Paar Matthias, Klein Dieter, Salmons Brian, Günzburg Walter H, Renner Matthias, Portsmouth Daniel
Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, Vienna, Austria.
BMC Mol Biol. 2009 Feb 9;10:8. doi: 10.1186/1471-2199-10-8.
The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design.
In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed.
Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.
基于鼠白血病病毒（MLV）的具有复制能力的逆转录病毒（RCR）载体技术的近期出现，为基因传递提供了令人兴奋的新工具，尽管已实现的载体效率进步也伴随着一系列新挑战。例如，额外转基因序列的表达增加了病毒基因组的长度，这可能导致复制效率降低，进而导致载体基因组不稳定。这就需要努力分析此类载体复制过程中重组体出现的速率和机制，以提供有助于改进RCR载体设计的数据。
在本研究中，我们对基于MLV的RCR载体在细胞培养和体内预先形成的皮下肿瘤中传播后的包装载体基因组和前病毒DNA进行了详细的分子分析。通过应用实时PCR和实时RT-PCR分析，定量分析了MLV毒株、转基因位置和宿主细胞类型对载体重组体出现速率的影响。通过PCR进一步表征单个突变体，并通过测序确定与这些突变体相关的核苷酸序列和结构基序。我们的数据表明，病毒毒株、载体设计和宿主细胞会影响主要载体突变体的出现速率，但不影响体外的潜在重组机制。然而，相比之下，观察到在细胞培养和体内实体瘤中出现的测序突变体相关的RNA二级结构基序存在差异。
我们的数据进一步证明，基于莫洛尼氏MLV毒株且在3'UTR区域包含转基因盒的基于MLV的RCR载体优于基于Akv-MLV和/或在LTR的U3区域包含转基因盒的载体。在体内实体瘤中获得的数据与我们自己以及先前发表的体外感染细胞的数据之间观察到的差异，证明了评估设计用于癌症基因治疗的载体在体内和体外的重要性。
