiently increase TERT levels. This is opposed to traditional methods, such as plasmids or viruses, which are constitutively expressed.
Previously, her group reported that the muscle stem cells of boys with Duchenne Muscular Dystrophy, a disorder characterized by a deficiency of dystrophin, had telomeres that were much shorter than those of boys without the disease. The complete etiology of the disease has been unclear, however their report suggests that dystrophin deficiency combined with increased oxidative stress leads to the erosion of the telomere. And it is the erosion of the telomere that ultimately causes cardiac failure and death.
Drawing from this conclusion, in their most recent paper Ramunas et al. sought to test the validity of using TERT encoding mRNA modified with 5-methyl-CTP and pseudo-UTP to artificially extend telomeres. Combined, these modifications reduce innate immune recognition of the mRNA while pseudo-UTP imparts increased nuclease resistance and increases translation.
First, they demonstrated that transfection of human fibroblasts and myoblasts with TERT mRNA increases telomerase activity in a dose-dependent manner. This activity peaked at 24 hours and returned to baseline 48 hours after transfection.
Next, Ramunas and colleagues looked at cell proliferation. A consequence of increased telomerase activity is an increase in cell proliferation capacity. Cell proliferation can be measured by looking at population doubling (PD), the total number of times the cells in the population have doubled from a specific time point. In fibroblasts, the treatment increased proliferative capacity by an additional 28 ± 1.5 PDs with an overall increase of 2.7 X 108 cells beyond untreated cells. In myoblasts, the PD was increased by 3.4 ± 0.4 leading to a 10-fold increase in total cell count.
The authors then measured telomere length and found that treatment extended telomeres up to 0.9 kb. Three months after treatment the telomere length had decreased, however they were still increased relative to untreated cells. This result was expected due to the transient nature of the treatment.
Additional analysis showed that the frequency distribution of chromosome length was shifted. While untreated cells showed a sharp peak at shorter lengths, those that received the TERT mRNA had a more bimodal distribution, indicating that more chromosomes carry longer telomeres.
Finally, Ramunas et al. notes that unlike immortal cell lines, the mRNA transfected cell lines eventually stopped proliferating and expressed markers of senescence similar to untreated cells. This underscores the fact that the treatment is less liklely to lead to a cancerous phenotype and may be useful in a clinical setting. Combined, this data shows that cellular treatment with TERT mRNA may be a useful tool for both research and therapeutic applications.
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