Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Gene Ther. 2021 Dec;28(12):729-739. doi: 10.1038/s41434-020-0182-4. Epub 2020 Aug 17.
Adeno-associated viral vectors (AAV) are unique in their ability to transduce a variety of both dividing and nondividing cells, with significantly lower risk of random genomic integration and with no known pathogenicity in humans, but their role in ex vivo regional gene therapy for bone repair has not been definitively established. The goal of this study was to test the ability of AAV vectors carrying the cDNA for BMP-2 to transduce human mesenchymal stem cells (MSCs), produce BMP-2, and induce osteogenesis in vitro as compared with lentiviral gene therapy with a two-step transcriptional amplification system lentiviral vector (LV-TSTA). To this end, we created two AAV vectors (serotypes 2 and 6) expressing the target transgene; eGFP or BMP-2. Transduction of human MSCs isolated from bone marrow (BMSCs) or adipose tissue (ASCs) with AAV2-eGFP and AAV6-eGFP led to low transduction efficiency (BMSCs: 3.57% and 8.82%, respectively, ASCs: 6.17 and 20.2%, respectively) and mean fluorescence intensity as seen with FACS analysis 7 days following transduction, even at MOIs as high as 10. In contrast, strong eGFP expression was detectable in all of the cell types post transduction with LV-TSTA-eGFP. Transduction with BMP-2 producing vectors led to minimal BMP-2 production in AAV-transduced cells 2 and 7 days following transduction. In addition, transduction of ASCs and BMSCs with AAV2-BMP-2 and AAV6-BMP-2 did not enhance their osteogenic potential as seen with an alizarin red assay. In contrast, the LV-TSTA-BMP-2-transduced cells were characterized by an abundant BMP-2 production and induction of the osteogenic phenotype in vitro (p < 0.001 vs. AAV2 and 6). Our results demonstrate that the AAV2 and AAV6 vectors cannot induce a significant transgene expression in human BMSCs and ASCs, even at MOIs as high as 10. The LV-TSTA vector is significantly superior in transducing human MSCs; thus this vector would be preferable when developing an ex vivo regional gene therapy strategy for clinical use in orthopedic surgery applications.
腺相关病毒载体（AAV）的独特之处在于能够转导多种分裂和非分裂细胞，其随机基因组整合的风险显著降低，并且在人类中没有已知的致病性，但它们在骨修复的体外区域基因治疗中的作用尚未得到明确确立。本研究的目的是测试携带 BMP-2 cDNA 的 AAV 载体转导人骨髓间充质干细胞（MSCs）、产生 BMP-2 并在体外诱导成骨的能力，与两步转录扩增系统慢病毒载体（LV-TSTA）的基因治疗进行比较。为此，我们构建了两种表达靶基因的 AAV 载体；绿色荧光蛋白（eGFP）或 BMP-2。用 AAV2-eGFP 和 AAV6-eGFP 转导从骨髓（BMSCs）或脂肪组织（ASCs）分离的人 MSCs，转导效率低（BMSCs：分别为 3.57%和 8.82%，ASCs：分别为 6.17%和 20.2%），7 天后通过流式细胞术分析检测到平均荧光强度，即使 MOI 高达 10。相比之下，用 LV-TSTA-eGFP 转导后，所有细胞类型都可检测到强烈的 eGFP 表达。用 BMP-2 产生载体转导后，转导细胞中 BMP-2 的产生量很少，转导后 2 天和 7 天。此外，用 AAV2-BMP-2 和 AAV6-BMP-2 转导 ASCs 和 BMSCs 并不能增强其成骨潜力，如茜素红测定法所示。相比之下，LV-TSTA-BMP-2 转导的细胞特征是大量产生 BMP-2，并在体外诱导成骨表型（p<0.001 与 AAV2 和 6 相比）。我们的结果表明，即使 MOI 高达 10，AAV2 和 AAV6 载体也不能在人 BMSCs 和 ASCs 中诱导明显的转基因表达。LV-TSTA 载体在转导人 MSCs 方面明显优越；因此，在开发用于骨科手术应用的体外区域基因治疗策略时，该载体将更可取。
