Fielding A K, Maurice M, Morling F J, Cosset F L, Russell S J
Cambridge Centre for Protein Engineering and Cambridge University Dept Haematology, MRC Centre, Cambridge, UK.
Blood. 1998 Mar 1;91(5):1802-9.
We previously reported that retroviral vectors displaying epidermal growth factor (EGF) as part of a chimeric envelope glycoprotein are sequestered upon binding to EGF receptor (EGFR)-positive target cells, leading to loss of infectivity. In the current study, we have displayed stem cell factor (SCF) on beta-galactosidase-transducing ecotropic and amphotropic retroviral vector particles as a factor Xa protease-cleavable N-terminal extension of the envelope glycoprotein. Viral incorporation of the SCF chimeric envelopes was demonstrated by immunoblotting of pelleted virions and their specific attachment to Kit receptors was demonstrated by flow cytometry. Gene transfer studies showed that when SCF was displayed on an amphotropic envelope, the infectivity of the SCF-displaying vectors was selectively inhibited on Kit-expressing cells, but could be restored by adding soluble SCF to block the Kit receptors or by cleaving the displayed SCF domain from the vector particles with factor Xa protease. The host range properties of EGF-displaying and SCF-displaying vectors were then compared in cell mixing experiments. When EGFR-positive cancer cells and Kit-positive hematopoietic cells were mixed and exposed to the different engineered vector particles, the cancer cells were selectively transduced by the SCF-displaying vector and the hematopoietic cells were selectively transduced by the EGF-displaying vector. Retroviral display of polypeptide growth factors can therefore provide the basis for a novel inverse targeting strategy with potential use for selective transduction of hematopoietic or nonhematopoietic cells (eg, cancer cells) in a mixed cell population.
我们之前报道过，作为嵌合包膜糖蛋白一部分展示表皮生长因子（EGF）的逆转录病毒载体，在与表皮生长因子受体（EGFR）阳性靶细胞结合后会被隔离，导致感染性丧失。在当前研究中，我们已在转导β-半乳糖苷酶的嗜亲性和双嗜性逆转录病毒载体颗粒上展示干细胞因子（SCF），作为包膜糖蛋白的Xa因子蛋白酶可切割的N端延伸。通过对沉淀病毒粒子进行免疫印迹证明了SCF嵌合包膜在病毒中的掺入，通过流式细胞术证明了它们与Kit受体的特异性结合。基因转移研究表明，当SCF展示在双嗜性包膜上时，展示SCF的载体在表达Kit的细胞上的感染性被选择性抑制，但可通过添加可溶性SCF阻断Kit受体或用Xa因子蛋白酶从载体颗粒上切割展示的SCF结构域来恢复。然后在细胞混合实验中比较了展示EGF和展示SCF的载体的宿主范围特性。当将EGFR阳性癌细胞和Kit阳性造血细胞混合并暴露于不同的工程化载体颗粒时，展示SCF的载体选择性转导癌细胞，展示EGF的载体选择性转导造血细胞。因此，多肽生长因子的逆转录病毒展示可为一种新型反向靶向策略提供基础，该策略可能用于在混合细胞群体中选择性转导造血或非造血细胞（如癌细胞）。
