Stover M L, Wang C K, McKinstry M B, Kalajzic I, Gronowicz G, Clark S H, Rowe D W, Lichtler A C
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Mol Ther. 2001 Apr;3(4):543-50. doi: 10.1006/mthe.2001.0293.
Gene therapy of bone would benefit from the availability of vectors that provide stable, osteoblast-specific expression. This would allow bone-specific expression of Col1a1 cDNAs for treatment of osteogenesis imperfecta. In addition, such a vector would restrict expression of secreted therapeutic proteins to the bone-synthesizing regions of the bone marrow after ex vivo transduction of marrow stromal cells and reintroduction of the cells into patients. Retrovirus vectors stably integrate into target cell genomes; however, long-term regulated expression from internal cellular promoters has not been consistently achieved. In some cases this is due to a stem cell-specific mechanism for transcriptional repression of retroviruses. We evaluated the ability of self-inactivating ROSA-derived vectors containing a bone-directed 2.3-kb rat Col1a1 promoter to display osteoblast-specific expression. In vitro expression was examined in bone marrow stromal cell cultures induced to undergo osteoblastic differentiation. In vivo expression was evaluated in chimeric mice derived from transduced embryonic stem cells. The results indicate that self-inactivating retrovirus vectors containing the Col1a1 promoter are not permanently inactivated in embryonic stem cells and are specifically expressed in osteoblasts in vivo and in vitro. Thus these vectors should be useful for bone-directed gene therapy.
骨基因治疗将受益于能够提供稳定的、成骨细胞特异性表达的载体。这将使I型胶原α1（Col1a1）cDNA在骨中特异性表达，用于治疗成骨不全症。此外，这种载体在体外转导骨髓基质细胞并将细胞重新引入患者体内后，会将分泌型治疗蛋白的表达限制在骨髓的骨合成区域。逆转录病毒载体可稳定整合到靶细胞基因组中；然而，尚未始终如一地实现来自内部细胞启动子的长期调控表达。在某些情况下，这是由于一种干细胞特异性机制对逆转录病毒进行转录抑制所致。我们评估了含有骨定向2.3kb大鼠Col1a1启动子的自失活ROSA衍生载体显示成骨细胞特异性表达的能力。在诱导进行成骨细胞分化的骨髓基质细胞培养物中检测体外表达。在源自转导胚胎干细胞的嵌合小鼠中评估体内表达。结果表明，含有Col1a1启动子的自失活逆转录病毒载体在胚胎干细胞中不会永久失活，并且在体内和体外的成骨细胞中特异性表达。因此，这些载体应可用于骨定向基因治疗。
