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Editors Selection IGR 22-2

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Comment by Derek Welsbie on:

111960 Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma, Karg MM; Lu YR; Refaian N et al., Cellular reprogramming , 2023; 25: 288-299

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Loss of epigenetic information has emerged as a key feature of aging and age-related disease (Yang et al., 2023). Seminal work by Shinya Yamanaka (Takahashi and Yamanaka, 2006) showed that overexpression of four transcription factors, OCT4, KLF4, SOX2 and MYC (OSKM), could fully rewind the epigenetic clock and reprogram adult somatic cells into pluripotent stem cells. Work by a collaborative team of investigators, led by David Sinclair, then showed that overexpression of just three of the four factors, minus MYC (OSK), could rescue some of the loss of epigenetic information without erasing cellular identify and generating undifferentiated stem cells (Lu et al., 2020). Using this approach, they showed improved axon regeneration in the mouse optic nerve crush model and improved retinal ganglion cell (RGC) function in the mouse microbead model of glaucoma. While exciting, there were several concerns with the study that were highlighted in an earlier edition of IGR (IGR 21-2). One of the senior authors from that study, Bruce Ksander, then set out to extend those findings by testing whether there was long-term visual improvement from OSK expression in the mouse microbead glaucoma model (Karg et al., 2023).

Karg et al. used intravitreally-injected adeno-associated virus (AAV) to express the OSK factors in mouse RGCs. Using both 'tet-on' and 'tet-off' designs, the expression of OSK factors could be controlled by systemic administration of the tetracycline analog, doxycycline. To test vision, the team turned to the optomotor reflex (OMR) in which the head of the mouse moves in response to a moving grating pattern. By varying the thickness of the vertical strips, it is possible to determine the resolution of vision (i.e., the minimal thickness before the mouse no longer recognizes that a grating exists and makes head movements). After showing a reduction in visual acuity at the four-week timepoint, the investigators transduced with AAV. This ensured that the team was looking at vision restoration and not the prevention of vision loss. Amazingly, with both the tet-on and tet-off strategies, there was rapid reversal of vision loss, claimed to be even better than the initial baseline. Unfortunately, extraordinary claims require extraordinary data and the study here was plagued by major omissions and inconsistencies. There was tremendous variability in gene expression between animals (with just a couple animals driving the entire effect), hints of tetracycline-regulated gene expression changes in the absence of the tetracycline activator, poor image quality, functional effects that were not sustained over time and a lack of structural data confirming the rescue (despite having measured structure with OCT). However, these are minor when compared to the fundamental problem ‐ there was no control OMR group (i.e., injury without OSK expression). Controls are a fundamental tenet of good science and simply showing that something gets better over time is inadequate. This is exacerbated by the fact that the OMR is a subjective test in which the investigator determines whether or not a head movement 'counts'. No masking was reported and, without controls, masking is not even possible at many of the timepoints. The team did attempt to show one control, that the effect in the tet-on arm was lost upon the discontinuation of doxycycline and regained by the reintroduction of doxycycline. However, that change in OMR was not obviously different than the normal day-to-day variability in uninjured 'saline' animals. Finally, the authors did use a complementary approach to measure RGC function, the pattern electroretinogram (PERG) but, unexplainably, there was no experimental PERG group (i.e., injury with OSK expression). For both the PERG and OMR, either there were never comparison groups or, perhaps more likely given their obviousness and the fact that all the tests can be run on the same animals, the data were acquired and then omitted because it complicated the conclusion of vision restoration. In any case, neither the OMR nor the PERG data can be interpreted without the relevant comparators.

The idea that OSK expression can be used to reverse aging and restore vision is very enticing. However, it is not clear that there has been a single, rigorous demonstration of this concept. The field desperately needs independent validation using appropriate controls.

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