Laboratory of Neurobiology, Biomedicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
Department of Molecular Biology and Genetics - Molecular Nutrition, Aarhus University, Aarhus, Denmark.
J Neurochem. 2021 Feb;156(3):290-308. doi: 10.1111/jnc.14982. Epub 2020 Mar 10.
Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood-brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease Niemann Pick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non-mitotic monocultures with a biological effect on NPC2-deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa-cells, known to persist in a mitotic state. When co-cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non-mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non-viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects. Cover Image for this issue: https://doi.org/10.1111/jnc.15050.
治疗许多影响中枢神经系统 (CNS) 的疾病都很复杂，因为有几种治疗方法无法穿过血脑屏障 (BBB)。通过基因修饰脑毛细血管内皮细胞 (BCEC)，可以将 BCEC 变成重组蛋白工厂，从而克服 BBB 的限制。这将导致蛋白质向大脑和外周循环分泌，这在遗传疾病中尤为相关，例如溶酶体贮积症 (LSD)，其中细胞在中枢神经系统和外周都普遍受到影响。在这里，我们研究了原代大鼠脑毛细血管内皮细胞 (rBCEC) 的转染，以合成和分泌 NPC2 重组蛋白，该蛋白在溶酶体胆固醇贮积病尼曼-皮克 C2 型中缺乏。我们证明，在非有丝分裂单核培养物中，21%的 BCEC 中 NPC2 基因明显诱导和蛋白分泌，这对 NPC2 缺陷型成纤维细胞有生物学作用，如胆固醇沉积的 Filipin III 染色变化所证实。相比之下，在已知持续有丝分裂状态的 HeLa 细胞中的转染效率为 75%。当在保持 BBB 特性的条件下与原代大鼠星形胶质细胞共培养时，有 7%的 BCEC 被转染，这清楚地表明，非有丝分裂 BCEC 的极化条件诱导 BBB 特性会影响转染效率和分泌方向性。总之，非病毒基因治疗 rBCEC 导致蛋白分泌，这为 NPC2 靶向中枢神经系统内的细胞提供了一种方法，因为存在 BBB 而无法到达这些细胞。然而，获得高转染效率对于达到足够的治疗效果至关重要。本期的封面图片：https://doi.org/10.1111/jnc.15050.
