A third-generation lentivirus vector with a conditional packaging system
A third-generation lentivirus vector with a conditional packaging system
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DOI：
10.1128/jvi.72.11.8463-8471.1998
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发表时间：
1998-11-01
影响因子：
5.4
通讯作者：
Naldini, L
中科院分区：
文献类型：
--
作者：
Dull, T;Zufferey, R;Naldini, L
源自人类免疫缺陷病毒（HIV）的载体是体内基因传递的高效工具。然而，它们的生物安全性是主要关注点。在此我们利用HIV基因组的复杂性为慢病毒载体提供新的生物安全特性。除了结构基因，HIV还包含两个对HIV复制至关重要的调节基因，tat和rev，以及四个编码关键毒力因子的辅助基因。我们先前报道过，I型HIV辅助开放阅读框对于慢病毒载体的高效基因转导是可有可无的。我们现在证明，通过在载体转录本上游放置组成型启动子，可以抵消对tat基因的需求。由包含这种嵌合长末端重复序列（LTR）的构建体产生的载体在体内以非常高的效率转导神经元，无论它们是否在Tat存在的情况下产生，当包装构建体中的rev基因也被删除时，gag和pol的表达严格依赖于反式的Rev互补。通过联合使用单独的非重叠Rev表达质粒和5' LTR嵌合转移构建体，我们实现了高转导效率载体的最佳产量（高达10⁷转导单位[TU]/ml和10⁴ TU/ng的p24）。这种第三代慢病毒载体仅使用一部分HIV基因：gag、pol和rev。此外，源自HIV的构建体以及它们之间的任何重组体都依赖于上游元件和反式互补来表达，因此在载体生产细胞之外是无功能的。这种分裂基因组、条件性包装系统基于现有的病毒序列，并作为一种防止产生有复制能力的重组体的内置装置。虽然载体的实际生物安全性最终将在体内得到验证，但这里提出的改进设计应有助于慢病毒载体的测试。
Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type I accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat, when the rer gene was also deleted from the packaging construct, expression of gag and pal was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24), This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pal, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.