AAV9 gene therapy with TRF1 telomere protective protein in adult and old mice prolongs mouse health span

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-05-2018Los telómeros son estrucruras nucleoprotéicas que se encuentran en los extremos de los cromosomas lineales, y que consisten en repeticiones en tándem de la secuencia de ADN, TTAGGG y un complejo de seis proteínas, llamado shelterina, que protege al ADN telomérico y evita que estos sean reconocidos como rupturas de doble cadena de ADN, lo que desencadenaría una respuesta de daño al ADN, que podría conducir a la apoptosis o senescencia celular. Los telómeros están ubicados encima de las regiones codificantes y protegen a los cromosomas de la degradación y fusiones cromosómicas. Sin embargo, debido a la replicación incompleta de los extremos de los cromosomas lineares eucarióticos, los telómeros se acortan por cada ciclo celular, y a una longitud críticamente corta se comprometería la viabilidad celular. TRF1 es un componente esencial de complejo shelterina, con funciones importantes en la protección y la replicación de los telómeros. Se ha demostrado que la deficiencia de TRF1 en el contexto de diferentes tejidos de ratón conduce a la pérdida del homeostasis del tejido debido a la función deteriorada de las células madre. Publicaciones recientes sugieren que los niveles de TRF1 disminuyen durante el envejecimiento celular in vitro inducido a través de los pases de células. Además, la posterior sobreexpresión de TRF1 en estas células "envejecidas" en pasajes tardíos redujo el daño del ADN en los telómeros y disminuyó la senescencia, lo que sugiere que la pérdida de TRF1 relacionada con la edad puede contribuir al fenotipo de envejecimiento in vitro. Por lo tanto, en nuestro trabajo primero abordamos si disminución de los niveles de TRF1 se asocia con el envejecimiento in vivo en ratones. Para ello, medimos los niveles de TRF1 en tejidos de diferentes edades y encontramos que los niveles de TRF1 disminuyen durante el envejecimiento del organismo tanto en ratones como en humanos. Para evaluar si la pérdida natural de TRF1 durante el envejecimiento contribuye al empeoramiento relacionado con la edad, sobre-expresamos TRF1 a través de la terapia génica con el gen de TRF1 en ratones adultos (1 año de edad) y viejos (2 años de edad) y estudiamos sus efectos sobre el proceso de envejecimiento. Para tal fin, utilizamos el vector serotipo 9 del virus adeno-asociado no integrativo (AAV9), que transduce la mayoría de los tejidos del ratón, permitiendo así, una sobreexpresión moderada y transitoria de TRF1. La terapia génica con AAV9-TRF1 evitó significativamente, la disminución en las funciones neuromusculares y cognitivas, la tolerancia a la glucosa, el mantenimiento de la grasa subcutánea y la anemia crónica, todos ellos factores relacionados con la edad. Adicionalmente, aunque el tratamiento con AAV9-TRF1 no afectó significativamente la longitud media de los telómeros, encontramos una menor abundancia de telómero cortos y daño del ADN asociado a los telómeros en algunos tejidos. En resumen, estos resultados sugieren que el rescate de los niveles de TRF1, que disminuyen de forma natural durante el envejecimiento del ratón, mediante el uso de la terapia génica con AAV9-TRF1 resultaría en una mejor salud del rató

Telomerase gene therapy ameliorates the effects of neurodegeneration associated to short telomeres in mice

Neurodegenerative diseases associated with old age such as Alzheimer's disease present major problems for society, and they currently have no cure. The telomere protective caps at the ends of chromosomes shorten with age, and when they become critically short, they can induce a persistent DNA damage response at chromosome ends, triggering secondary cellular responses such as cell death and cellular senescence. Mice and humans with very short telomeres owing to telomerase deficiencies have an earlier onset of pathologies associated with loss of the regenerative capacity of tissues. However, the effects of short telomeres in very low proliferative tissues such as the brain have not been thoroughly investigated. Here, we describe a mouse model of neurodegeneration owing to presence of short telomeres in the brain as the consequence of telomerase deficiency. Interestingly, we find similar signs of neurodegeneration in very old mice as the consequence of physiological mouse aging. Next, we demonstrate that delivery of telomerase gene therapy to the brain of these mice results in amelioration of some of these neurodegeneration phenotypes. These findings suggest that short telomeres contribute to neurodegeneration diseases with aging and that telomerase activation may have a therapeutic value in these diseases

Small extracellular vesicles from young adipose-derived stem cells prevent frailty, improve health span, and decrease epigenetic age in old mice.

Aging is associated with an increased risk of frailty, disability, and mortality. Strategies to delay the degenerative changes associated with aging and frailty are particularly interesting. We treated old animals with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals, and we found an improvement in several parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function, as well as an important decrease in frailty. ADSC-sEVs induced proregenerative effects and a decrease in oxidative stress, inflammation, and senescence markers in muscle and kidney. Moreover, predicted epigenetic age was lower in tissues of old mice treated with ADSC-sEVs and their metabolome changed to a youth-like pattern. Last, we gained some insight into the microRNAs contained in sEVs that might be responsible for the observed effects. We propose that young sEV treatment can promote healthy aging

The effect of rapamycin on bovine oocyte maturation success and metaphase telomere length maintenance

Dr P. Kordowitzki was supported by KNOW consortium (Poland MS&HE, Decision No. 05-1/KNOW2/2015) and by a special statutory fund of DRI&P IAR&FR PAS in OlsztynMaternal aging-associated reduction of oocyte viability is a common feature in mammals, but more research is needed to counteract this process. In women, the first aging phenotype appears with a decline in reproductive function, and the follicle number gradually decreases from menarche to menopause. Cows can be used as a model of early human embryonic development and reproductive aging because both species share a very high degree of similarity during follicle selection, cleavage, and blastocyst formation. Recently, it has been proposed that the main driver of aging is the mammalian target of rapamycin (mTOR) signaling rather than reactive oxygen species. Based on these observations, the study aimed to investigate for the first time the possible role of rapamycin on oocyte maturation, embryonic development, and telomere length in the bovine species, as a target for future strategies for female infertility caused by advanced maternal age. The 1nm rapamycin in vitro treatment showed the best results for maturation rates (95.21±4.18%) of oocytes and was considered for further experiments. In conclusion, rapamycin influenced maturation rates of oocytes in a concentration-dependent manner. Our results also suggest a possible link between mTOR, telomere maintenance, and bovine blastocyst formation.S

Gene therapy with the 1 telomere gene rescues decreased 1 levels with aging and prolongs mouse health span

Altres ajuts: Research in Maria Blasco's laboratory is funded by the Ministerio de Economía y Competitividad, Plan Nacional I+D+I (SAF2013-45111-R), and by the Fundación Botín.The shelterin complex protects telomeres by preventing them from being degraded and recognized as double-strand breaks. 1 is an essential component of shelterin, with important roles in telomere protection and telomere replication. We previously showed that 1 deficiency in the context of different mouse tissues leads to loss of tissue homeostasis owing to impaired stem cell function. Here, we show that 1 levels decrease during organismal aging both in mice and in humans. We further show that increasing 1 expression in both adult (1-year-old) and old (2-year-old) mice using gene therapy can delay age-associated pathologies. To this end, we used the nonintegrative adeno-associated serotype 9 vector (9), which transduces the majority of mouse tissues allowing for moderate and transient 1 overexpression. 9-1 gene therapy significantly prevented age-related decline in neuromuscular function, glucose tolerance, cognitive function, maintenance of subcutaneous fat, and chronic anemia. Interestingly, although 9-1 treatment did not significantly affect median telomere length, we found a lower abundance of short telomeres and of telomere-associated damage in some tissues. Together, these findings suggest that rescuing naturally decreased 1 levels during mouse aging using 9-1 gene therapy results in an improved mouse health span

Gene therapy with the TRF1 telomere gene rescues decreased TRF1 levels with aging and prolongs mouse health span

The shelterin complex protects telomeres by preventing them from being degraded and recognized as double-strand DNA breaks. TRF1 is an essential component of shelterin, with important roles in telomere protection and telomere replication. We previously showed that TRF1 deficiency in the context of different mouse tissues leads to loss of tissue homeostasis owing to impaired stem cell function. Here, we show that TRF1 levels decrease during organismal aging both in mice and in humans. We further show that increasing TRF1 expression in both adult (1-year-old) and old (2-year-old) mice using gene therapy can delay age-associated pathologies. To this end, we used the nonintegrative adeno-associated serotype 9 vector (AAV9), which transduces the majority of mouse tissues allowing for moderate and transient TRF1 overexpression. AAV9-TRF1 gene therapy significantly prevented age-related decline in neuromuscular function, glucose tolerance, cognitive function, maintenance of subcutaneous fat, and chronic anemia. Interestingly, although AAV9-TRF1 treatment did not significantly affect median telomere length, we found a lower abundance of short telomeres and of telomere-associated DNA damage in some tissues. Together, these findings suggest that rescuing naturally decreased TRF1 levels during mouse aging using AAV9-TRF1 gene therapy results in an improved mouse health span.We would like to thank Rosa Serrano for mouse care and the confocal microscopy unit and the histopathology unit of CNIO for their assistance. The viral vectors were produced by the lab of Fatima Bosch. Fatima Bosch is an ICREA Academia recipient, Generalitat de Catalunya, SpainS