Addressing recombinant AAV9 as a novel therapeutic strategy for gene correction of Niemann Pick's type C2 disease in the CNS
Authors
Hauerslev, Lissa Vestergaard ; Andersen, Ida Køgs ; Johansen, Lonnie Møller
Term
4. term
Publication year
2019
Submitted on
2019-05-30
Pages
42
Abstract
Niemann-Pick type C2 (NPC2) is a fatal disease with neurological involvement caused by mutations in the NPC2 gene, and the neurotropic vector AAV9 is a potential vehicle to deliver a functional NPC2 gene to the central nervous system. This study aimed to evaluate AAV9-based gene therapy for NPC2 by testing a recombinant AAV9-NPC2-2A-GFP construct in a non-contact in vitro blood–brain barrier (BBB) model composed of porcine brain endothelial cells and primary rat astrocytes. Transduction was assessed 10 days after virus addition by detecting GFP with immunocytochemistry and RT-qPCR; potential therapeutic effect was examined by ELISA for secreted NPC2; and potential detrimental effects were evaluated by transendothelial electrical resistance (TEER) and immunostaining of ZO-1 and GFAP. GFP was not detected in endothelial cells or astrocytes, and secreted NPC2 was below the detection limit. TEER values were significantly lower in AAV9-treated barriers than in untreated controls from days 1 to 5, and GFAP staining suggested increased astrocyte reactivity; no differences were observed for ZO-1. Overall, AAV9-NPC2-2A-GFP did not transduce the BBB model and compromised barrier integrity in this in vitro setting.
Niemann-Picks type C2 (NPC2) er en dødelig sygdom med neurologisk involvering forårsaget af mutationer i NPC2-genet, og den neurotrofe vektor AAV9 kan potentielt bruges til at levere et funktionelt NPC2-gen til centralnervesystemet. Dette studie havde til formål at vurdere AAV9-baseret genterapi mod NPC2 ved at teste et rekombinant AAV9-NPC2-2A-GFP-konstrukt i en in vitro blod-hjerne-barriere (BHB) uden kontakt, bestående af porcine hjerneendotelceller og primære rotteastrocytter. Transduktion blev vurderet 10 dage efter virusaddition via detektion af GFP med immuncytokemi og RT-qPCR; mulig terapeutisk effekt blev undersøgt ved ELISA for secerneret NPC2; potentielle skadevirkninger blev vurderet ved trans-endotel elektrisk modstand (TEER) og immunfarvning af ZO-1 og GFAP. GFP blev ikke påvist i endotelceller eller astrocytter, og secerneret NPC2 lå under detektionsgrænsen. TEER var signifikant lavere i AAV9-behandlede barrierer end i ubehandlede fra dag 1 til dag 5, og GFAP-farvning tydede på øget astrocytreaktivitet; der sås ingen forskelle i ZO-1. Samlet set transducerede AAV9-NPC2-2A-GFP ikke BHB-modellen og kompromitterede barriereintegriteten i denne in vitro-opsætning.
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