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Gene therapy targeting of choroidal disease and AAV transcytosis through the outer blood retina barrier epithelium
Author(s) -
Layton C.,
Dhungel B.,
Andrzejewski S.,
Jayachandran A.,
Murali A.,
RamloganSteel C.,
Steel J.
Publication year - 2017
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2017.02683
Subject(s) - transcytosis , retinal pigment epithelium , retina , tight junction , epithelium , retinal , choroid , biology , microbiology and biotechnology , chemistry , medicine , ophthalmology , pathology , endocytosis , cell , biochemistry , neuroscience
Purpose The retinal pigment epithelium ( RPE ) forms the relatively impermeable outer blood‐retinal barrier, which, when intact, prevents intraocular gene therapy vectors from effectively targeting choroidal diseases such as choroidal melanoma or choroiditis. This study hypotheses that AAV vector optimisation may effectively penetrate this ocular barrier. Methods Monolayers of aRPE ‐19 and primary human RPE were established on 24 well plate transwell inserts and integrity confirmed with stable transepithelial resistance ( TEER ) of 35±5 Ω/cm 2 and resistance to passage of FITC ‐Dextran (400 kDa) at 4 weeks. 10^8±1 vgs of AAV serotypes 1, 2, 3, 4, 5, 6, and 8 were added to the apical surface and media collected from the basal surface at various time points. Viral concentrations were measured by qPCR . Results AAV serotype 6 moved through the intact monolayer of both aRPE ‐19 and primary human RPE . 41.5%. 47.4% and 60.4% of AAV ‐6 viral particles moved through the monolayer at 30 minutes, 3 hours and 5 hours respectively. Blocking RPE exocytosis with tannic acid prevented movement of AAV 6 through the monolayer. All other serotypes were unable to penetrate the RPE monolayer, with less than 10% penetrance at 24 hours. Conclusions These results indicate that AAV 6 passes through a model of the outer blood retina barrier by active intracellular transport. Additionally, this is the first report of AAV ‐6 transcytosis across any barrier epithelium. AAV 6 may be a promising tool to deliver therapeutic genes for ocular diseases which lay beneath the retinal epithelium.