Boychenko Stanislav, Abdullina Alina, Laktyushkin Viktor S, Brovin Andrew, Egorov Alexander D
Gene Therapy Department, Science Center for Translational Medicine, Sirius University of Science and Technology, 354340 Sirius, Russia.
Resource Center for Cell Technologies, Laboratory Complex, Sirius University of Science and Technology, 354340 Sirius, Russia.
Pharmaceuticals (Basel). 2024 Sep 18;17(9):1227. doi: 10.3390/ph17091227.
BACKGROUND/OBJECTIVES: Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry.
Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated.
AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5-2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 10 to 1.6 × 10 VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue.
Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes.
背景/目的：腺相关病毒（AAV）被广泛用作哺乳动物细胞基因递送的病毒载体。我们通过使用IncuCyte S3的活细胞成像显微镜和流式细胞术，重点研究了表达绿色荧光蛋白（GFP）的AAV2/5、2/6、2/8和2/9在前脂肪细胞中的转导效率。
评估了3T3-L1前脂肪细胞中的三种转导模式：3T3-L1前脂肪细胞中的AAV转导、进一步分化为成熟脂肪样细胞的转导以及分化的3T3-L1脂肪细胞的转导。在体内研究中，我们将AAV2/6、AAV2/8和AAV2/9注射到C57BL6小鼠的脂肪组织中，并评估了AAV2/6与AAV2/8和AAV2/9的转导能力。
AAV2/6在3T3-L1前脂肪细胞中表现出最高的转导效率，在病毒浓度为2×10至1.6×10病毒基因组/细胞的范围内，其效率比AAV2/5和AAV2/8高1.5至2倍。AAV2/5和AAV2/8的转导效率彼此相似。在巨细胞病毒（CMV）启动子下GFP的表达在长达20天内保持稳定。AAV转导三天后3T3-L1分化的诱导并未改变GFP表达水平，并且AAV2/6表现出最佳的转导效率。AAV2/6表现出转导成熟脂肪细胞的能力。这些结果在C57BL6小鼠的体内研究中得到证实。AAV2/6在腹股沟和肩胛间脂肪组织上具有最高的转导活性。
因此，与AAV2/5、AAV2/8和AAV2/9相比，AAV2/6在3T3-L1脂肪细胞和体内脂肪组织中均表现出更高的转导效率，这证明了其与AAV2/8和AAV2/9一起用于向脂肪细胞进行基因递送的可用性。
