Journal: Journal of Neurochemistry
Article Title: A novel strategy for delivering N iemann‐ P ick type C2 proteins across the blood–brain barrier using the brain endothelial‐specific AAV‐BR1 virus
doi: 10.1111/jnc.15621
Figure Lengend Snippet: Viral vector distribution of AAV‐BR1‐NPC2‐eGFP and barrier integrity following transduction of an in vitro blood–brain barrier (BBB) model using primary mouse brain endothelial cells (mBECs) in non‐contact co‐culture with primary mouse glial cells. (a) The expression of two important tight junction proteins (Zonula occludens 1 (ZO1) and Claudin 5 (CLD5)) was evaluated in the mBECs using immunocytochemistry. Transduced cells expressing enhanced green fluorescent protein (eGFP) (green) are found both 2 and 4 days after virus administration with the majority of positive cells being present after 4 days. In both cases, transduced mBECs maintain a robust tight junction expression (red) similar to that observed in the respective non‐transduced controls (CTRL). Nuclei are counterstained with DAPI (blue). Scale bar 25 μm. (b) Barrier integrity of primary mBECs in non‐contact co‐cultures with primary mouse glial cells, measured as TEER, is not affected by the transduction of mBECs. Data were analyzed using a REML mixed‐effects model with Sidaks multiple comparisons revealing no significant differences between TEER values of transduced mBECs ( n = 25 culture inserts) and non‐transduced CTRL ( n = 17 culture inserts). Data are presented as mean ± SD. (c) Viral vectors can cross the in vitro BBB model following transduction with 10 10 viral vectors per culture insert added to the upper chamber, representing the blood side of the BBB. 24 h post‐transduction remaining viral particles in the cell medium (presented as the percentage of viral genomes (vg) in relation to the total number of vg added to the upper chamber) are mainly present in the upper chamber, while only a very small fraction is present in the lower chamber representing the brain side. The majority (98%) of vg is taken up by the cells. Data are presented as mean ± SD ( n = 6 culture inserts). (d) Viral vectors primarily transduced mBECs in the in vitro BBB model when analyzing the presence of viral genomes (vg) within the primary mBECs and glial cells 24 h after vector administration. Data are presented as mean ± SD ( n = 4 number of DNA samples obtained from three inserts for mBECs and one well for glial cells for data).
Article Snippet: All cells were incubated with primary antibodies diluted in blocking buffer for 1 h. mBECs were incubated with a primary goat anti‐GFP antibody (Abcam, #ab6673) diluted 1:500 combined with either mouse anti‐zonula occludens (ZO1) (Invitrogen, #339100), rabbit anti‐claudin 5 (CLD5) (Merck KGaA, #SAB4502981), or rabbit anti‐NPC2 (Novusbio, #NBP1‐84012) primary antibodies diluted 1:250 in blocking buffer.
Techniques: Plasmid Preparation, Transduction, In Vitro, Co-Culture Assay, Expressing, Immunocytochemistry, Virus
