Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter

BackgroundInternal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.ResultsA series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.ConclusionThe existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

背景 逆转录病毒载体中的内部聚合酶III启动子已被广泛用于在不同位置和方向表达短RNA序列，如核酶、RNA适体或短干扰RNA抑制剂。然而，这些启动子在反向时的稳定性尚未得到严格评估。 结果 构建了一系列携带U6 + 1启动子以及3种不同的HIV - 1逆转录酶特异性RNA适体和1种对照适体的逆转录病毒载体，所有适体均为反向。经过穿梭包装后，用每种载体转导CD4 +细胞系CEMx174，筛选绿色荧光蛋白（GFP）表达细胞，并用HIV - 1进行攻击。我们在这些转导的细胞群中未观察到对HIV - 1复制的抑制。从转导的CEMx174细胞中对U6 + 1启动子 - RNA适体抑制剂盒进行PCR扩增，以及从转染的Phoenix（双嗜性）包装细胞中进行逆转录PCR（RT - PCR）扩增，结果显示出两种不同的产物：一种是预期大小的全长产物，另一种是截短产物。全长PCR产物的序列与预测的扩增子序列相同。然而，对截短产物进行测序发现U6启动子中有一个139bp的缺失。这种缺失降低了U6启动子的转录活性。对U6启动子反义方向缺失序列的分析表明存在共有剪接供体、剪接受体和分支点序列。 结论 当U6启动子在逆转录病毒载体中反向表达时，存在一个隐蔽剪接位点，在包装过程中会产生缺失，这可能会限制该启动子用于表达小RNA抑制剂的效用。