Molecular Therapy. Nucleic Acids最新文献

Inner ear hair cell (HC) damage is irreversible in mammals, but it has been shown that supporting cells (SCs) have the potential to differentiate into HCs. Serpine2, a serine protease inhibitor, encodes protease nexin 1, and this has been suggested to be a factor that promotes HC regeneration. In this study, we overexpressed Serpine2 in inner ear SCs cultured in two- and three-dimensional systems using the adeno-associated virus-inner ear (AAV-ie) vector, which promoted organoid expansion and HC differentiation. Overexpression of Serpine2 in the mouse cochlea through the round window membrane (RWM) injection promoted SC proliferation and HC regeneration, and the regenerated HCs were found to be derived from Lgr5⁺ SCs. Regenerated HCs have electrophysiological properties that are similar to those of native HCs. Notably, Serpine2 overexpression promoted HC survival and restored hearing of neomycin-damaged mice. In conclusion, our findings indicate that Serpine2 overexpression promotes HC regeneration and suggests that the utilization of inner ear progenitor cells in combination with AAVs might be a promising therapeutic target for hearing restoration.

There is an urgent need for agents that promote health and regeneration of cells and tissues, specifically to treat diseases of the aging nervous system. Age-associated nervous system degeneration and various diseases are driven by many different biochemical stresses, often making it difficult to target any one disease cause. Our laboratory has previously identified DNA aptamers with apparent regenerative properties in murine models of multiple sclerosis by selecting aptamers that bind oligodendrocyte membrane preparations. Here, we selected from vast libraries of molecules (∼10¹⁴ unique DNAs) those with the ability to bind cultured human SH-SY5Y neuroblastoma cells as a neuronal model, followed by screening for aptamers capable of eliciting biological responses, with validation of binding in differentiated SH-SY5Y, human induced pluripotent stem cell (iPSC)-derived sensory neurons, and human embryonic stem cell (hESC)-derived cortical neurons. This demonstrates a proof-of-concept workflow to identify biologically active aptamers by cycles of cell selection.

CHD6, a member of the chromodomain helicase DNA-binding protein family, has been implicated in various diseases and tumors. However, its precise binding model of CHD6 on regulatory functional genes remains poorly understood. In this study, we discovered sharp peaks of CHD6, as the first member of CHD family for housekeeping process, binding only to the promoter region of genes in the C4-2 cell line. These genes, with conserved sharp CHD6 peaks across tumor cells, likely represent housekeeping genes ADNP and GOLGA5. Genes with sharp CHD6 peaks exhibit stable and low expression levels, sharing epigenetic features similar to housekeeping genes. Furthermore, this regulatory model also exists in both HEK293 cells and cardiomyocytes. Overall, the results of this study demonstrate that CHD6 binds to the promoter regions of housekeeping genes, regulating their histone modifications, chromatin structure, and gene expression.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) can be achieved by ectopic expression of cardiac transcription factors (TFs) via viral vectors. However, risks like genomic mutations, viral toxicity, and immune response limited its clinical application. Transactivation of endogenous TFs emerges as an alternative approach that may partially mitigate some of the risks. In this study, we utilized a modified CRISPRa/dCas9 strategy to transactivate endogenous reprogramming factors MEF2C, GATA4, and TBX5 (MGT) to induce iCMs from both mouse and human fibroblasts. We identified single-guide RNAs (sgRNAs) targeting promoters and enhancers of the TFs capable of activating various degrees of endogenous gene expression. CRISPRa-mediated Gata4 activation, combined with exogenous expression of Mef2c and Tbx5, successfully converted fibroblasts into iCMs. Despite extensive sgRNA screening, transactivation of Mef2c and Tbx5 via CRISPRa remained less effective, potentially due to de novo epigenetic barriers. While future work and refined technologies are needed to determine whether cardiac reprogramming could be achieved solely through CRISPRa activation of endogenous factors, our findings provide proof of concept that reliance on exogenous TFs for reprogramming can be reduced through CRISPRa-mediated activation of endogenous factors, particularly Gata4, offering a novel strategy to convert scar-forming fibroblasts into iCMs for regenerative purposes.