Improved foamy virus vectors with minimal viral sequences
Improved foamy virus vectors with minimal viral sequences
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DOI：
10.1006/mthe.2002.0672
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发表时间：
2002-09-01
影响因子：
12.4
通讯作者：
Russell, DW
中科院分区：
文献类型：
--
作者：
Trobridge, G;Josephson, N;Russell, DW
泡沫病毒（FV）载体显示出基因治疗应用的前景。然而，现有的FV载体要么保留野生型病毒基因组的显著部分，要么以低滴度产生。我们描述了一个瞬时共转染系统，产生高滴度的FV载体与最小的顺式作用区。这些载体基因组在gag、pol、env和bel 1 -3辅助基因以及LTR U3区域中具有缺失，但保留必需的2.5 kb顺式作用区域。此外，将终止密码子引入剩余的gag序列中以防止病毒肽的表达并消除Gag-Pol融合蛋白的显性负效应。尽管这些缺失的泡沫状（DeltaPhi）载体用我们先前的包装构建体以相对低的滴度产生，但是我们设计了用于Gag、Pol和Env表达的单独的辅助质粒，其允许我们通过四质粒瞬时转染常规地产生具有超过10（5）转导单位/ml的滴度的无辅助、未浓缩的载体原液。然后通过超离心将DeltaPhi载体储备液浓缩至超过10（7）个转导单位/ml的滴度。含有9.2-kb转基因盒的DeltaPhi载体以超过10（5）个转导单位/ml的未浓缩滴度产生，证明了这些缺失的载体对于大治疗基因的效用。
Foamy virus (FV) vectors show promise for gene therapy applications. However, existing FV vectors either retain a significant portion of the wild-type virus genome or are produced at low titers. We describe a transient cotransfection system that produces high-titer FV vectors with minimal cis-acting regions. These vector genomes have deletions in the gag, pol, env, and bel1-3 accessory genes, as well as the LTR U3 region, but retain an essential 2.5-kb cis-acting region. In addition, stop codons were introduced into the remaining gag sequences to prevent expression of viral peptides and to eliminate dominant-negative effects of a Gag-Pol fusion protein. Although these deleted foamy (DeltaPhi) vectors were produced at relatively low titers with our prior packaging construct, we designed separate helper plasmids for Gag, Pol, and Env expression that allowed us to routinely produce helper-free, unconcentrated vector stocks with titers of over 10(5) transducing units/ml by four-plasmid transient transfection. The DeltaPhi vector stocks were then concentrated by ultracentrifugation to titers over 10(7) transducing units/ml. A DeltaPhi vector containing a 9.2-kb transgene cassette was produced at unconcentrated titers of over 10(5) transducing units/ml, demonstrating the utility of these deleted vectors for large therapeutic genes.