Gene Therapy-Mediated Partial Reprogramming Extends Lifespan and Reverses Age-Related Changes in Aged Mice

Abstract

Aging is a complex progression of changes best characterized as the chronic dysregulation of cellular processes leading to deteriorated tissue and organ function. Although aging cannot currently be prevented, its impact on life- and healthspan in the elderly can potentially be minimized by interventions that aim to return these cellular processes to optimal function. Recent studies have demonstrated that partial reprogramming using the Yamanaka factors (or a subset; OCT4, SOX2, and KLF4; OSK) can reverse age-related changes in vitro and in vivo. However, it is still unknown whether the Yamanaka factors (or a subset) are capable of extending the lifespan of aged wild-type (WT) mice. In this study, we show that systemically delivered adeno-associated viruses, encoding an inducible OSK system, in 124-week-old male mice extend the median remaining lifespan by 109% over WT controls and enhance several health parameters. Importantly, we observed a significant improvement in frailty scores indicating that we were able to improve the healthspan along with increasing the lifespan. Furthermore, in human keratinocytes expressing exogenous OSK, we observed significant epigenetic markers of age reversal, suggesting a potential reregulation of genetic networks to a younger potentially healthier state. Together, these results may have important implications for the development of partial reprogramming interventions to reverse age-associated diseases in the elderly.

Keywords: AAV; OSK; age reversal; epigenetic; gene therapy.

FIG. 1.

Partial reprogramming with TRE-OSK leads to increased lifespan and improved frailty scores in very old mice. (a) Schematic of the constructs, virus, and injection route used in the study. (b) Kaplan–Meier curves for 124-week WT mice injected with AAV9.TRE-OSK and AAV9.hEF1ɑ-rtTA4 (both 1E12 vg/animal) through the retro-orbital route, and induced with 1 week on/off doxycycline paradigm (TRE-OSK) showed median lifespan extension of remaining life by 109% compared with either doxycycline-treated control animals (Control-Dox) or to historical published Jax data for Bl6/J mice (Jax historical). Red arrow at top indicates AAV injections. Mantel Cox Log rank test, **p < 0.05. (c) Graph shows remaining lifespan of individual mice (after injections at week 124) for data shown in (b). Two-tailed unpaired t-test; **p < 0.05. (d) FI, the compound score of 28 different health parameters (range 0–1 in 0.5 increments), showed significant reduction in FI for TRE-OSK mice at 142 weeks of age (18 weeks after injections) as compared with Control-Dox mice. Student's unpaired t-test, **p < 0.05. AAV, adeno-associated virus; FI, frailty index; WT, wild-type.

FIG. 2.

Partial reprogramming with TRE-OSK leads to age reversal as assessed by DNA methylation age. (a) Measurement of DNA methylation age acceleration in mouse liver (left panel) and heart (right panel) from control doxycycline-treated mice (Control-Dox) or TRE-OSK mice using LUC epigenetic clocks trained on the indicated tissues. Age acceleration is the difference between clock age and chronological age (Supplementary Table S3) *p < 0.05, p = 0.0139 for liver and p = 0.0414 for heart by unpaired t-test. (b) Human keratinocytes isolated from the scalp of a 65-year-old male patient transduced with lentivirus at two different MOIs (0.5 and 1.0) expressing OSK showed epigenetic age reversal as compared with control GFP transduced or nontransduced (WT) cells. n = 2 technical repeats for each group. One-way ANOVA with Holm–Šídák's multiple comparisons test. ***p < 0.001. LUC, Lifespan Uber Correlation; ns, not significant.

FIG. 3.

AAV9-CMV-OSK expression in tissues of young and aged mice. qPCR of Oct4, Sox2, and Klf4 from 8-week-old (Young) and 82-week-old (Old) mice retro-orbitally injected with the AAV9 1.7e11 vg/mouse and uninfected controls, six different tissues were collected 12 weeks after injection. Level of each gene is normalized to that of controls receiving PBS. Multiple Mann–Whitney test with Holm–Šídák's multiple comparisons. **p < 0.05. PBS, phosphate buffered saline.