Capitalizing on liver tropism of adeno-associated viral (AAV) vectors, intravenous vector administration is commonly used to genetically modify hepatocytes, a strategy currently in clinical trials for a number of liver-based hereditary disorders. Although hepatocytes are known to exhibit extensive phenotypic heterogeneity influenced by liver zonation and dietary cycle, there is little data available for the tropism capacity, as well as the potential transcriptional dysregulation, of AAV vectors for specific liver cell types. To assess these issues, we employed single-cell RNA sequencing of the mouse liver after intravenous administration of the liver tropic AAVrh.10 vector to characterize cell-specific AAV-mediated transgene expression and transcriptome dysregulation. Wild-type 8-week-old male C57Bl/6 mice under normal feed cycle were randomly divided into three groups and intravenously administered phosphate-buffered saline (PBS), AAVrh.10Null (no transgene), or AAVrh.10mCherry (marker gene). Overall, a total of 46,500 liver cells were sequenced. The single-cell transcriptomic profiles were grouped into three separate clusters of hepatocytes (Ttr-enriched “Hep1,” Tat-enriched “Hep2,” and Alb-enriched “Hep3”) and multiple other cell types. The hepatocyte diversity was driven by glucose and lipid homeostasis signaling. Assessment of the transgene expression demonstrated that AAVrh.10 is primarily Hep1-tropic, with a 10-gene signature positively correlated with AAVrh.10-mediated transgene expression. The transgene expression was less in Hep2 and Hep3 cells with a high receptor tyrosine kinase phenotype. Importantly, AAVrh.10 vector interactions with the liver markedly altered the transcriptional patterns of all cell types, with modified genes enriched in pathways of complement and coagulation cascade, cytochrome P450, peroxisome, antigen processing and presentation, and endoplasmic reticulum protein processing. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.

中文翻译：

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静脉注射 AAVrh.10 载体后小鼠肝细胞特异性向性和转录失调的单细胞转录组分析。


利用腺相关病毒（AAV）载体的肝脏趋向性，静脉注射载体通常用于对肝细胞进行基因改造，这是目前针对许多肝脏遗传性疾病的临床试验中的一种策略。尽管已知肝细胞表现出受肝脏分区和饮食周期影响的广泛表型异质性，但关于特定肝细胞类型的 AAV 载体的向性能力以及潜在的转录失调的数据很少。为了评估这些问题，我们在静脉注射肝向 AAVrh.10 载体后对小鼠肝脏进行单细胞 RNA 测序，以表征细胞特异性 AAV 介导的转基因表达和转录组失调。将正常喂养周期下的野生型8周龄雄性C57Bl/6小鼠随机分为三组，静脉注射磷酸盐缓冲盐水（PBS）、AAVrh.10Null（无转基因）或AAVrh.10mCherry（标记基因） 。总体而言，总共对 46,500 个肝细胞进行了测序。单细胞转录组谱分为三个独立的肝细胞簇（富含Ttr的“Hep1”、富含Tat的“Hep2”和富含Alb的“Hep3”）和多种其他细胞类型。肝细胞多样性是由葡萄糖和脂质稳态信号驱动的。转基因表达的评估表明，AAVrh.10 主要是 Hep1 亲性的，10 基因特征与 AAVrh.10 介导的转基因表达呈正相关。具有高受体酪氨酸激酶表型的 Hep2 和 Hep3 细胞中转基因表达较少。重要的是，AAVrh。10载体与肝脏的相互作用显着改变了所有细胞类型的转录模式，修饰的基因富含补体和凝血级联、细胞色素P450、过氧化物酶体、抗原加工和呈递以及内质网蛋白加工的途径。这些观察结果提供了对 AAV 介导的肝脏基因转移的肝细胞特异性后果的见解，远远超出了众所周知的载体传递转基因的器官特异性表达。