Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Q1 Biochemistry, Genetics and Molecular Biology
Shaomian Yao, Weiqiong Rong, Yuanying Yuan
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引用次数: 1

Abstract

Background: 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.

Methods: hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) 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.

Results: 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.

Conclusions: 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.

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腺相关病毒(AAV)基因转染人骨髓干细胞(hBMSCs)的优化
背景:有效地将核酸传递到哺乳动物细胞中是评估基因功能的过表达基因的必要条件。人骨髓干细胞(hBMSCs)是研究最多的组织源性干细胞。腺相关病毒(aav)已被用于将DNA传递到造血干细胞中,用于各种目的。目前的文献报道,通过将AAV基因传递到hBMSCs中可以实现高达65%的转导效率。进一步提高效率是必要的,也是可能的。本研究测试了AAV血清型的选择,以便高效地将DNA递送到hBMSCs中。方法:用不同供者的hBMSCs感染不同血清型的含有CMV启动子驱动的增强型绿色荧光蛋白(eGFP)报告基因的aav。每隔5天监测感染细胞的绿色荧光。在感染后的指定时间点收集细胞,进行反转录聚合酶链反应(RT-PCR)和定量反转录聚合酶链反应(qRT-PCR),评估eGFP mRNA的转录情况。结果:AAV血清型的转导效率顺序为:AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV- dj。在感染的多重性(multiplicity of infection, MOI)大于100K时,AAV2几乎可以实现100%的转导。在20K ~ 50K MOI下,90%以上的细胞可转导。在moi为10K和15K时,约有80%的转导。RT-PCR分析显示,在aav感染后第5天至第30天可以检测到eGFP mRNA。来自不同患者的hBMSCs的转导效率的差异表明了供者与供者之间的差异,并且在aav2感染后第15天通常可以看到eGFP mRNA的增加。感染后第30天检测到最大eGFP转录。结论:在100K或更高的MOI下,AAV2和AAV2.7m8可以有效地将转基因传递到hBMSCs中,转导效率接近100%,并持续表达一个月以上。然而,在转导效率和转基因表达方面存在供体间的差异,特别是在MOIs小于100K时。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Stem cell investigation
Stem cell investigation Biochemistry, Genetics and Molecular Biology-Developmental Biology
CiteScore
5.80
自引率
0.00%
发文量
9
期刊介绍: The Stem Cell Investigation (SCI; Stem Cell Investig; Online ISSN: 2313-0792) is a free access, peer-reviewed online journal covering basic, translational, and clinical research on all aspects of stem cells. It publishes original research articles and reviews on embryonic stem cells, induced pluripotent stem cells, adult tissue-specific stem/progenitor cells, cancer stem like cells, stem cell niche, stem cell technology, stem cell based drug discovery, and regenerative medicine. Stem Cell Investigation is indexed in PubMed/PMC since April, 2016.
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