rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector.
OBJECTIVE
To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy.


METHODS
trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA.


RESULTS
After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy.


CONCLUSIONS
The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.
Lai
Chooi-May
CM
Centre for Ophthalmology and Visual Science, The University of Western Australia, Crawley, Western Australia, Australia.
Estcourt
Marie J
MJ
Wikstrom
Matthew
M
Himbeck
Robyn P
RP
Barnett
Nigel L
NL
Brankov
Meliha
M
Tee
Lisa B G
LB
Dunlop
Sarah A
SA
Degli-Esposti
Mariapia A
MA
Rakoczy
Elizabeth P
EP
D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D004195:Disease Models, Animal; D004596:Electroretinography; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005451:Fluorescein Angiography; D019084:Fluorescent Antibody Technique, Indirect; D015316:Genetic Therapy; D005822:Genetic Vectors; D007111:Immunity, Cellular / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D015861:Retinal Neovascularization / Q000276:immunology* / Q000473:pathology / Q000628:therapy*; D012171:Retinal Vessels / Q000378:metabolism / Q000473:pathology; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics* / Q000276:immunology
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C501163:Flt1 protein, mouse; D040281:Vascular Endothelial Growth Factor Receptor-1