Yao Shaomian, Rong Weiqiong, Yuan Yuanying
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
Stem Cell Investig. 2023 Feb 2;10:3. doi: 10.21037/sci-2022-042. eCollection 2023.
Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs.
hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein () reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription.
The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection.
We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.
有效地将核酸导入哺乳动物细胞对于过表达基因以评估基因功能至关重要。人骨髓干细胞（hBMSCs）是研究最多的组织来源干细胞。腺相关病毒（AAVs）已被用于将DNA导入hBMSCs以实现各种目的。当前文献报道，通过AAV基因导入hBMSCs可实现高达65%的转导效率。效率的进一步提高是必要且有可能的。本研究测试了一系列AAV血清型用于高效地将DNA导入hBMSCs。
来自不同供体的hBMSCs用不同血清型的携带由CMV启动子驱动的增强绿色荧光蛋白（）报告基因的AAVs进行感染。每隔五天监测感染细胞中的绿色荧光。在感染后的指定时间点收集细胞用于逆转录聚合酶链反应（RT-PCR）和定量逆转录聚合酶链反应（qRT-PCR）以评估eGFP mRNA转录。
结果表明AAV血清型的转导效率顺序为AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ。AAV2在感染复数（MOI）大于100K时可实现近100%的转导。在20K至50K的MOI下，超过90%的细胞可被转导。在10K和15K的MOI下可见约80%的转导。RT-PCR分析表明在AAV感染后第5天至第30天可检测到eGFP mRNA。不同患者的hBMSCs观察到的转导效率差异表明供体间存在变异性，并且在AAV2感染后第15天之后通常可见eGFP mRNA增加。在感染后第30天检测到最大的eGFP转录。
我们得出结论，MOI为100K或更高的AAV2和AAV2.7m8可以有效地将转基因导入hBMSCs，转导效率高达近100%，并能持续表达一个多月。然而，转导效率和转基因表达存在供体间差异，尤其是在MOI小于100K时。
