Van Damme An, Thorrez Lieven, Ma Ling, Vandenburgh Herman, Eyckmans Jeroen, Dell'Accio Francesco, De Bari Cosimo, Luyten Frank, Lillicrap David, Collen Désiré, VandenDriessche Thierry, Chuah Marinee K L
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Herestraat 49, Building O&N1, Leuven B-3000, Belgium.
Stem Cells. 2006 Apr;24(4):896-907. doi: 10.1634/stemcells.2003-0106. Epub 2005 Dec 9.
Human bone marrow (BM) mesenchymal stem/progenitor cells are potentially attractive targets for ex vivo gene therapy. The potential of lentiviral vectors for transducing BM mesenchymal cells was examined using a self-inactivating vector that expressed the green fluorescent protein (GFP) from an internal cytomegalovirus (CMV) promoter. This vector was compared with oncoretroviral vectors expressing GFP from the CMV promoter or a modified long-terminal repeat that had been optimized for long-term expression in stem cells. The percentage of GFP-positive cells was consistently higher following lentiviral versus oncoretroviral transduction, consistent with increased GFP mRNA levels and increased gene transfer efficiency measured by polymerase chain reaction and Southern blot analysis. In vitro GFP and FVIII expression lasted for several months post-transduction, although expression slowly declined. The transduced cells retained their stem/progenitor cell properties since they were still capable of differentiating along adipogenic and osteogenic lineages in vitro while maintaining high GFP and FVIII expression levels. Implantation of lentivirally transduced human BM mesenchymal cells using collagen scaffolds into immunodeficient mice resulted in efficient engraftment of gene-engineered cells and long-term transgene expression in vivo. These biocompatible BM mesenchymal implants represent a reversible, safe, and versatile protein delivery approach because they can be retrieved in the event of an unexpected adverse reaction or when expression of the protein of interest is no longer required. In conclusion, efficient gene delivery with lentiviral vectors in conjunction with the use of bioengineered reversible scaffolds improves the therapeutic prospects of this novel approach for gene therapy, protein delivery, or tissue engineering.
人骨髓间充质干/祖细胞是体外基因治疗潜在的有吸引力的靶点。使用一种自失活载体检测了慢病毒载体转导骨髓间充质细胞的潜力，该载体从内部巨细胞病毒（CMV）启动子表达绿色荧光蛋白（GFP）。将该载体与从CMV启动子或为在干细胞中进行长期表达而优化的修饰长末端重复序列表达GFP的逆转录病毒载体进行比较。慢病毒转导后GFP阳性细胞的百分比始终高于逆转录病毒转导，这与通过聚合酶链反应和Southern印迹分析测得的GFP mRNA水平增加和基因转移效率提高一致。转导后体外GFP和FVIII表达持续数月，尽管表达缓慢下降。转导的细胞保留了其干/祖细胞特性，因为它们在体外仍能够沿着成脂和成骨谱系分化，同时保持高GFP和FVIII表达水平。使用胶原支架将慢病毒转导的人骨髓间充质细胞植入免疫缺陷小鼠体内，导致基因工程细胞有效植入并在体内长期表达转基因。这些生物相容性骨髓间充质植入物代表了一种可逆、安全且通用的蛋白质递送方法，因为在发生意外不良反应或不再需要感兴趣蛋白质的表达时可以将其取出。总之，慢病毒载体的高效基因递送与生物工程可逆支架的使用相结合，改善了这种用于基因治疗、蛋白质递送或组织工程的新方法的治疗前景。
