Wilcox D A, Olsen J C, Ishizawa L, Griffith M, White G C
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9654-9. doi: 10.1073/pnas.96.17.9654.
Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.
血小板黏附受体整合素αIIbβ3在巨核细胞中的特异性表达，是由αIIb启动子的调控元件所致，这些调控元件在巨核细胞生成早期可指导高水平的选择性基因转录。为了开发转基因靶向表达的方法，我们用受人类整合素αIIb启动子（核苷酸-889至+35）控制的鼠白血病病毒（MuLV）载体转导人CD34+外周血细胞。将编码整合素β3亚基的Pl(A2)同种异体抗原形式（Pro(33)）的天然cDNA亚克隆到该构建体（-889Pl(A2)β3）中，并转导到内源性合成Pl(A1)β3（Leu(33)）的细胞中，作为检测前病毒衍生β3的标志物。首先在细胞系中检测该载体将Pl(A2)β3表达靶向巨核细胞的能力。用人抗Pl(A2)同种血清进行免疫印迹分析，在转导的前巨核细胞中检测到Pl(A2)β3的合成；然而，在转导的上皮细胞中未检测到Pl(A2)β3蛋白。用-889Pl(A2)β3病毒粒子转导人造血CD34+细胞，并用巨核细胞生长和发育因子诱导其分化。在子代巨核细胞中形成了杂合αIIbβ3复合物，其中检测到前病毒衍生的Pl(A2)β3与内源性αIIb亚基相关联。另一个编码大肠杆菌β-半乳糖苷酶的αIIb启动子驱动的MuLV载体（-889nlacZ）用于证明转基因表达被选择性地靶向转导的CD34+细胞的巨核细胞后代。这些研究证明了使用αIIb启动子驱动MuLV载体将造血CD34+细胞进行基因转移以在发育中的巨核细胞和血小板中靶向转基因表达的可行性，并表明了其在血小板疾病人类基因治疗中的潜在应用。
