Pulmonary fibrosis is a fatal lung condition that can be associated with lung cells exhibiting short telomeres, the protective structures at the ends of chromosomes. Researchers in Spain now report on the use of an adeno-associated virus (AAV) vector-based gene therapy to rebuild telomeres and effectively cure mice with a form of idiopathic pulmonary fibrosis (IPF) that mirrors the disease in humans. Reporting in eLife, the team, led by Maria A. Blasco, Ph.D., and Paula Martínez, Ph.D., at the Spanish National Cancer Centre (CNIO) Molecular Oncology Program’s Telomeres and Telomerase Group, suggests that the studies represent proof-of-principle that the same approach could be used to treat pulmonary fibrosis associated with short telomeres in people. Their paper is entitled “Therapeutic Effects of Telomerase in Mice with Pulmonary Fibrosis Induced by Damage to the Lungs and Short Telomeres.”

“The most relevant aspect of our work is that it suggests a potentially viable and effective solution to a real clinical problem, pulmonary fibrosis, for which there is still no treatment,” Martínez says. “The only approved treatments for pulmonary fibrosis to date have no curative effects, as they target a symptom and not the cause of fibrosis. Our therapy is based on correcting the molecular cause of pulmonary fibrosis in patients with short telomeres, introducing into the cells of damaged lung tissue the only enzyme capable of lengthening telomeres, telomerase.”

Mammalian telomeres are effectively structural caps comprising TTAGGG repeats bound by a protein complex known as shelterin, which protects the ends of chromosomes. Maintaining the length and integrity of telomeres is the job of the enzyme telomerase, which is composed of two subunits, the telomerase reverse transcriptase (Tert) and an RNA component (Terc), which is used as the template for adding the telomeric repeats to chromosome ends.

Gradual telomere shortening is a natural process that occurs as we age and cells repeatedly divide. Once a critically short telomere length has been reached, the cell is triggered to undergo senescence or apoptosis. “Indeed, this progressive shortening of telomeres with increasing age is considered one of the hallmarks of aging both in mice and humans,” the eLife authors write. Research also suggests that it may be possible to target telomere shortening as an approach to preventing or delaying age-related disorders. Preclinical studies have previously demonstrated the use of an AAV9 vector-based Tert gene therapy to lengthen telomeres, delay aging, increase lifespan and reduce age-related diseases in adult mice. 

As well as being a hallmark of cell aging, telomere shortening is also a feature of a number of diseases, including IPF in humans. Tert and Terc mutations can be a feature of both familial and sporadic cases of the lung disorder, although some sporadic cases of IPF are associated with short telomeres, even though there is no evidence of telomerase mutations, the CNIO researchers note. To investigate whether telomerase gene therapy could feasibly represent an approach to treating IPF in human patients with shortened telomeres, the Spanish team first had to test the approach in an animal model, and this meant developing a new mouse model that accurately mirrored the disease in humans. Recapitulating human pulmonary fibrosis in mice has traditionally involved inducing lung damage in animals by administering bleomycin to represent environmental damage. However, in contrast with IPF in human patients, these animals go into remission within a few weeks and do not develop telomere shortening.

The CNIO team previously developed a mouse with short telomeres, which could represent the foundation of a new, more representative IPF model. They found that administering low doses of bleomycin—which would normally be insufficient to cause pulmonary fibrosis in wild-type mice—to these telomerase-deficient animals resulted in what they call full-blown progressive pulmonary fibrosis. “…this model shows that short telomeres are at the molecular origin of pulmonary fibrosis and could represent a useful preclinical tool to test the challenging hypothesis of whether therapeutic strategies based on telomerase activation maybe effective in the treatment of the disease,” the team suggests.

The researchers were then able to test an AAV9-Tert gene therapy in these short-telomere IPF mice. The treatment resulted in significantly reduced inflammation, rapid regression of pulmonary fibrosis, and improved pulmonary function within just 1–3 weeks. Even more encouragingly, a significant percentage of the treated IPF mice were completely cured within just eight weeks.

Further investigation showed that the AAV9 vector preferentially targeted and promoted increased proliferation of regenerative alveolar type II cells (ATII) cells in the lungs, and was associated with telomere lengthening and reduced DNA damage, apoptosis, and senescence. “As a consequence, Tert-treated mice show a better pulmonary function as well as decreased inflammation and decreased fibrosis (lower collagen depots),” the authors write. “These results are in line with the notion that short telomeres can impair the ability of stem cells to regenerate tissues, and with recent findings suggesting that IPF is the result of defective regeneration upon repetitive epithelial cell injury.”

Interestingly, genetic analysis showed that AAV9-Tert expression in fibrotic lungs led to downregulation of pathways involved in fibroblast activation, and in particular, downregulation of the transforming growth factor-β (TGF-β) pathway, the researchers state. “Indeed, gene expression analysis of isolated ATII cells from Tert-treated mice cells showed downregulation of p53 signaling, apoptotic pathways, and of several inflammation related pathways, including the TGF-β, NF-469 KappaB, IL-2 [interleukin-2],  and TNF [tumor necrosis factor] signaling pathways as early as early as 1 week after Tert treatment. Dampening of inflammation upon Tert treatment was further demonstrated by decreased levels of a large number of cytokines already at 3 weeks post-viral treatment that were maintained all throughout the experiment.”

The authors stress that in contrast with currently approved IPF treatments, pirfenidone and nintedanib, which can’t help reverse disease, the AAV9-Tert therapy speeds regression of established pulmonary fibrosis in mice. “We would like to propose here that this might be due to the fact that AAV9-Tert therapy targets one of the molecular causes of the disease, namely short telomeres, which in turn we show here that results in decreased DNA damage and improved proliferative potential of the ATII cells, and subsequently in decreased fibrosis and inflammation,” they conclude. “ …we observe that telomerase gene therapy reverses the fibrotic process in mice, which suggests that it could be effective in human patients, opening a new therapeutic opportunity towards the treatment of this disease,” states Juan Manuel Povedano, Ph.D., co-first author of the eLife paper.

And given that telomere shortening is an indicator of organism aging, Blasco points out that the new studies represent “the first time that pulmonary fibrosis has been treated as an age-related disease, looking for rejuvenating the affected tissues.”

Work is now needed to translate the technology used in the mouse model into a prototype human therapy. “The strategy devised by the CNIO group is very encouraging,” concurs co-researcher Fàtima Bosch, Ph.D., at the Centre of Animal Biotechnology and Gene Therapy, Autonomous University of Barcelona. “…although we are still far from reaching the clinic, we are already generating gene therapy vectors for human therapy”.

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