Dataset Information

Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors.

ABSTRACT: Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an ex vivo system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associated virus (AAV) vector AAV-DJ as a powerful tool to transduce mouse and human liver ductal organoids. Via AAV-DJ-mediated up- or down-regulation of target genes, we successfully manipulated cholangiocyte-to-hepatocyte differentiation. We induced differentiation by overexpressing the hepatocyte-specifying regulator hepatocyte nuclear factor 4? (HNF4?) and blocked differentiation by stimulating Notch signaling or interfering with Smad signaling. Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators. We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine.

SUBMITTER: Wei J

PROVIDER: S-EPMC6755792 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors.

Wei Jinsong J Ran Gai G Wang Xin X Jiang Ning N Liang Jianqing J Lin Xinhua X Ling Chen C Zhao Bing B

The Journal of biological chemistry 20190731 38

Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an <i>ex vivo</i> system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associat ...[more]

PMID: 31366731

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Project description:Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

Dataset Information

Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors.

Publications

Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors.

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