A Safer Path to Cellular Rejuvenation: Endogenous Oct4 Activation via CRISPR/dCas9 in Progeria Mouse Models

Although transient expression of OSKM can ameliorate cellular markers of aging these approaches risk cancer formation during cellular dedifferentiation. Oct4 is fundamental in induction and maintenance of the pluripotency network that is key for reprogramming initiation (Buganim et al., 2012) and its reactivation plays a critical role in removal of somatic epigenetic landscape facilitating epigenetic reprogramming (Wu and Schöler, 2014). A recent breakthrough study published in Aging Cell has shown that activating the endogenous expression of Oct4 alone is sufficient for rejuvenating cells in a progeria mouse model (Kim et al., 2023).

In this study, the authors harnessed a mutant dead Cas9 (dCas) that performs RNA-dependent DNA binding in the absence of endonuclease activity (Brezgin et al., 2019; Qi et al., 2013) and is fused to a functional domain that can activate expression through binding to a single-guide RNA (sgRNA) (Perez-Pinera et al., 2013). From testing 10 sgRNAs targeting Oct4 promoter regions, the authors identified an sgRNA targeting the Oct4 proximal promoter to be the most efficient for transcriptional activation of endogenous Oct4 without off-target effects.

The system was temporal and overcomes limitations of classic gene overexpression for induction of transgenesis, such as vector size. The effect was rapid and reversible and expression levels remained high, thus serving as a promising system for partial reprogramming that involves reversible induction of expression.

In vitro, the authors demonstrated that the dCas9-Oct4 activator can efficiently trigger epigenetic remodeling (Hansson et al., 2012; Polo et al., 2012), which is essential in the initial stages of OSKM-mediated cellular reprogramming. The activator upregulates H3K9me3 and downregulates H4K20me3 at the protein level, both of which are crucial for maintaining heterochromatin structure during cellular senescence. Interestingly, these effects were not observed with exogenous Oct4 expression alone. The authors also showed that the dCas9-Oct4 activator effectively alters the mRNA expression levels of mesenchymal-epithelial transition-related (MET-related) genes, which are often used to measure the early stages of reprogramming.

The activator promotes dedifferentiation of skin fibroblasts by inducing MET and suppressing EMT through endogenous Oct4 activation. In line with a landmark study by Ocampo et al. (2016), the authors demonstrated that activating endogenous Oct4 using the dCas9 activator in aged mouse fibroblasts transduced with mutant laminA/C (LMNA) effectively ameliorates several hallmarks of aging in vitro. These include reducing DNA double-strand breaks, downregulating stress response and senescence-related genes, and restoring H3K9me3 and H4K20me3 to youthful levels.

The authors confirmed that in vivo delivery of the AuNPs/dCas9 complex in a progeric mouse results in the expression of endogenous Oct4 in various tissues after 1 week, with ∼30%–40% of Oct4 positive cells. The authors further assessed the tumorigenic potential of endogenous Oct4 overexpression. Their results confirmed that endogenous Oct4 expression was transient in vivo, peaking at day 7 and going back to basal levels after 14 days in the aorta.

Tumor incidence was reduced in comparison with the exogenous OSKM induction in tetO-OSKM mouse model, and tumor-related gene expression remained at basal levels in the liver. In addition, transient Oct4 overexpression extended median lifespan by 5 weeks (+25%) and maximum lifespan by 13 weeks (+45%) of the progeric mice. Overall, body weight and appearance, including a reduction in the spine curvature in the progeria mice, were improved in the progeric model compared with nonprogeric wild-type controls.

The authors then investigated the aorta ultrastructure of the progeric animals, which is known to be one of the major causes of death in this syndrome. The histological analysis demonstrated an enhancement in the ultrastructure of aortic tissue, which included a restoration of the aortic tissue thickness phenotype. Finally, dCas9-Oct4 activator treatment tended to reduce the level of progerin at the protein level, thus participating in the rescue of the progeric phenotype.

Overall, the findings summarized in Figure 1 suggest that CRISPR/dCas9-mediated transient activation of endogenous Oct4 expression alone can efficiently alleviate age-associated phenotypes, providing new insights into the potential use of this approach as a longevity intervention that mitigates oncogenic risks associated with the in vivo reprogramming.

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FIG. 1.

Schematic summarizing of the findings of this study. Activation of endogenous Oct4 through CRISPR/dCas9 activator leads to the amelioration of some age-related hallmarks in vitro, including histones marks remodeling, DNA damage reduction, MET activation, and senescence inhibition. In vivo, this partial reprogramming through endogenous Oct4 also contributes to rescuing the pathological phenotype of progeric animals, including lifespan extension, aorta remodeling, and progerin inhibition. MET, mesenchymal-epithelial transition.