HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency.
HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency.
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DOI：
10.1038/s41598-021-91309-w
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发表时间：
2021-06-08
影响因子：
4.6
通讯作者：
Swanson CM
中科院分区：
文献类型：
--
作者：
Sertkaya H;Ficarelli M;Sweeney NP;Parker H;Vink CA;Swanson CM
许多用于基因治疗的慢病毒载体来源于HIV-1。最佳载体基因组将仅包括转导效率和基因表达所需的病毒序列，以使插入患者基因组中的外源序列的量最小化。然而，目前还不清楚是否所有的HIV-1序列在载体基因组是必不可少的。为了确定需要哪些病毒序列，我们进行了系统的缺失分析，结果表明，大部分的gag区和超过50%的env区可以被删除。由于慢病毒载体的剪接谱表征不佳，我们使用长读段测序来确定典型和隐蔽剪接位点的使用。删除env序列的特定区域减少了每个转录本的剪接事件的数量，并增加了未剪接基因组的比例。最后，将gag中的大缺失与重新定位3' R下游的Rev-应答元件以防止其逆转录相结合，表明可以从整合的载体基因组中除去HIV-1序列的1201个核苷酸，而基本上不损害转导效率。总的来说，这允许创建包含最少HIV-1序列的慢病毒载体基因组，这可以提高安全性并将更少的病毒序列转移到患者的DNA中。
Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.
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作者：
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