Rasmussen Charlotte Laurfelt Munch, Frederiksen Signe Frost, Heegaard Christian Würtz, Thomsen Maj Schneider, Hede Eva, Laczek Bartosz, Körbelin Jakob, Wüstner Daniel, Thomsen Louiza Bohn, Schwaninger Markus, Jensen Ole N, Moos Torben, Burkhart Annette
Neurobiology Research and Drug Delivery, Department of Health Science and Technology, Aalborg University, Selma Lagerlöfts Vej 249, 9260, Gistrup, Denmark.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.
Fluids Barriers CNS. 2025 Jan 31;22(1):13. doi: 10.1186/s12987-025-00621-4.
Patients with the genetic disorder Niemann-Pick type C2 disease (NP-C2) suffer from lysosomal accumulation of cholesterol causing both systemic and severe neurological symptoms. In a murine NP-C2 model, otherwise successful intravenous Niemann-Pick C2 protein (NPC2) replacement therapy fails to alleviate progressive neurodegeneration as infused NPC2 cannot cross the blood-brain barrier (BBB). Genetic modification of brain endothelial cells (BECs) is thought to enable secretion of recombinant proteins thereby overcoming the restrictions of the BBB. We hypothesized that an adeno-associated virus (AAV-BR1) encoding the Npc2 gene could cure neurological symptoms in Npc2-/- mice through transduction of BECs, and possibly neurons via viral passage across the BBB.
Six weeks old Npc2-/- mice were intravenously injected with the AAV-BR1-NPC2 vector. Composite phenotype scores and behavioral tests were assessed for the following 6 weeks and visually documented. Post-mortem analyses included gene expression analyses, verification of neurodegeneration in Purkinje cells, determination of NPC2 transduction in the CNS, assessment of gliosis, quantification of gangliosides, and co-detection of cholesterol with NPC2 in degenerating neurons.
Treatment with the AAV-BR1-NPC2 vector improved motor functions, reduced neocortical inflammation, and preserved Purkinje cells in most of the mice, referred to as high responders. The vector exerted tropism for BECs and neurons resulting in a widespread NPC2 distribution in the brain with a concomitant reduction of cholesterol in adjacent neurons, presumably not transduced by the vector. Mass spectrometry imaging revealed distinct lipid alterations in the brains of Npc2-/- mice, with increased GM2 and GM3 ganglioside accumulation in the cerebellum and hippocampus. AAV-BR1-NPC2 treatment partially normalized these ganglioside distributions in high responders, including restoration of lipid profiles towards those of Npc2+/+ controls.
The data suggests cross-correcting gene therapy to the brain via delivery of NPC2 from BECs and neurons.
患有遗传性疾病尼曼-皮克C2型病（NP-C2）的患者会出现溶酶体胆固醇蓄积，从而导致全身和严重的神经症状。在小鼠NP-C2模型中，原本成功的静脉注射尼曼-皮克C2蛋白（NPC2）替代疗法无法缓解进行性神经变性，因为注入的NPC2无法穿过血脑屏障（BBB）。脑内皮细胞（BEC）的基因改造被认为能够使重组蛋白分泌，从而克服血脑屏障的限制。我们假设，编码Npc2基因的腺相关病毒（AAV-BR1）可以通过转导BEC，并可能通过病毒穿过血脑屏障转导神经元，从而治愈Npc2-/-小鼠的神经症状。
对6周龄的Npc2-/-小鼠静脉注射AAV-BR1-NPC2载体。在接下来的6周内评估综合表型评分和行为测试，并进行视觉记录。死后分析包括基因表达分析、浦肯野细胞神经变性的验证、中枢神经系统中NPC2转导的测定、胶质增生的评估、神经节苷脂的定量以及在退化神经元中胆固醇与NPC2的共检测。
用AAV-BR1-NPC2载体治疗改善了大多数小鼠的运动功能，减轻了新皮质炎症，并保护了浦肯野细胞，这些小鼠被称为高反应者。该载体对BEC和神经元具有嗜性，导致NPC2在大脑中广泛分布，同时相邻神经元中的胆固醇减少，这些神经元可能未被该载体转导。质谱成像显示Npc2-/-小鼠大脑中存在明显的脂质改变，小脑和海马体中GM2和GM3神经节苷脂积累增加。AAV-BR1-NPC2治疗使高反应者的这些神经节苷脂分布部分恢复正常，包括脂质谱恢复到Npc2+/+对照的水平。
数据表明通过从BEC和神经元递送NPC2对大脑进行交叉校正基因治疗。
