Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence (“spacer”). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22^phox, was achieved.

合成的microRNA（miRNA）小基因（SMIG）在分子治疗中具有重大潜力；但是，仍然需要确定它们的最佳体系结构。我们先前已经优化了miRNA发夹的茎结构，以实现有效的基因敲低。在这里，我们研究了由聚合酶II依赖性启动子驱动的SMIG的整体结构。当将miRNA发夹直接放置在启动子后面时，与包含插入序列（“间隔子”）的构建体相比，基因敲低效率低下。间隔子序列与敲低效率和串联潜力有关：基于GFP的序列（即使是截短的或包括终止密码子的序列）也特别有效。相反，基于CD4内含子序列的相似长度的间隔子是完全无效的。间隔子序列影响miRNA稳态水平，而不影响转录本稳定性。我们证明，使用优化的间隔子，可有效表达多达五个针对两个不同基因的连锁发夹，并能够敲除它们各自的靶标。含有CCR5基因敲低的SMIG的造血干细胞的移植证明了持续性我们方法的体内功效。总而言之，我们定义了可优化SMIG效率的功能。根据这些结果，可以实现对目标基因（如HIV共同受体CCR5和NADPH氧化酶亚基p22 ^phox）的优化敲除。