Kurpad C, Mukherjee P, Wang X S, Ponnazhagan S, Li L, Yoder M C, Srivastava A
Department of Microbiology & Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
J Hematother Stem Cell Res. 1999 Dec;8(6):585-92. doi: 10.1089/152581699319740.
Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.
人细小病毒B19基因从病毒p6启动子（B19p6）的表达仅限于正在经历红系分化的原代人造血细胞。我们已经证明，在重组细小病毒基因组的背景下，该启动子在已建立的人红系细胞系中不发生表达（Ponnazhagan等人，《病毒学杂志》69:8096 - 8101，1995年）。然而，在原代人骨髓细胞中可以很容易地检测到该启动子的大量表达（Wang等人，《美国国家科学院院刊》92:12416 - 12420，1995年；Ponnazhagan等人，《普通病毒学杂志》77:1111 - 1122，1996年）。在本研究中，我们研究了B19p6启动子在原代人骨髓来源的CD34 +造血祖细胞分化为髓系和红系谱系过程中的表达模式。用重组腺相关病毒2（AAV）载体转导CD34 +细胞，这些载体含有在以下启动子/增强子控制下的β - 半乳糖苷酶（lacZ）基因：巨细胞病毒启动子（vCMVp-lacZ）、B19p6启动子（vB19p6-lacZ）、带有来自人β - 珠蛋白基因簇基因座控制区（LCR）的上游红系细胞特异性增强子元件（HS-2）的B19p6启动子（vHS2-B19p6-lacZ）以及带有HS-2增强子的人β - 珠蛋白基因启动子（vHS2-βp-lacZ）。在感染后48小时或体外红系分化3周后评估转基因表达。虽然感染后48小时巨细胞病毒启动子的高水平表达随时间降低，但B19p6和β - 珠蛋白启动子的低水平表达在红系分化后显著增加。此外，在造血祖细胞分析中，巨细胞病毒启动子在髓系或红系细胞来源的集落中的表达水平没有显著差异。另一方面，B19p6和β - 珠蛋白启动子的表达仅限于红系细胞集落。这些数据进一步证实B19p6启动子是红系细胞特异性的，并表明重组AAV - B19杂交载体可能在一般人类血红蛋白病的基因治疗中，特别是在镰状细胞贫血和β - 地中海贫血的基因治疗中证明是有用的。
