Secretion of recombinant protein towards the central nervous system (CNS) induced by viral gene modification of brain endothelial cells (BCECs), represents a novel strategy for drug delivery to the CNS, by circumventing the blood-brain-barrier (BBB). A relevant application of the strategy is the lysosomal storage disease, Niemann-Picks type C2, as it is a genetic disease caused by a deficiency of the soluble protein NPC2, resulting in accumulation of intracellular cholesterol, which causes both visceral symptoms and progressive neurodegeneration. Furthermore, the lysosomal storage has been observed to cause increased lysosomal biogenesis, with increased expression of lysosomal genes. Intravenous NPC2 replacement has been observed to reverse visceral but not CNS symptoms, due to an inability to cross the BBB. The aim of this study was, therefore, to investigate if BCECs could be genetically modified using a viral vector to induce secretion of recombinant NPC2 protein towards both the CNS and the circulation without compromising the barrier integrity in an in vitro BBB model and whether this could reverse cholesterol accumulations in NPC2 mutant fibroblasts.
NPC2 expression lentiviral vectors were produced and used to for gene modification of and in vitro BBB model. Gene modification efficiency was evaluated based on flow cytometry and immunocytochemical staining of NPC2. The barrier integrity was continuously measured based on trans-endothelial electrical resistance (TEER). Conditioned medium from transduced cells was used to treat NPC2 mutant human fibroblasts and cholesterol accumulations were examined by Filipin III staining of cholesterol. Furthermore, the relative gene expression of the lysosomal genes Lamp1 and MCOLN1 was examined in wildtype and NPC2 mutant fibroblast with or without NPC2 replacement.
The in vitro BBB model was successfully transduced with produced NPC2 expression lentivira without compromising the barrier integrity in BBB model. Furthermore, the lentiviral vector seemed to induce a more stable long-term recombinant NPC2 expression compared to a non-viral vector. Cholesterol accumulations in NPC2 mutant fibroblasts were reversed following treatment with conditioned medium from genetically modified cells but a similar effect was observed following treatment with conditioned medium from non-modified cell due to an unexpectedly high natural secretion of endogenous NPC2. Signs of a therapeutic effect from secreted recombinant NPC2 was, however, seen from treatment with conditioned medium from the top chamber of the genetically modified BBB model. Moreover, MCOLN1 gene expression was increased in NPC2 mutant fibroblasts compared to wildtype, and seemed to decrease following NPC2 replacement, while no significant differences were observed for Lamp1 expression.