Reversal of aging-induced and glaucoma-induced vision loss by in vivo epigenetic reprogramming

Abstract

Purpose : DNA methylation (Horvath epigenetic clock) is a biomarker of aging, but whether accumulating epigenetic signals simply denote age or regulate aging is unclear. In vitro reprogramming using the four Yamanaka genes (OSKM) removes epigenetic signals and dedifferentiates mature cells into iPS cells. However, in vivo OSKM reprogramming produces teratomas. We hypothesize that transient reprogramming in vivo, using only three Yamanaka genes (OSK) will partially reprogram cells and remove epigenetic signals, reversing age and restoring youthful cellular function without dedifferentiation or tumor formation.

Methods : Intravitreal injection of Dox-inducible AAV2-OSK (Oct4, Sox2, Klf4) resulted in retinal ganglion cell (RGCs) specific OSK expression. In the microbead-induced mouse glaucoma model, optomotor reflex (OMR) and pattern ERG (pERG) were measured at 3 wks, followed by intravitreal AAV2-OSK. At 4 wks post OSK treatment, OMR, pERG, and axon counts were performed. Negative controls: AAV-OSK-off, vehicle only, and no treatment. In aging, baseline OMR and pERG were measured in 4 and 12-month-old mice followed by intravitreal AAV2-OSK. 4 wks post OSK treatment, OMR and pERG were repeated. Specific sh-RNA knocked-down Tet 1,2 demethyl transferases. FACS sorted RGCs were used for RNA-seq and epigenetic clock analysis.

Results : Microbead injection induced elevated IOP for 4 wks, causing significantly reduced OMR, pERG, and axon counts in control groups. Transient OSK treatment, initiated after glaucoma-induced injury had occurred, significantly increased axon density (50.85±2.2 vs 42.21±1.2 axons 10⁴/mm²) and restored OMR (0.28±.01 vs 0.22±.01 cyc/deg) and pERG (7.04±.56 vs 4.07±.20 Amp μV) OSK-on vs OSK-off. In aged mice, transient OSK treatment increased OMR (0.32±.02 vs 0.22±.02 cyc/deg) and pERG (5.0±.5 vs 2.6±.2 Amp μV) that was blocked by knock-down of Tet 1,2 indicating a dependence on epigenetic reprogramming. RNA-seq analysis of purified RGCs from OSK treated and control mice (young vs old) indicated reprogrammed genes were predominantly in the sensory pathway. OSK treatment significantly reduced RGC age as measured by the epigenetic clock.

Conclusions : These data indicate DNA methylation is not just a correlate of age, but a regulator of aging. Old cells retain a “youthful” record of epigenetic information that can be accessed by partial reprogramming to restore tissue function and reverse aging.

This is a 2020 ARVO Annual Meeting abstract.