Department of Molecular Cell Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands.
Hum Gene Ther. 2013 Jan;24(1):78-96. doi: 10.1089/hum.2012.069. Epub 2012 Dec 11.
Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.
整合缺陷型慢病毒载体（IDLVs）在基础和临床前基因转移研究中得到了越来越多的应用。然而，与整合有效的慢病毒载体相比，IDLV 的转基因表达谱通常较低。我们假设，IDLV 的游离体性质使其成为涉及染色质重塑的组蛋白去乙酰化的表观遗传沉默的首选靶标。因此，使用携带各种顺式作用元件和转录单元组件的载体，通过包装构建体进行组装，这些包装构建体编码野生型或 I 型突变体 D116N 整合酶部分。在存在和不存在组蛋白去乙酰化酶（HDAC）抑制剂丁酸钠和曲古抑菌素 A 的情况下，评估每个载体类别的转导水平和转基因产物产量。为了研究靶细胞复制状态的作用，我们在生长停滞的人骨髓间充质干细胞和有丝分裂后合胞体肌管中进行了实验。我们发现，IDLVs 会受到 HDAC 的急性影响，而不受其遗传组成或靶细胞复制率的影响。有趣的是，由于 HDAC 抑制，IDLV 转基因表达的挽救程度因载体骨架和细胞类型而异。最后，通过染色质免疫沉淀定量 PCR（ChIP-qPCR）研究组蛋白修饰发现，与同基因整合有效的载体模板相比，IDLV 基因组上的常染色质标记数量较少。这些发现支持了这样的观点，即 IDLV 构成了涉及组蛋白去乙酰化的表观遗传沉默的首选靶标，这有助于抑制其全部转录潜力。我们的数据提供了如何最优化滴定和部署这些有前途的游离体基因传递载体的线索。
