Title: Reprogramming to recover youthful epigenetic information and restore vision

URL Source: http://www.nature.com/articles/s41586-020-2975-4

Markdown Content:
## Data availability

RRBS data for DNA methylation analysis and RNA sequencing data are available in the BioSample database (NCBI) and under BioProject [PRJNA655981](http://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA655981). Illumina Human Methylation EPIC array data are available in the Gene Expression Omnibus (GEO) database (NCBI) and under [GSE147436](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147436). All other relevant data that support the findings of this study are available from the corresponding author upon reasonable request.[Source data](http://www.nature.com/articles/s41586-020-2975-4#Sec50) are provided with this paper.

## Code availability

The code for determining methylation ageing signatures is provided in the Supplementary Information.

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## Acknowledgements

We thank A. Wagers, R. Mostoslavsky, Y. Shi, A. Das, A. Pogoutse, C. Petty, A. Coffey, B. Zhang, P. Dmitriev, K. Booher, E. Chen, J. Wang, D. Vogel, M. Thompson, A. Jacobi and S. Hou for advice and assistance; and Y. Weng, H. Song and F. Wang for reagents and mice. The work was supported by the Harvard Medical School Epigenetics Seed Grant and Development Grant; The Paul F. Glenn Foundation for Medical Research; a gift from E. Schulak; NIH awards R01AG019719 and R37AG028730 (to D.A.S.), R01EY026939 and R01EY021526 (to Z.H.), R01AG067782 and R01GM065204 (to V.N.G.) and R01AG065403 (to M.E.L. and V.N.G.). We thank Boston Children’s Hospital Viral Core, which is supported by NIH5P30EY012196; and Schepens Eye Institute Core facilities, supported by NEI-P30EY003790. X.T. was supported by NIH award K99AG068303 and by NASA Postdoctoral Fellowship 80NSSC19K0439; D.L.V. was supported by NIH training grant T32AG023480; J.-H.Y. was partially supported by National Research Foundation of Korea (2012R1A6A3A03040476); B.R.K. was partially supported by the St Vincent de Paul Foundation and by NEI awards R24EY028767 and R01EY025794; and M.S.G.-K. by NEI award R21EY030276. We thank P. F. Glenn for his mentorship and support of ageing research.

## Author information

Author notes

1.   These authors contributed equally: Benedikt Brommer, Xiao Tian, Anitha Krishnan, Margarita Meer

2.   These authors jointly supervised this work: Meredith S. Gregory-Ksander, Bruce R. Ksander, Zhigang He, David A. Sinclair

### Authors and Affiliations

1.   Department of Genetics, Blavatnik Institute, Paul F. Glenn Center for Biology of Aging Research, Harvard Medical School, Boston, MA, USA

Yuancheng Lu,Xiao Tian,Daniel L. Vera,Qiurui Zeng,Doudou Yu,Michael S. Bonkowski,Jae-Hyun Yang,Michael B. Schultz,Alice E. Kane,Karolina Chwalek,Luis A. Rajman&David A. Sinclair

2.   Department of Neurology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

Benedikt Brommer,Chen Wang,Songlin Zhou&Zhigang He

3.   Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

Benedikt Brommer,Anitha Krishnan,Chen Wang,Songlin Zhou,Emma M. Hoffmann,Margarete M. Karg,Ekaterina Korobkina,Meredith S. Gregory-Ksander,Bruce R. Ksander&Zhigang He

4.   Schepens Eye Research Institute of Mass Eye and Ear, Harvard Medical School, Boston, MA, USA

Anitha Krishnan,Emma M. Hoffmann,Margarete M. Karg,Ekaterina Korobkina,Meredith S. Gregory-Ksander&Bruce R. Ksander

5.   Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Margarita Meer&Vadim N. Gladyshev

6.   Department of Pathology, Yale School of Medicine, New Haven, CT, USA

Margarita Meer&Morgan E. Levine

7.   Department of Genetics, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

Noah Davidsohn&George M. Church

8.   Department of Molecular Biology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

Konrad Hochedlinger

9.   Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

Steve Horvath

10.   Laboratory for Ageing Research, Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, New South Wales, Australia

David A. Sinclair

Authors

1.   Yuancheng Lu
2.   Benedikt Brommer
3.   Xiao Tian
4.   Anitha Krishnan
5.   Margarita Meer
6.   Chen Wang
7.   Daniel L. Vera
8.   Qiurui Zeng
9.   Doudou Yu
10.   Michael S. Bonkowski
11.   Jae-Hyun Yang
12.   Songlin Zhou
13.   Emma M. Hoffmann
14.   Margarete M. Karg
15.   Michael B. Schultz
16.   Alice E. Kane
17.   Noah Davidsohn
18.   Ekaterina Korobkina
19.   Karolina Chwalek
20.   Luis A. Rajman
21.   George M. Church
22.   Konrad Hochedlinger
23.   Vadim N. Gladyshev
24.   Steve Horvath
25.   Morgan E. Levine
26.   Meredith S. Gregory-Ksander
27.   Bruce R. Ksander
28.   Zhigang He
29.   David A. Sinclair

### Contributions

Y.L. and D.A.S. conceived the project. Y.L., X.T. and D.A.S. wrote the manuscript with input from all co-authors. Y.L. was involved in all experiments and analyses. M.S.B. and J.-H.Y. provided early training to Y.L. B.B., C.W., Q.Z., D.Y., S.Z. and Z.H. contributed to the optic nerve crush studies and imaging. A.K., D.Y., Q.Z., E.M.H., E.K., M.S.G.-K. and B.R.K. contributed to the glaucoma and ageing studies. M.M.K. and B.R.K. performed OCT imaging and analysis. M.M. and V.N.G. conducted ribosomal DNA methylation age analysis for mouse RGCs. M.E.L. developed the DNA methylation ageing signature. D.L.V. performed the RNA sequencing and gene association analysis. X.T. conducted human neuron experiments. S.H. conducted the human methylation clock analysis. X.T., J.-H.Y. and K.H. helped with the work on transgenic mouse fibroblasts. M.S.B., X.T., M.B.S., A.E.K. and L.A.R. helped with systemic AAV9 experiments. N.D. and G.M.C. helped with plasmid constructs and AAV9 production. K.C. helped with grant applications and project management.

### Corresponding author

Correspondence to [David A. Sinclair](mailto:david_sinclair@hms.harvard.edu).

## Ethics declarations

### Competing interests

D.A.S. is a consultant to, inventor of patents licensed to, board member of and equity owner of Iduna Therapeutics, a Life Biosciences company developing epigenetic reprograming therapies. D.A.S. is an advisor to Zymo Research, an epigenetics tools company. Additional disclosures are at [https://genetics.med.harvard.edu/sinclair/people/sinclair-other.php](https://genetics.med.harvard.edu/sinclair/people/sinclair-other.php). Y.L., L.A.R. and S.H. are equity owners of Iduna Therapeutics, a Life Biosciences company. D.L.V. is an advisor to Liberty Biosecurity. M.S.B. is a shareholder in MetroBiotech. K.C. is an equity owner in Life Biosciences and affiliates. N.D. and G.M.C. are co-founders of Rejuvenate Bio. Disclosures for G.M.C. can be found at [http://arep.med.harvard.edu/gmc/tech.html](http://arep.med.harvard.edu/gmc/tech.html). M.E.L. is a bioinformatics advisor to Elysium Health. Y.L., N.D. and D.A.S. are inventors on patents arising from this work (WO/2020/069373 and WO/2020/069339), filed by the President and Fellows of Harvard College. The other authors declare no competing interests.

## Additional information

**Peer review information**_Nature_ thanks Andrew Huberman, Hongjun Song, Yasuhiro Yamada and the anonymous reviewer(s) for their contribution to the peer review of this work.Peer reviewer reports are available.

**Publisher’s note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## Extended data figures and tables

### [Extended Data Fig. 1 Effectiveness and safety of OSK reprogramming.](http://www.nature.com/articles/s41586-020-2975-4/figures/5)

**a**, Experimental outline for testing the effects of OSKM and OSK on gene expression in fibroblasts from young and old transgenic(TG) mice. **b**, **c**, Expression of OSKM (**b**, R26 rtTA; Col1a1 OSKM, _n_=3 biological replicates each condition) and OSK (**c**, R26 rtTA; Col1a1 OKS-mCherry, _n_=3 and 8 biological replicates) rescue age-associated transcriptional changes without inducing _Nanog_ mRNA. mo, month(s). qPCR primers are listed in Supplementary Table [7](http://www.nature.com/articles/s41586-020-2975-4#MOESM1). **d**, AAV-ubiquitinC (UbC)-rtTA and AAV-TRE-Luc vectors for measuring tissue distribution. **e**, Luciferase imaging of WT mice 2 months after intravenous injection (retro-orbital) of AAV9-UbC-rtTA;TRE-Luc (1.0 × 10 12 gene copies total). DOX was delivered in drinking water (1 mg ml−1) for 7 days to +DOX mice. **f**, Luciferase imaging of the eye (Ey), brain (Br), pituitary gland (Pi), heart (He), thymus (Th), lung (Lu), liver (Li), kidney (Ki), spleen (Sp), pancreas (Pa), testis (Te), adipose (Ad), muscle (Mu), spinal cord (SC), stomach (St), small intestine (In) and caecum (Ce) 2 months after retro-orbital injection of AAV9-UbC-rtTA;TRE-Luc followed by treatment with DOX for 7 days. The luciferase signal was primarily in the liver. Imaging the same tissues with a longer exposure time (right) with the liver removed revealed a strong signal in the pancreas. **g**, Toxicity and safety studies in young and old mice after in vivo delivery of OSK-expressing AAVs. In **h**, **i**, at the age of 5 months, mice were intravenously injected with AAV9-rtTA;TRE-OSK (3 and 7 × 10 11 gene copies of AAV per mouse). After 1 month, mice remained untreated (−DOX) or were treated with DOX (+DOX) for 18 months. WT mice were not injected with AAV. In **j**–**n**, at the age of 21 months, mice were injected intravenously with 5 × 10 11 gene copies of AAV9-rtTA and 7 × 10 11 of either AAV9-TRE-GFP (GFP) or TRE-OSK (OSK) per mouse. After 1 month, GFP, OSK, and non-injected WT mice were treated with DOX for 10 months. **h**, SOX2 expression in the liver of WT mice 2 months after intravenous delivery of OSK-expressing AAV9s with or without a month of DOX induction, and in the liver of OSK transgenic mice, 129S1/C57BL/6J mixed background. Uncropped scans in are shown in Supplementary Fig. [1](http://www.nature.com/articles/s41586-020-2975-4#MOESM1). **i**, Body weight of WT mice, OSK transgenic mice, and AAV-mediated OSK-expressing mice with or without DOX in the first 4 weeks (left; _n_=5, 3, 6, 4, 4 and 3 mice) and after 17 months (right, _n_=5, 3, 6 and 4 mice). **j**, Examples of liver sections from WT or GFP mice showing the infection of AAV9. Scale bar, 100 μm. **k**, KLF4 and GFP protein levels in the livers of WT, GFP and OSK mice at 32 months of age. * indicates high OSK expression, + indicates induced protein expression levels in livers of OSK transgenic mice. Uncropped scans are shown in Supplementary Fig. [1](http://www.nature.com/articles/s41586-020-2975-4#MOESM1). **l**, Tumour incidence in WT, GFP, and OSK mice at age 32 months after 10 months of DOX induction. **m**, **n**, Liver tumour scores (**m**) and white blood cell counts (**n**) for WT, GFP and OSK groups at age 32 months after 10 months of DOX induction. OSK mice were defined as either high expression (indicated by * in **k**) or low expression (WT, _n_=11 mice; GFP, _n_=10 mice; OSK high, _n_=7 mice; OSK low, _n_=8 mice). For **m**, **n**, there was no difference between the groups using one-way ANOVA. All data are presented as mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM12)

### [Extended Data Fig. 2 Normal architecture and absence of tumours in the retina after long-term OSK expression mediated by AAV2 delivery.](http://www.nature.com/articles/s41586-020-2975-4/figures/6)

**a**, **b**, Representative wholemount retina display of RBPMS (a RGC marker) and _Klf4_ immunofluorescence showing (**a**) expression from the AAV2 Tet-Off system can be turned off by DOX in drinking water (2 mg ml−1, 3 days), and (**b**) expression from the AAV2 Tet-On system can be turned on by DOX (2 mg ml−1, 2 days). Scale bars, 1 mm; _n_=4 retinas for each condition. **c**, Corresponding retinal wholemount images stained for RBPMS and _Klf4_ are shown for each group tested: top, no injection, _n_=6; middle, −OSK (intravitreal injection of AAV2-rtTA;TRE-OSK without DOX induction), 10 months post-injection, _n_=3; bottom, +OSK (intravitreal injection of AAV2-tTA;TRE-OSK with DOX induction), 15 months post-injection, _n_=6. All retinas are from 16-month-old mice, showing similar expression within the group. Scale bars, 100 μm. **d**, Volume intensity projection of en-face OCT (optical coherence tomography) retinal image with a white line indicating the location of **e**. **e**, Representative retinal cross-section B-scan images. White box indicates the location of the high magnification scans in **f** (retinal layers: GCL,ganglion cell layer; INL,inner nuclear layer; ONL,outer nuclear layer and choroid). Videos of complete retinal cross-section B-scan images of the entire globe are provided as Supplementary Videos[1](http://www.nature.com/articles/s41586-020-2975-4#MOESM5)–[3](http://www.nature.com/articles/s41586-020-2975-4#MOESM7). **g**, Low- and high-power representative images of haemotoxylin and eosin (H&E)-stained cross-sections of corresponding eyes, verifying retinal layers. **h**, Quantitative measurements of retinal thickness, there was no difference between the groups at any location using two-way ANOVA with Bonferroni correction (_n_=6, 3 and 6, respectively). **i**, Immunosuppressed NOD scid gamma mice received a subretinal injection of approximately 10,000 human retinoblastoma tumour cells. The OCT image shows a small retinal tumour and increased retinal thickness 14 days post-injection, demonstrating the ability of the OCT scan to detect tumours. *Rb indicates retinoblastoma.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM13)

### [Extended Data Fig. 3 Polycistronic OSK induces long-distance axon regeneration post-injury without RGC proliferation.](http://www.nature.com/articles/s41586-020-2975-4/figures/7)

**a**, Proliferating cells in the optic nerve (for example, glial cells) in BrdU-injected mice as a positive control (_n_=2 nerves). BrdU staining co-localized with Ki67, a proliferation marker. **b**, Representative retina wholemount staining shows OSK-expressing RGCs do not stain for BrdU in the first or second week after crush injury; _n_=4 retinas. Scale bars, 100 μm. **c**, Imaging of optic nerves showing regenerating and sprouting axons with or without OSK AAV treatment, 12 weeks post-crush (wpc); _n_=2 nerves. Scale bars, 200 μm. **d**, Whole-nerve imaging showing CTB-labelled regenerative axons at 16 wpc in WT mice with intravitreal injection of AAV2-tTA;TRE-OSK (_n_=2 nerves). Scale bars, 200 μm. **e**, Survival of RBPMS-positive cells in the RGC layer transduced with different AAV2s, 16 dpc (_n_=6, 4, 4, 4, 4, 4, 8 and 4 eyes). All data are mean±s.e.m. **f**, **g**, Representative immunofluorescence (**f**) and sub-population proportion (**g**) of wholemount retinas transduced with a polycistronic AAV vector expressing _Oct4_, _Sox2_ and _Klf4_ in the same cell. White arrows designate triple-positive cells. _n_=3 retinas. Scale bars, 100 μm. **h**, **i**, Immunofluorescence (**h**) and sub-population proportion (**i**) of wholemount retinas transduced with AAVs separately encoding _Oct4_, _Sox2_ and _Klf4_. Red, blue, and green arrows designate single-positive cells, with a white arrow marking a triple-positive cell, and other arrows marking double-positive cells. _n_=3 retinas. Scale bar, 100 μm. One-way ANOVA with Bonferroni correction in **e**, with comparisons to d2EGFP shown.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM14)

### [Extended Data Fig. 4 Regenerative and pro-survival effects of OSK are RGC-specific and cell-autonomous.](http://www.nature.com/articles/s41586-020-2975-4/figures/8)

**a**, Effect of OSK expression on RGC survival in young (1-month-old, _n_=8), adult (3-month-old, _n_=5), and old (12-month-old, _n_=8) mice after optic-nerve crush-injury compared to expression of d2EGFP as a negative control (_n_=6, 5 and 6, respectively). **b**, Axon regeneration after OSK expression compared to d2EGFP controls in young (1-month-old, _n_=5, 6), adult (3-month-old, _n_=6), and old (12-month-old, _n_=4, 5) mice, 2 wpc. **c**, Number of RGCs in the intact, 2 wpc or 5 wpc retinas of 12-month-old mice expressing GFP (AAV2-tTA;TRE-d2EGFP, _n_=7, 6 and 6, respectively) or OSK (AAV2-tTA;TRE-OSK, _n_=5, 8 and 6, respectively). **d**, Axon regeneration in 12-month-old mice with OSK AAV or control AAV (d2EGFP) treatment, 5 wpc (_n_=5 nerves). **e**, Schematic of retinal structure showing Vglut2-Cre mice selectively expressing Cre in excitatory neurons such as RGCs, whereas Vgat-Cre mice selectively express Cre in inhibitory amacrine and horizontal cells. **f**, Schematic of the FLEx (flip-excision) Cre-switch system. AAV2-FLEx-tTA is inverted by Cre to express tTA and therefore induces OSK only in Cre-positive cells. **g**, Confocal image stack demonstrating delivery of AAV2-FLEx-tTA;TRE-OSK to intact Vglut2-Cre transgenic retinas, resulting in RGC-specific OSK expression (top) and robust axon regeneration in the optic nerve (bottom). White arrows indicate RBPMS+ (AP2-)-labelled RGCs that express _Klf4_ (green). _n_=4 independent replicates. **h**, Confocal image stack demonstrating delivery of AAV2-FLEx-tTA;TRE-OSK to intact Vgat-Cre transgenic retinas, resulting in amacrine-specific OSK expression (top) and poor axon regeneration in the optic nerve (bottom). White arrows indicate AP2+ (RBPMS-)-labelled amacrine cells that express _Klf4_ (green). _n_=4 independent replicates. **i**, Representative image of AAV-expressing or non-expressing RGCs in intact and crushed retinas 2 wpc with AAVs expressing d2EGFP or OSK. d2EGFP: AAV2-tTA;TRE-d2EGFP, _n_=6 retinas; OSK: AAV2-tTA;TRE-OSK, _n_=8 retinas. **j**, RGC survival rate (crushed/intact) of d2EGFP- (_n_=6 eyes) or _Klf4_-expressing cells (_n_=8 eyes) and their surrounding non-expressing cells indicating a cell-autonomous pro-survival effect of OSK-expressing RGCs after crush, 2 wpc. **k**, Frequency of d2EGFP- or _Klf4_-positive RGCs pre- or 2 weeks post-injury (_n_=4, 6, 6 and 8 eyes). Two-way ANOVA with Bonferroni correction in **a–d**, **j**; one-way ANOVA with Bonferroni correction in **k**. Scale bars (**g**–**i**), 100 μm. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM15)

### [Extended Data Fig. 5 OSK activates _Stat3_ in the absence of mTOR activation or global demethylation.](http://www.nature.com/articles/s41586-020-2975-4/figures/9)

**a**, Representative images of retinal wholemounts transduced with AAV2-tTA (−OSK) or AAV2-tTA;TRE-OSK (+OSK) in the presence or absence of crush injury after 3 days. Retinal wholemounts immunostained for _pStat3_, _Klf4_ and RBPMS. _n_=2 retinas each condition. **b**, Representative images of retinal wholemounts transduced with d2EGFP- or OSK-encoding AAV2 in the presence or absence of a crush injury. Retinal wholemounts immunostained for RBPMS and mTOR activation marker phosphorylated S6 (pS6). _n_=4 retinas for each condition. **c**, Percentage of pS6-positive RGCs in intact and crushed samples (_n_=4 retinas for each condition). **d**, Representative images of d2EGFP in retina expressed from the Tet-On AAV system. No GFP expression was observed in the absence of DOX. GFP expression reached peak levels 2 days after DOX induction and remained at a similar level at day 5 after induction. _n_=2 retinas each condition. **e**, Representative images of retinal d2EGFP expression using the Tet-Off AAV system with various durations of DOX treatments (2 mg ml−1). Once pre-treated with DOX to suppress expression (on DOX), GFP was sparse even on day 8 after DOX withdrawal, lower than peak expression (Never DOX). _n_=2 retinas each condition. **f**, Axon regeneration at 2 or 4 wpc in response to OSK induction either pre- or post-injury (_n_=4, 5, 5, 4 and 4 eyes, respectively). **g**, Correlation between ribosomal DNA methylation(DNAm) age and chronological age of sorted mouse RGCs (1 month, _n_=6; 12 months, _n_=2; 30 months, _n_=5), with the light blue region representing the confidence interval. _P_ value of the linear regression is calculated by two-sided _F_-test of overall significance. In agreement with previous studies, DNA methylation age estimates of neurons tend to be lower than their chronological age but remain correlated (see[Methods](http://www.nature.com/articles/s41586-020-2975-4#Sec10)). **h**, Average DNA methylation levels across the mouse genome in RGCs from different ages and treatments, based on 703,583 shared CpG sites from RRBS of all samples (combined strands), _n_=6, 8, 2, 8, 6, 4, 8, 8, 6, 5, 6, 4 and 5, respectively. **i**, Correlation of DNA methylation at each CpG site versus age (_x_-axis; 1 month, 12 months, 30 months) and versus injury (_y_-axis; intact, injured GFP). The heat map represents the number of sites located in each block of value coordinates. Pearson’s correlation coefficient, _r_=0.34, _P_<1e−200. **j**, Hierarchical clustered heat map of methylation levels of 4,106 CpGs that significantly changed in RGCs after crush injury (intact vs injured GFP, _q_<0.05) and the effect of OSK. **k**, Top biological processes associated with the 698 CpGs that were significantly altered by both injury and OSK. Two-way ANOVA with Bonferroni correction in **c**, **f**. Scale bars (**a**, **b**, **d**, **e**), 100 μm. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM16)

### [Extended Data Fig. 6 Protective and regenerative effect of OSK is dependent on TET1 and TET2.](http://www.nature.com/articles/s41586-020-2975-4/figures/10)

**a**, Mouse _Tet1_, _Tet2_ and _Tet3_ mRNA levels with or without OSK expression in RGCs (_n_=6 biological replicates each condition). The _P_ value indicated with an asterisk was calculated using an unpaired one-tailed _t_-test. **b**, Representative images of retinal wholemounts transduced with AAV2-tTA;TRE-OSK in combination with a AAV2-shRNA-YFP (yellow fluorescent protein) having either a scrambled sequence (sh-Scr) or a hairpin sequence to knockdown _Tet1_ (sh-Tet1) or _Tet2_ (sh-Tet2) expression, at titre ratio 5:5:1. Retinal wholemounts immunostained for _Klf4_. _n_=3 retinas for each condition. **c**, Quantification of shRNA-YFP AAV transduction in OSK-expressing RGCs (_n_=3 retinas for each condition). **d**, Mouse _Tet1_, _Tet2_ and _Tet3_ mRNA levels with sh-Scr (_n_=5), sh-Tet1 (_n_=4) or sh-Tet2 (_n_=5) YFP AAV2 in RGCs in the presence of OSK expression. **e**, **f**, Quantification of axon regeneration (**e**, _n_=4 eyes each condition) and RGC survival (**f**, _n_=10, 7 and 9 eyes) at 2 wpc in retinas co-transduced with AAV2- tTA;TRE-OSK;shRNA. **g**, Mouse _Stat3_ mRNA levels after knockdown using sh-Scr (_n_=5), sh-Tet1 (_n_=4) or sh-Tet2 (_n_=5) in RGCs in the presence of OSK expression. **h**, Cre-dependent Tomato expression in RGCs after intravitreal AAV2-Cre injection of Tomato reporter mice (Rosa-CAG-lox-STOP-lox-Tomato), and the co-expressed frequency of Cre and _Klf4_ (_n_=3 eyes). **i**, **j**, RGC survival (**i**) and representative longitudinal sections of regenerating axons in longitudinal sections (**j**) in response to OSK expression (AAV2-tTA;TRE-OSK, _n_=5 for each condition) compared to no expression (saline, _n_=3 and 4), 16 days after crush injury in _Tet2_ _flox/flox_ mice injected with saline (Tet2 WT) or AAV2-Cre (Tet2 cKO). Scale bars (**b**, **h** and **j**), 100 μm. Two-way ANOVA in **a**, **d**, **i**; unpaired two-tailed Student’s _t_-test in **g**; one-way ANOVA in **e**, **f**. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM17)

### [Extended Data Fig. 7 OSK-induced axon regeneration and survival require non-global active DNA demethylation through thymine DNA glycosylase.](http://www.nature.com/articles/s41586-020-2975-4/figures/11)

**a**, Representative images of retinal wholemounts transduced with sh-Scr-H2B-GFP or sh-TDG-H2B-GFP AAV2 s for 4 weeks, demonstrating that knockdown of thymine DNA glycosylase (TDG) increased levels of 5-hydroxymethylcytosine (5-hmC). _n_=4 retinas for each condition. **b**, Representative retinal wholemount images and images of longitudinal sections through the optic nerve showing CTB-labelled regenerative axons in WT mice, 16 dpc after an intravitreal injection of AAV2-tTA ;TRE-OSK in combination with AAV2-sh-Scr (sh-Scr) or AAV2-sh-TDG (sh-TDG) at titre ratio 5:5:1. _n_=4 retinas for each condition. **c**, **d**, Quantification of regenerating axons (**c**) and RGC survival (**d**) in OSK-treated mice 16 dpc with AAVs carrying sh-Scr or sh-TDG (_n_=4 nerves for each condition). **e**, Representative image of retinal wholemounts transduced with AAV2-tTA;TRE-OSK. Retinal wholemounts were immunostained for 5-methylcytosine (5-mC) and _Klf4_, showing a lack of global demethylation in OSK expressing RGCs. _n_=3 retinas. **f**, Representative images of retinal wholemounts transduced with AAV2 vectors encoding the HA-TET1 catalytic domain (TET1-CD) or its catalytic mutant (TET1-mCD) for 4 weeks, demonstrating that overexpression of TET1-CD decreases global 5-mC levels. _n_=3 retinas for each condition. **g**, **h**, Quantification of axon regeneration (**g**) and RGC survival (**h**) at 2 wpc in retinas transduced without or with AAV2 vectors encoding HA-TET1 CD mutant or HA-TET1 CD (_n_=3, 4 and 3 eyes). **i**, A schematic diagram illustrating passive demethylation and TDG-dependent active DNA demethylation. Scale bars (**a**, **b**, **e**, **f**), 100 μm. One-way ANOVA with Bonferroni’s multiple comparison test in **c**, **g**, **h**; unpaired two-tailed Student’s _t_-test in **d**. There was no difference between the groups in **g** and **h** using two-way ANOVA and one-way ANOVA, respectively. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM18)

### [Extended Data Fig. 8 OSK induces axon regeneration and reversal of DNA methylation age in human neurons.](http://www.nature.com/articles/s41586-020-2975-4/figures/12)

**a**, mRNA levels of mouse _Oct4_, _Sox2_ and _Klf4_ in human neurons transduced with vectors packaged by AAV-DJ, a recombinogenic hybrid capsid that is efficient for in vitro transduction. −OSK: AAV-DJ-tTA (_n_=3); +OSK: AAV-DJ-tTA;TRE-OSK (_n_=3). **b**, Percentage of cells in S phase, as measured by propidium iodide (PI)-staining (_n_=4). **c**, FACS profiles of G1, S and G2 phases in undifferentiated SH-SY5Y cells and differentiated cells transduced with −OSK and +OSK vectors. **d**, Experimental outline for testing axon regeneration in human neurons after vincristine (VCS) damage. **e**, **f**, DNA methylation (DNAm) age of human neurons without damage (intact), and 1 or 9 days after VCS damage in the absence (**e**) or presence (**f**) of OSK expression, measured using the skin and blood clock suited to in vitro studies (see[Methods](http://www.nature.com/articles/s41586-020-2975-4#Sec10)). The linear regression _P_ value in **e** (_P_=0.55) indicates nonlinear DNA methylation age changes, and in **f** (_P_=0.008) indicates a continuous decrease in DNA methylation age (_n_=3, 3 and 6). **g**, Average DNA methylation levels among 850,000 probes from the EPIC array in human neurons without damage (intact), and 1 or 9 days after VCS damage in the absence or presence of OSK expression (_n_=3, 3 and 6). **h**, **i**, Representative images (**h**, similar results were confirmed in two series of experiments) and quantification (**i**) of neurite area at different time points after VCS damage (_n_=6, 7, 5, 5, 7 and 5; 2 independent experiments). Cells were not passaged after damage to avoid cell–cell contact for quantifying maximum axon regeneration. **j**–**l**, Human _TET1_, _TET2_ and _TET3_ mRNA level with scrambled shRNA (sh-Scr) or sh-Tet2 AAV in human neurons in the presence or absence of OSK expression (_n_=4; 2 independent experiments). **m**, Representative images of human neurons in each AAV treated group, 9 days after VCS damage. Similar results were confirmed in three series of experiments. **n**–**p**, Neurite area (**n**), axon number (**o**) and axon length (**p**) in each AAV-treated group 9 days after VCS damage (_n_=20, 21, 24 and 23; 3 independent experiments). **q**, Mouse _Oct4_ mRNA levels (from OSK AAV) in human neurons with sh-Scr or sh-Tet2 AAV and in presence or absence of OSK AAV (_n_=4). **r**, The effect of mTOR inhibition by rapamycin (Rap,10 nM) on axon regeneration of differentiated neurons with or without OSK (_n_=18, 19, 13 and 11; 2 independent experiments). **s**, S6 phosphorylation levels in human neurons 5 days after treatment with rapamycin (Rap,10 nM). Similar results were seen in two independent experiments. Uncropped scans are shown in Supplementary Fig. [1](http://www.nature.com/articles/s41586-020-2975-4#MOESM1). **t**, Neurite area of neurons expressing TET1 catalytic domain (TET1-CD) or its catalytic mutant (TET1-mCD) 9 days after VCS damage (_n_=24, 28 and 21; 2 independent experiments). One-way ANOVA with Bonferroni’s multiple comparison test in **b**, **e**–**g** and **t**; two-way ANOVA with Bonferroni’s multiple comparison test in **a**, **i**, **j–l** and **n**–**r**. All bar graphs are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM19)

### [Extended Data Fig. 9 Vision restoration and regenerative effect of OSK rely on functional improvement of existing RGCs.](http://www.nature.com/articles/s41586-020-2975-4/figures/13)

**a**, Axon density and representative photomicrographs of PPD-stained optic nerve cross-sections, 4 weeks after microbead or saline injection (baseline, _n_=5 eyes each condition). Scale bars, 25 μm. **b**, Quantification of RGCs and representative confocal microscopic images from retinal flat-mounts stained with anti-Brn3a (red), an RGC-specific marker, and DAPI (4′,6-diamidino-2-phenylindole, blue), a nuclear stain, 4 weeks after microbead or saline injection (baseline, _n_=5 eyes each condition). Scale bar, 100 μm. **c**, Axon density and representative micrographs from PPD-stained optic nerve cross-sections, 4 weeks after AAV2 or PBS injection (treated, _n_=9, 7, 6 and 8 eyes). Scale, 50 μm. **d**, Quantification of RGCs and representative confocal microscopic images 4 weeks after PBS or AAV injection (treated, _n_=7, 5, 6 and 5 eyes). Scale bar, 100 μm. **e**, PERG measurement at different ages 4 weeks after −OSK (_n_=16, 14 and 11 eyes) or +OSK treatment (_n_=20, 12 and 14 eyes). Similar results from 2 independent experiments are combined. **f**, Visual acuity in 18-month-old mice treated with −OSK (_n_=11 eyes) or +OSK (_n_=14 eyes) AAV for 4 weeks. **g**, **h**, Axon (**g**; _n_=4, 6, 10 and 9 nerves) and RGC (**h**; _n_=5, 4, 10 and 8 retinas) density in 4- and 12-month-old-mice, 4 weeks after −OSK or +OSK AAV injection. **i**, Scatter plot of OSK-induced changes and age-associated changes in mRNA levels in RGCs, with differentially expressed genes labelled. Gene selection criteria are in Methods. **j**, Hierarchical clustered heat map showing relative mRNA levels of age-associated sensory perception genes in FACS-sorted RGCs from untreated young (5-month-old) or old (12-month-old) mice or old mice treated with either −OSK or +OSK AAV. Sensory genes were extracted from the mouse Sensory Perception (GO:0007600) category of the Gene Ontology database. Gene selection criteria are in Methods. −OSK: AAV2-rtTA;TRE-OSK for **c**–**h**, AAV2-TRE-OSK for **i**, **j**; +OSK: AAV2-tTA;TRE-OSK for **c**–**j**. Unpaired two-tailed Student’s _t_-test in **a**, **b**, **f**; one-way ANOVA with Bonferroni’s multiple comparison test in **c**, **d**; two-way ANOVA with Bonferroni correction in **e**, **g**, **h**. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM20)

### [Extended Data Fig. 10 OSK expression in old RGCs restores youthful epigenetic signatures.](http://www.nature.com/articles/s41586-020-2975-4/figures/14)

**a**, **b**, Top biological processes based on transcriptome data that were either lower expressed in old compared to young RGCs and reversed by OSK (**a**), or higher expressed in old RGCs compared to young and reversed by OSK (**b**). **c**, Heat map showing relative mRNA levels of genes involved in the negative regulation of neural projection development, among the 464 differentially expressed genes during ageing. The accumulation of the gene _Efemp1_ during ageing is suspected to have a role in diseases of the retina. **d**, RGC _Efemp1_ mRNA levels measured by qPCR (relative to GAPDH) compared between young mice, old mice, and old mice treated with −OSK or +OSK AAV. Old RGCs with sh-Scr, sh-Tet1 or sh-Tet2 knockdown combined with +OSK AAV are included for comparison (_n_=7, 6, 5, 4, 5, 5 and 6 eyes). **e**, Principal component 1 value of 1 month, 12 month and 30 month RGC training samples in the PCA analysis. Values are standardized to have a mean=0 and s.d.=1 (_n_=6, 2 and 6). **f**, DNA methylation ageing signatures of 6-week-old RGCs isolated from axon-intact retinas infected with GFP-expressing AAV, or from axon-injured retinas infected with GFP- or OSK- expressing AAV at 4 dpc (_n_=4, 4 and 8 eyes). **g**, Top biological processes associated with the 1,226 signature CpG sites. **h**, **i**, Transcription factor (TF) binding (**h**) and histone modifications (**i**) specifically enriched at the 1,226 signature CpG sites, compared to five sets of randomly selected CpGs. **j**, Correlation of _Tet1_ and _Tet2_ knockdown-induced changes in methylation (5-mC and 5-hmC together) at the selected CpGs. _r_=0.4, _P_=2.53e−45. **k**, Delta value of ribosomal DNA methylation age (months) of 12-month-old RGCs infected for 4 weeks with +OSK (_n_=5 retinas). Values are relative to the average of RGCs infected with −OSK AAV. **l**, Ribosomal DNA methylation age (months) of 12-month-old OSK-treated RGCs infected for 4 weeks with sh-Tet1 or sh-Tet2 (_n_=4, 5 retinas). Values are relative to the average of RGCs infected with sh-Scr. **m**, PERG amplitudes in old (12-month-old) mice treated with −OSK, +OSK or +OSK together with either sh-Scr or sh-Tet1/sh-Tet2-mediated knockdown for 4 weeks (_n_=8, 7, 5, 6 and 6 eyes). **n**, Working model. The loss of youthful epigenetic information during ageing and injury (including genome-wide changes to DNA methylation, acceleration of the DNA methylation clock, and disruption of youthful gene expression patterns) causes a decline in tissue function and regenerative capacity. OSK-mediated reprogramming recovers youthful epigenetic information, reverses the DNA methylation clock, restores youthful gene expression patterns, and improves tissue function and regenerative capacity, a process that requires active DNA demethylation by TET1/TET2 and TDG. The PRC2 complex may serve to recruit TET1 and TET2 to specific sites in the genome, and DNA methylation by DNA methyltransferases (DNMTs) may be important as well. One-way ANOVA with Bonferroni’s multiple comparison test in **d**–**f**, **m**. All data are mean±s.e.m.

[Source data](http://www.nature.com/articles/s41586-020-2975-4#MOESM21)

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### Cite this article

Lu, Y., Brommer, B., Tian, X. _et al._ Reprogramming to recover youthful epigenetic information and restore vision. _Nature_**588**, 124–129 (2020). https://doi.org/10.1038/s41586-020-2975-4

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*   Received: 31 July 2019

*   Accepted: 22 October 2020

*   Published: 02 December 2020

*   Version of record: 02 December 2020

*   Issue date: 03 December 2020

*   DOI: https://doi.org/10.1038/s41586-020-2975-4