摘要
HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies.
摘要译文
HIV-1衍生的慢病毒载体已用各种包膜糖蛋白对其宿主范围进行了伪型。以前，我们发现衍生自αRossRiver病毒（RRV）的包膜糖蛋白可以伪造慢病毒载体并介导各种上皮和成纤维细胞衍生的细胞系的有效转导。在这项研究中，我们使用编码增强的绿色荧光蛋白（EGFP）的RRV型型载体研究了造血细胞的转导。 RRV介导的人CD34 +脐带血细胞和祖细胞的转导非常低，即使在100个感染的多重性下（0.4％EGFP阳性祖细胞菌落）。在多种造血细胞系中也观察到效率低下的转导。然而，将两种衍生的红血病衍生的细胞系和被驱动到巨噬细胞样分化的单核细胞被中度转导。用对照VSV-G-PESUDYTY型慢病毒载体转导造血细胞通常是有效的，但在刺激淋巴细胞细胞系或原代鼠骨髓细胞后，意外降低了三倍。同样，测试的造血细胞系基本上是与腺相关2型（AAV）载体的不可允许的，并且这不受谱系，活性或分化的影响。用蛋白酶对允许的293个细胞的处理表明，RRV-和VSV-G-PESEDYTYPECTECTOR的转导部分部分取决于细胞表面蛋白的存在。这些结果表明，RRV糖蛋白在介导造血细胞转导的能力严重受限，这可能是由于特定受体需求与VSV-G和AAV不同的受体需求所致。相反，用VSV糖蛋白转导的细胞活化影响越来越广泛地相信。我们的发现表明，需要对每种应用进行仔细评估所采用的包膜糖蛋白和培养条件。此外，RRV-PESUDYTY型向量的独特限制宿主范围可能有助于设计新型细胞选择性转导策略。
Christoph A. Kahl[1];Karen Pollok[2];Laura S. Haneline[2][3];Kenneth Cornetta[1][3][4]. Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions[J]. Molecular Therapy, 2005,11(3): 470-482