Telomere dysfunction drives pulmonary fibrosis and offers new therapeutic strategies

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27 de mayo de 2016La fibrosis pulmonar idiopática (FPI) es una enfermedad degenerativa de los pulmones. Los pacientes presentan una supervivencia media tras el diagnóstico de 2 a 3 años. La pérdida de células alveolares tipo 2 (ATII) y la pérdida de la capacidad regenerativa de los pulmones son características de la enfermedad. Mutaciones en los componentes de la enzima telomerasa o en proteínas relacionadas con la protección de los telómeros se han encontrado tanto en casos esporádicos como familiares. Telómeros críticamente cortos o disfuncionales pueden comprometer la regeneración tisular y son un factor de riesgo para el desarrollo de FPI. Nuevas estrategias y tratamientos son necesarios, sin embargo, la falta de modelos murinos que recapitulen la enfermedad humana han obstaculizado nuevos avances al respecto. Durante esta Tesis Doctoral hemos generado dos modelos murinos independientes de FPI producida por disfunción telomérica. En un modelo, se llevó a cabo la deleción de Trf1 en células ATII para producir una disfunción telomérica severa. En un segundo modelo, ratones con telómeros críticamente cortos, deficientes en telomerasa, fueron sometidos a un agente inductor de daño pulmonar, la bleomicina. En ambos modelos se desarrolló fibrosis pulmonar debido a daño telomérico. En ambos modelos también se observó la pérdida de función pulmonar debido al reemplazamiento de diferentes tipos celulares por tejido fibrótico e infiltrado inflamatorio. En conclusión, ambos modelos proporcionan una prueba del papel causal del daño en el ADN telomérico en el desarrollo de la FPI e identifican a los telómeros como nuevas dianas terapéuticas. El modelo de FPI inducido por telómeros cortos (ratones deficientes para Tert tratados con bleomicina) se utilizó posteriormente para comprobar la eficacia terapéutica de la re-expresión de telomerasa. Para ello usamos una estrategia de terapia génica con el vector AAV9 portando el gen Tert de la subunidad catalítica de la telomerasa. Encontramos que AAV9-Tert transduce preferentemente las células ATII. Se realizaró un seguimiento radiológico tras el tratamiento con AAV9-Tert y reveló una reversión de la fibrosis y remisión total en el 50% de los casos a las 7 semanas tras el diagnóstico, mientras que los animales tratados con el vector vacío seguían mostrando focos de fibrosis severa. Esta regresión fue asociada con un incremento de la longitud telomérica en células ATII, una disminución del daño en el ADN y disminución de la apoptosis y de la senescencia. El tratamiento con AAV9-Tert incrementó la proliferación de las células ATII y resultó en cambios en la expresión génica indicativos de la disminución de fibrosis e inducción de regeneración. Los resultados obtenidos en esta Tesis indican que la terapia génica con telomerasa constituye un tratamiento efectivo para la FPI asociada a telómeros cortos.Idiopathic pulmonary fibrosis (IPF) is a degenerative disease of the lungs with an average survival post-diagnosis of 2-3 years. Loss of alveolar type II (ATII) cells and impairment of the regenerative capacity of the lung are also characteristic of the disease. Mutations in components of the telomere-maintenance enzyme telomerase or in proteins important for telomere protection are found both in familiar and sporadic IPF cases. Critically short or dysfunctional telomeres can impair tissue regeneration and have been established as a risk factor to develop IPF. New therapeutic targets and treatments are necessary; however, the lack of mouse models that faithfully recapitulate the human disease has hampered new advances. Here, we generated two independent mouse models, which develop IPF owing to telomere dysfunction. Severe telomere dysfunction in the absence of telomere shortening was induced by Trf1 deletion in ATII cells leading to pulmonary fibrosis. Mice deficient for telomerase presenting critically short telomeres were treated with low dosage of bleomycin to induce IPF. Bleomycin inoculation synergized with short telomeres provoking fibrosis in the lungs. We show that both mouse models develop pulmonary fibrosis through induction of telomere damage. Loss of lung function owing to replacement of lung cell types by fibrotic and inflammatory infiltrates was observed in both mouse models. In conclusion, those mouse models provide proof of principle of the causal role of DNA damage stemming from dysfunctional telomeres in IPF development and identifying telomeres as promising targets for new treatments. The mouse model of pulmonary fibrosis owing to short telomeres generated (Tert-deficient mice subjected to a low bleomycin dose) was used to test the therapeutic efficacy of telomerase activation using AAV9 virus carrying the telomerase Tert gene. We find that AAV9-Tert preferentially targets ATII cells. AAV9-Tert treatment following radiological diagnosis of pulmonary fibrosis resulted in significant reversal of fibrosis and full remission in 50% of the mice 7 weeks after diagnosis, while all the mice treated with the empty vector still showed severe fibrosis. Reversal of fibrosis in AAV9-Tert treated mice was associated with an increase in telomere length in ATII cells, decreased DNA damage, and decreased apoptosis and senescence. AAV9-Tert treatment increased the percentage of proliferating ATII cells and lead to gene expression changes indicative of decreased fibrosis and increased regeneration. These results obtained during this Thesis indicate that telomerase gene therapy is effective in the treatment of pulmonary fibrosis provoked or associated with short telomeres

Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres

Pulmonary fibrosis is a fatal lung disease characterized by fibrotic foci and inflammatory infiltrates. Short telomeres can impair tissue regeneration and are found both in hereditary and sporadic cases. We show here that telomerase expression using AAV9 vectors shows therapeutic effects in a mouse model of pulmonary fibrosis owing to a low-dose bleomycin insult and short telomeres. AAV9 preferentially targets regenerative alveolar type II cells (ATII). AAV9-Tert-treated mice show improved lung function and lower inflammation and fibrosis at 1-3 weeks after viral treatment, and improvement or disappearance of the fibrosis at 8 weeks after treatment. AAV9-Tert treatment leads to longer telomeres and increased proliferation of ATII cells, as well as lower DNA damage, apoptosis, and senescence. Transcriptome analysis of ATII cells confirms downregulation of fibrosis and inflammation pathways. We provide a proof-of-principle that telomerase activation may represent an effective treatment for pulmonary fibrosis provoked or associated with short telomeres.We are indebted to D Megias for microscopy analysis, to J Mun˜ oz and F Garcı´a for hydroxiproline analysis as well as to CNIO Histopathological Unit. The research was funded by project SAF2013- 45111-R of Societal Changes Programme of the Spanish Ministry of Economics and Competitiveness (MINECO) co-financed through the European Fund of Regional Development (FEDER), Fundacio´n Botı´n and Banco Santander (Santander Universities Global Division) and Roche Extending the Innova- tion Network Program (EIN) Academia Partnering Programme.S