VP of R&D Shenzhen GenTurn Life Co. Ltd., China (People's Republic)
Abstract: Partial reprogramming (PR) is an emerging therapeutic approach to rejuvenate cells via epigenetic remodeling. However, methods to precisely constrain PR from altering cell identity in clinical applications remain elusive. To address this challenge, we established initiation phase partial reprogramming (IPR) involving two key techniques to retain cellular reprogramming in the initiation phase, thus extending the time window for rejuvenating cells without losing their identities. Firstly, we built an AI-assisted protein design pipeline to generate a liquid-liquid phase separation (LLPS)-deficient OCT4 which loses the ability to promoting cellular reprogramming towards late stages while continuously exerting rejuvenation effects. Secondly, we built an AI-assisted circular mRNA (circRNA) design pipeline to generate two multi-gene circRNA vectors, one co-expresses the wild-type OCT4, SOX2, and KLF4 (OSK), and the other co-expresses the LLPS-deficient OCT4, SOX2, and KLF4 (O’SK). The two vectors were capsulated with LNP and tested to reprogram human skin fibroblasts derived from aged donors. DdPCR examination of the embryonic stem cell-related gene (ESRG) showed that OSK induced ESRG expression quickly in the cells, neither did O'SK, indicating that O'SK is safer than OSK. The epigenetic ages were reduced 10 years by OSK and 14 years by O'SK. The SA-β-Gal-positive cells were decreased 80% by OSK and 92% by O'SK. Single-cell RNA-seq and ATAC-seq data revealed a novel set of transcription factors (TFs) related to cell survival and proliferation, rather than the pluripotency-related TFs, elevated in rejuvenated cells, indicating that the rejuvenation and dedifferentiation processes were controlled by different molecular mechanisms. Furthermore, the two circRNA vectors were examined in UV-radiated human skin tissues. The histological analysis showed 72%, 138%, 191%, and 64% recoveries in elastin, collagen I, III, and IV in the O'SK-treated group, and merely 45%, 75%, 65%, and 36% recoveries in the OSK-treated group, respectively. The rejuvenation effects of O’SK were much better than those of OSK. Collectively, these results demonstrate an augmented cell rejuvenation effect through IPR induced by the LLPS-deficient OCT4, thus offering a novel therapeutic strategy for reversing age-related diseases.
