Gene therapy to the blood–brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease

Abstract

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.

The blood-brain barrier studied in vitro across species

“…The addition of the BBB induction factor cAMP is also known to be able to increase the expression of tight junctions proteins [28,54]. Furthermore, when comparing previously published immunolabelings of ZO-1 in rBECs cultured in monolayers in well plates, without the presence of neither astrocytes nor tight junctions inducing factors, the organization of ZO-1 seems to be highly affected by the addition of the tight junction inducing factors [55], suggesting that cAMP and HC is also important for the organization of this protein.…”

“…However, setting up the triple culture model is complex, which is probably why this model is not commonly used, despite it has been developed and characterized for all three species [22,28,29,31,57]. The mono-culture and the co-culture models are on the contrary easy to construct and therefore also the preferred models across different laboratories [32,33,37,38,40,55,[58][59][60][61]. Future analysis of the significance of pericytes in in vitro BBB models should include analysis of the vesicular transport in the BECs as the presence of pericytes during embryogenesis decreases the vesicular transport and contribute to the formation of tight junctions [10,62].…”

The blood-brain barrier studiedin vitroacross species

“…The addition of the BBB induction factor cAMP is also known to be able to increase the expression of tight junctions proteins [28,62]. Furthermore, when comparing previously published immunolabelings of ZO-1 in rBECs cultured in monolayers in well plates, without the presence of neither astrocytes nor tight junctions inducing factors, the organization of ZO-1 seems to be highly affected by the addition of the tight junction inducing factors [63], suggesting that cAMP and HC is also important for the organization.…”

“…However, setting up the triple culture model is complex, which is probably why this model is not that commonly used, despite it has been developed and characterized for all three species [22,28,29,31,65]. The mono-culture and the co-culture models are on the contrary easy to construct and therefore also often the preferred models across different laboratories [32,33,42,43,45,49–51,63,66].…”

Human iPSC‐Derived Blood‐Brain Barrier Models: Valuable Tools for Preclinical Drug Discovery and Development?

“…Additionally, gene therapy is discussed as a promising technology to restore functionality in diseased tissue. Recently, primary rodent BCECs were successfully transduced targeting the lysosomal cholesterol storage disease Niemann Pick type C2 (Hede et al., 2019). To target AD‐ and PD‐related BBB symptoms, there is a need to first understand the consequence of disease‐associated genetic variants.…”