Unlocking the Therapeutic Potential of Hibernation-Induced Metabolic Reprogramming

Summary: Hibernation is a natural adaptation that allows certain mammals, such as the 13-lined ground squirrel, to survive extreme environmental stress through profound metabolic suppression and a shift from carbohydrate to lipid-based energy metabolism. These animals maintain vital organ function despite profound hypothermia, prolonged fasting, and sustained immobility, providing mechanistic insights with translational potential for organ preservation, thrombosis prevention, and targeted metabolic reprogramming for therapeutic purposes. However, the mechanisms by which these metabolic adaptations can be elucidated and translated into therapeutic strategies remain insufficiently characterized, posing a major barrier to their clinical application. To address this knowledge gap, we collected and performed transcriptomic analysis of liver tissue from hibernating versus active 13-lined ground squirrel. Notably, our pathway network analysis of transcriptomic data recapitulated the metabolic shift observed in hibernating squirrels, showing a marked transcriptional suppression of various biosynthetic pathways, while genes involved in fatty acid β-oxidation remained upregulated. These findings suggest that lipid catabolism is selectively maintained to support essential energy demands during deep metabolic suppression. Leveraging these insights, we constructed a focused CRISPR activation (CRISPRa) library and delivered it into human iPSC-derived hepatocytes (iHep) equipped with a dCas9-SAM system to enable precise and programmable transcriptional activation. These iHep are then subjected to glucose-deprived, lipid-enriched culture conditions designed to mimic the metabolic stress of hibernation. Through an unbiased killing curve selection screen, we aim to identify candidate genes that confer enhanced survival, metabolic plasticity, and adaptation to energy substrate shifts under these physiologically relevant stress conditions. To evaluate the in vivo relevance of candidate genes, we established a Cas9-expressing rat model that enables tissue-specific gene modulation. Using lipid nanoparticle (LNP)-mediated delivery of synthetic guide RNAs (gRNAs), we achieved efficient and selective gene knockout in the liver. In parallel, we employed a liver-targeted mRNA-LNP delivery platform to mediate transient, tissue-specific expression of exogenous genes in primary human hepatocytes. Together, these complementary in vivo systems allow for precise and programmable gene modulation in the liver, serving as a critical translational bridge between in vitro findings and physiological function. Ultimately, this work seeks to uncover master transcriptional regulator of energy metabolism, with wide-ranging implications for metabolic disease therapy, organ preservation, and even long-duration spaceflight requiring induced metabolic suppression. Jiahui Hou, Rodrigo M Florentino, Lanuza A P Faccioli, Zhiping Hu, Annalisa M Baratta, Bo Yang, Yiyue Sun, Deepak Nagrath, Sarah J Hainer, Hatice Ulku Osmanbeyoglu, and Alejandro Soto-Gutierrez