Accumulation of short/damaged telomeres with increasing age is considered one of the main sources of aging-associated DNA damage capable of causing loss of the regenerative capacity of tissues and systemic organismal aging both in humans and mice with impaired telomerase activity (Armanios et al, 2007; Blasco et al, 1997; Flores et al, 2005; Garcia-Cao et al, 2006; Herrera et al, 1999; Mitchell et al, 1999; Schoeftner et al, 2009; Tsakiri et al, 2007; Vulliamy et al, 2001; Yamaguchi et al, 2005). Telomerase activation is envisioned as a potential strategy to rejuvenate tissues and to treat diseases characterized by premature telomere shortening (de Jesus et al, 2011; Jaskelioff et al, 2011). Interestingly, even though mouse telomeres are much longer than human telomeres at younger ages in spite of their shorter life spans, recent evidence suggest that mouse telomeres suffer a dramatic shortening at old ages and that telomere length can be rate-limiting for mouse longevity (Flores et al, 2008; Garcia-Cao et al, 2006). This is further supported by the fact that telomerase activation can delay normal mouse aging in cancer resistant mice (Tomas-Loba et al, 2008). However, with the exception of mice genetically engineered to be cancer resistant, increased telomerase expression is associated with a higher susceptibility to develop cancer both in mice and humans (Artandi et al, 2002; Canela et al, 2004; Gonzalez-Suarez et al, 2001; McKay et al, 2008; Rafnar et al, 2009). Notably, in these studies increased TERT expression is forced since early embryo development through germ line modifications, which may favour the expansion of cancerous cells and the development of cancer later in life. Gene therapy approaches are currently envisioned as a way to deliver genes into adult tissues in order to correct genetic defects or diseases, however, to our knowledge have never been envisioned as a valid strategy to delay aging or extend longevity. Here, we show that increased TERT expression later in life (adult and old mice) by using a gene therapy strategy has rejuvenating effects without increasing cancer risk. In particular, TERT interventions based on adeno-associated vectors (AAV9-mTERT) at 1 or 2 years of age are able to re-activate telomerase activity in a wide range of tissues and have beneficial effects on different aspects of mouse health, including delayed osteoporosis, improved epithelial barrier fitness, improved metabolic function, and improved neuromuscular coordination. The fact that AAV9-TERT treatment of old mice efficiently reactivated telomerase in the lungs offers a therapeutic opportunity for some cases of human pulmonary fibrosis associated with presence of short telomeres owing to telomerase mutations (Armanios et al, 2007; Tsakiri et al, 2007). Importantly, AAV9-mTERT treated mice do not develop more cancer, illustrating the safety of this type of strategy. This is likely to be related to the fact that AAV vectors are non-integrative, and therefore mTERT over-expression will be lost in highly proliferating cells. In addition, AAV preferentially targets post-mitotic cells from peripheral tissues, which are considered more resistant to cancer than the highly proliferative ones, and could explain the tissue contribution for healthspan amelioration. Indeed, the limited gene transfer to some tissues (such as the brain) could account some of the modest effects observed.
Our TERT-based gene therapy of aging also improved several molecular markers of aging. On one hand, and as expected from the canonical function of TERT as the catalytic component of telomerase, mice treated with AVV9-mTERT vectors showed telomere elongation in a variety of tissues, which was concomitant with a significant decrease in the abundance of short telomeres, in turn responsible for chromosomal aberrations (Hemann et al, 2001; Samper et al, 2001). The increase in telomere length in quiescent/differentiated tissues could be related to prevention of stress-dependent telomere shortening (Sahin et al, 2011; Sharma et al, 2003), or to re-population of tissues by cells with longer telomeres (Hao et al, 2005). In particular, we and others have previously demonstrated that TERT over-expression could be acting on the pool of adult stem cells present in tissues, leading to their mobilization and subsequent tissue regeneration (Flores et al, 2005; Sarin et al, 2005). On the other hand, in agreement with the proposed role of TERT as activator of genes of the Wnt pathway (Park et al, 2009), tissues with increased TERT expression also showed increased expression of active β-catenin, as well as of its target gene cyclinD1, which in turn may mediate some of the known effects of TERT improving stem cell mobilization (Flores et al, 2005; Reya & Clevers, 2005; Sarin et al, 2005). Increased cyclinD1 was also paralleled by generally lower levels of p16 expression in some mouse tissues, a further sign of extended renewal and proliferative capacities associated with TERT expression. However, mice treated with a catalytically inactive TERT (AAV9-mTERT-DN), and despite showing increased CyclinD1 mRNA levels as well as decrease p16 levels in the tissues tested, in agreement with the proposed role of TERT regulating the Wnt pathway in a telomerase activity-independent manner (Park et al, 2009), were not paralleled by a rescue of short telomeres and increased health span and longevity. These could indicate that the non-canonical role of telomerase could not impact on the health status per se, but could have a synergistic effect in the context of a telomerase positive background.
Finally, by looking at markers of metabolic and mitochondrial fitness, we show here that normal aging comprises similar metabolic changes to those observed in aging produced by accelerated telomere shortening, as well as demonstrate that increased healthspan through telomerase overexpression is associated with protection from metabolic decline.
In conclusion, we provide proof-of-principle for the feasibility of anti-aging interventions on adult/old mammals. Aged organisms accumulate telomere-derived DNA damage and we show that it is possible to repair or delay the accumulation of this type damage through telomerase gene therapy. This has direct consequences on the health of the aged organisms including an increase in the average and maximal lifespan.