Correlation of gene expression changes and cell signalling pathways in insulin-expressing mouse liver cells transduced with an integrating adeno-associated viral vector

Type 1 Diabetes (T1D) is a chronic, metabolic disorder for which current treatments are unable to prevent the onset of complications. Previously, we used an adeno-associated viral vector (AAV8) to deliver furin-cleavable human insulin (INS-FUR) to the livers of diabetic non-obese diabetic (NOD) mice to reverse T1D. Use of the traditional AAV8-INS-FUR vector was not able to bring about normoglycaemia, however this vector, coupled with a transposon system, in the AAV8/piggyBac-INS-FUR vector, was able to do so. The aim of this study was to investigate the transcriptomic profiles of diabetic, AAV8-INS-FUR transduced, and AAV8/piggyBac-INS-FUR transduced livers and compared these to normal liver to identify genetic differences resulting from delivery of the AAV8/piggyBac-INS-FUR vector which produced normoglycaemia. Differen-tial gene expression was determined by RNA-Seq analysis and the differentially expressed genes from each treatment were mapped on to cellular pathways to determine cellular signalling and downstream effects of the treatments. We observed distinct differences between the piggyBac transduced and diabetic models, particularly in terms of metabolic function and the upregulation of key pancreatic markers in the liver of piggyBac transduced animals. The success of the piggyBac vector in achieving normoglycemia through stable transduction was evident, however further advancements are necessary to achieve complete pancreatic transdifferentiation of liver cells. RNA was extraction from 1) non-diabetic untreated liver 2) diabetic,non-treated liver, 3) AAV8-INS-FUR transduced liver 4) AAV8/piggyBac transduced liver

1型糖尿病（Type 1 Diabetes, T1D）是一种慢性代谢紊乱性疾病，当前治疗手段均无法阻止其并发症发作。此前本团队曾利用腺相关病毒载体（adeno-associated viral vector, AAV8）将弗林蛋白酶可切割的人胰岛素（INS-FUR）递送至糖尿病非肥胖糖尿病（NOD）小鼠的肝脏，以逆转1型糖尿病。传统AAV8-INS-FUR载体无法实现血糖正常化，但将该载体与转座子系统结合构建的AAV8/piggyBac-INS-FUR载体则可达成此目标。本研究旨在探究糖尿病小鼠肝脏、经AAV8-INS-FUR转导的肝脏以及经AAV8/piggyBac-INS-FUR转导的肝脏的转录组谱，并将其与正常肝脏进行比对，以识别由可实现血糖正常化的AAV8/piggyBac-INS-FUR载体递送所引发的基因表达差异。研究通过RNA测序（RNA-Seq）分析确定差异基因表达情况，并将各处理组的差异表达基因映射至细胞通路，以明确各处理方式的细胞信号传导及下游效应。结果显示，piggyBac转导组与糖尿病模型组之间存在显著差异，尤其体现在代谢功能以及piggyBac转导动物肝脏中关键胰腺标志物的上调方面。piggyBac载体通过稳定转导实现血糖正常化的效果已得到验证，但要实现肝细胞完全的胰腺转分化，仍需进一步的技术优化。本实验提取的RNA样本来自以下4组：1）非糖尿病未处理肝脏；2）未接受治疗的糖尿病肝脏；3）经AAV8-INS-FUR转导的肝脏；4）经AAV8/piggyBac转导的肝脏