Konetschny C, Holzer G W, Falkner F G
Baxter BioScience/Vaccine AG Biomedical Research Center, A-2304 Orth/Donau, Austria.
J Virol. 2002 Feb;76(3):1236-43. doi: 10.1128/jvi.76.3.1236-1243.2002.
Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.
已知内含子和聚腺苷酸化（pA）位点可提高转录本稳定性和核质运输能力，并且通常存在于高效基因表达载体中。然而，标准逆转录病毒载体不允许包含此类序列元件，因为内部剪接位点和pA位点处的mRNA加工会干扰功能性全长载体基因组的产生。在本报告中，我们研究了杂交痘苗病毒/逆转录病毒载体转导逆转录病毒基因组内带有核RNA加工信号的复杂基因盒的能力。构建了一种逆转录病毒载体，其包含一个目的基因（人凝血因子IX [FIX] cDNA），包括一个内含子和一个内部pA位点。将修饰后的前病毒载体基因组克隆到痘苗病毒启动子下游，并插入痘苗病毒基因组中。用重组痘苗病毒载体感染包装细胞系，导致逆转录病毒颗粒分泌，细胞培养上清液的平均滴度为每毫升10(5) CFU。由于痘苗病毒系统中的细胞质转录和对核转录信号的不识别，获得了仍包含内含子的全长转录本。在逆转录病毒转导的细胞系中，FIX转录本在内部pA位点处终止。转录本进行了定量剪接，并且FIX被分泌。可以产生具有稳定单拷贝插入物且含有高效基因功能所需序列元件的重组细胞系。因此，描述了一种用于产生复杂逆转录病毒载体的相对简单的细胞质系统。转导含内含子基因盒的逆转录病毒载体可能在基因治疗应用中发挥进一步作用。
