Mitta Barbara, Rimann Markus, Ehrengruber Markus U, Ehrbar Martin, Djonov Valentin, Kelm Jens, Fussenegger Martin
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, ETH Hoenggerberg, HPT, CH-8093 Zurich, Switzerland.
Nucleic Acids Res. 2002 Nov 1;30(21):e113. doi: 10.1093/nar/gnf112.
In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (P(hCMV)) or the human elongation factor 1alpha (P(hEF1)(alpha)), (iii) P(hCMV-) or P(PGK-) (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5'U3, R-U5-psi+-SD, cPPT-RRE-SA and 3'LTR(DeltaU3) modules or placed within the 5'U3 (-78 bp) and 3'LTR(DeltaU3) (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5'LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3'LTR(DeltaU3) element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.
近年来，慢病毒表达系统在基因治疗领域赢得了无与伦比的声誉，因为它们能够将治疗性转基因导入多种难以转染/转导的靶组织（脑、造血系统、肝脏、肺、视网膜），且不会引发显著的体液免疫反应。我们克隆了一个类似构建试剂盒的自我失活慢病毒表达载体家族，该家族与最先进的包装和假型化技术兼容，除了必需的顺式作用慢病毒序列外，还包含：（i）无与伦比的多克隆位点，有多达29个用于限制性内切酶的独特位点，其中许多识别8碱基对基序；（ii）源自人巨细胞病毒立即早期启动子（P(hCMV)）或人延伸因子1α（P(hEF1)(α)）的强启动子；（iii）由P(hCMV-)或P(PGK-)（磷酸甘油酸激酶启动子）驱动的G418抗性标记或基于荧光蛋白的表达示踪剂；以及（iv）用于协调表达多达三个转基因的三顺反子表达盒。此外，我们设计了一系列经过大小优化的高度模块化慢病毒表达载体（pLenti Module），除了广泛的中央多克隆位点外，这些载体在5'U3、R-U5-psi+-SD、cPPT-RRE-SA和3'LTR(DeltaU3)模块中的任何一个两侧或置于5'U3（-78 bp）和3'LTR(DeltaU3)（8666 bp）内都有独特的限制性位点。pLentiModule能够在慢病毒表达载体家族成员之间直接进行盒式模块交换，并有助于设计不依赖于Tat的（用异源启动子元件替换5'LTR）、受调控的和可自我切除的前病毒（在3'LTR(DeltaU3)元件中插入响应性操纵子或LoxP）。我们通过将其转导至多种昆虫、鸡、小鼠和人类细胞系以及成年大鼠心肌细胞、大鼠海马切片和鸡胚中，验证了我们的慢病毒表达载体。这里描述的新型多功能构建试剂盒样载体系列自身以及与许多其他（非病毒）哺乳动物表达载体兼容，可直接交换关键组件（如启动子、LTR、抗性基因），并将协助基因治疗和组织工程领域开发专门用于最佳治疗重大人类疾病的慢病毒表达载体。
