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Mariana Santana Dias, Victor Guedes de Araujo, Rafael Linden, Hilda Petrs Silva; Modulation of gene transduction by rAAV2 in the rat retina after intravitreal injection. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5661.
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© ARVO (1962-2015); The Authors (2016-present)
Intravitreal injections are considered an easier and safer delivery route for retinal gene therapy compared to subretinal injections. They are, however, still inefficient in large eyes such as the primates’. rAAV can be degraded by ubiquitin-proteasome machinery, impairing transgene expression, and the proteosomal inhibitor bortezomib expressively enhanced rAAV2 transduction in all retinal layers through the vitreous in mice. In this work, we investigated whether bortezomib could enhance intravitreal transduction efficiency of rAAV2 in rat retinas, an animal with higher eye volume than mice.
Adult Lister-Hooded rats were intravitreally injected with either bortezomib 0.3 µM/1µL or vehicle, followed by injection of AAV2-GFP 109 vg. Rats were euthanized one month later, the eyes enucleated, fixed in 4% paraformaldehyde, and either processed for flat mount analysis of the ganglion cell layer (GCL) or for evaluation of all retinal layers in cryosections. Transduction efficiency was assessed by GFP immunoreaction and counting of GFP positive cells. The number of TOPRO-3 stained nuclei in GCL was quantified for all retinas.
Eyes infected with rAAV2 in the presence of bortezomib had a near 2 fold increase in transduced cells density in GCL (1768±185) when compared to control PBS (937.5±160 cells/mm2; n=6; p = 0.0068). Bortezomib did not alter the number of TOPRO-3 stained nuclei in GCL compared to control (4562±89 vs. 4616±153 cells/mm2; n=5-7; p=0.7479). In retinal layers located distally from the injection site, bortezomib enhanced transduction in outer nuclear layer (ONL) (13.3±0.6 vs. 3.9±1.7 cells/mm2; n=5; p = 0.0008), but yet with a maximum of only 1-2 transduced cells identified per field, and did not significantly increase transduction in inner nuclear layer (INL) (948±67.7 vs. 606.4±176.9 cells/mm2; n=5; p = 0.1089).
Bortezomib enhanced transduction by rAAV2 in GCL of the rat retina after intravitreal injection. The overall effect of bortezomib throughout the retina was, however, mild, especially in the light of previous results with this drug in mice. This highlighted the difficulty in obtaining expressive increase in transduction efficiency with rAAV2 in all the retina after intravitreal injections in eyes that are not as small as the mice’s, and pointed the rat as a potential more reliable model for studies aiming to enhance rAAV2 transduction through the vitreous.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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