Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

靶向 CNS 内皮细胞的基因载体可以操纵血脑屏障并纠正 CNS 中的遗传缺陷。由于基于腺相关病毒 (AAV) 的载体容量有限，因此控制基因表达的 DNA 序列必须短。此外，它必须对内皮细胞具有特异性，以避免脱靶效应。为了开发对脑内皮细胞具有选择性的改进调控序列，我们测试了 11 个（脑）内皮特异性基因的截短启动子与短调控元件的转录活性，即土拨鼠转录后调控元件 (W)、 CMV 增强子元件 (C) 和Tie2的第一个内含子的片段基因（S），通过转染三种物种的脑内皮细胞。调节元件和短启动子的四种组合（Cdh5、Ocln、Slc2a1和Slco1c1）通过该体外管道进行，显示出合适的活性。在小鼠中进行测试时，与常用的 CAG 启动子相比，调节序列 C -Ocln -W 和 C -Slc2a1 -SW 能够在脑内皮细胞中实现更强、更特异的基因表达。总之，新的调控元件有效地控制脑内皮细胞中的基因表达，并可能有助于用基因治疗载体特异性靶向血脑屏障。