Date: 8th December 2020
Ageing is a degenerative process that can lead to tissue and organ dysfunction. One possible cause of ageing is the accumulation of epigenetic noise, such as DNA methylation changes, that disrupts gene expression patterns and over time decreases tissue function and regenerative capacity. The central nervous system (CNS) is one such system that loses function and the ability to regenerate. Now, scientists have shown that gene therapy can reprogramme retina cells, restoring youthful DNA methylation patterns and transcriptomes, promoting axon regeneration after injury, and reversing vision loss in a mouse model of glaucoma and in aged mice.
In 2006, Shinya Yamanaka discovered that by adding just four genes into adult skin cells in mice, he could induce the cells to become like embryonic stem cells which he called induced pluripotent stem cells, or iPS cells. The now famous transcription factors, Oct4, Sox2, Klf4, and Myc (OSKM) are also known as the Yamanaka factors, and whilst they reliably create iPS cells, they can also cause unintended effects, which can lead to cells becoming cancerous.
The reprogramming by OSKM involves processes that erases cellular identity and resets DNA methylation age and previous work has suggested that OSKM may counteract normal ageing however, how to circumvent the cancerous side effects will be a crucial discovery for this research to translate.
Now scientists, from Harvard Medical School, US, led by David Sinclair have used the eye as a model CNS tissue, and have shown that ectopic expression of 3 of the Yamanaka factors Oct4, Sox2 and Klf4 can restore vision in mice by turning back the clock on aged eye cells in the retina to recapture youthful gene function, without the induction of tumours.
The team’s first aim was to reset the epigenome without erasing cell identity, which occurs when using all four Yamanaka factors. They decided to eliminate Myc from the process as it is an oncogene and is not required for the initiation of cellular programming. They used an adeno-associated virus (AAV) as a vehicle to deliver tightly controlled inducible expression of Oct4, Sox2 and Klf4 (OSK-AAV) into young and old mice.
To assess the safety of the therapy, they began by intravenously administering the OSK-AAV, and after 10-18 months of continuous OSK induction, there were no increased incidence of tumours and overall health remained unchanged in both groups of mice.
OSK therapy stimulated axon regeneration
Satisfied the therapy showed no long-term deleterious effects, the researchers then wanted to determine whether OSK could stimulate axon regeneration. Retinal ganglion cells (RGCs) of the CNS project axons away from the retina to form the optic nerve, and in neonates and embryos can regenerate upon injury – a phenomenon that is rapidly lost a few days after birth.
This time the team injected the OSK-AAV into the vitreous humour in an optic nerve crush-injury model mouse. Around 40% of the RGCs showed OSK expression once induced, and even after 15 months of continuous induction, OSK expression did not induce any tumours or structural changes in the retina.
By around 12-16 weeks post injury (two weeks post OSK-AAV injection), the team saw regenerating and sprouting RGC axon fibres, which was not seen when OSK expression was suppressed. Further studies revealed that all three factors were required for regeneration and single or dual administration of the factors was not suffucient. In addition, the age of the mice did not drastically alter the regeneration capacity, and successful regeneration was seen in both old and young mice.
The team then measured the DNA methylation age of RGCs and found upon injury RGCs experience an acceleration of DNA methylation age, which was reversed in OSK-treated mice. This reversal was enriched at genes that were associated with light detection and synaptic transmission, and was dependent on DNA demethylation.
OSK therapy to treat disease
Next, the team wanted to test whether OSK induction could restore the function of RGCs in a disease setting. As glaucoma is a leading cause of age-related blindness worldwide the team used a mouse model for this by increasing intraocular pressure unilaterally. Here, the OSK-treated glaucomatous eyes showed restored axon density equivalent to that in the non-glaucomatous eyes, with no evidence of RGC proliferation. Importantly, this was accompanied by a significant increase in visual acuity.
The discovery that OSK induction could effectively restore the DNA methylation age of RGCs after injury suggested that vision loss caused by natural ageing might be reversible too.
To address this the team treated elderly, 12-month-old mice, with diminishing vision due to normal ageing. Following treatment of the elderly mice, the gene expression patterns and electrical signals of the optic nerve cells were similar to young mice, and vision was restored. Restoration of visual acuity was not seen in 18-month-old mice, likely due to age-dependent increase in corneal opacity.
Conclusions and future applications
The team here show that OSK-AAV therapy can reverse the age of a complex tissue and restore its biological function in vivo, safely and without the induction of tumours. They use the eye as a model system, and demonstrated that ectopic expression of OSK can epigenetically restore aged neurons after injury and from natural ageing.
The work here represents the first example of vision-loss reversal after glaucomatous injury has occurred. Although it should be said that scientists have recently used gene therapy to deliver an adapter molecule, Protrudin, into the eye which stimulated axon regeneration of the damaged nerve fibres, and protected them from cell death after injury, offering another hope of a new treatment for glaucoma. However, vision restoration itself was not assessed in these studies.
Gene therapy has also been used to deliver a highly photosensitive multi-characteristic opsin (MCO1) protein into retina bipolar cells, bypassing degenerative photoreceptors and restoring vision in blind mice. In this case, the team were studying the effects of age-related macular degeneration rather than glaucoma. However, it would be interesting to determine whether OSK-AAV therapy could be targeted to the degenerative photoreceptors and restore these ageing cells.
The researchers are now trying to confirm their findings in further animal work, and hope to initiate clinical trials within two years to test the efficacy of the approach in people with glaucoma.
From a wider perspective the team suggest that epigenetic reprogramming, either by gene therapy or other means, could promote tissue repair and the reversal of age-related decline in humans, and that it could be used to treat a variety of age-related diseases affecting different organs. With aged populations currently at their highest in human history, this work may significantly contribute to tackling some of the world’s most pressing health issues associated with this phenomenon.
For more information please see the press release from Harvard Medical School
Lu, Y., B. Brommer, X. Tian, A. Krishnan, M. Meer, C. Wang, D. L. Vera, Q. Zeng, D. Yu, M. S. Bonkowski, J.-H. Yang, S. Zhou, E. M. Hoffmann, M. M. Karg, M. B. Schultz, A. E. Kane, N. Davidsohn, E. Korobkina, K. Chwalek, L. A. Rajman, G. M. Church, K. Hochedlinger, V. N. Gladyshev, S. Horvath, M. E. Levine, M. S. Gregory-Ksander, B. R. Ksander, Z. He and D. A. Sinclair (2020). “Reprogramming to recover youthful epigenetic information and restore vision.” Nature 588(7836): 124-129.
https://doi.org/10.1038/s41586-020-2975-4
