Effects of Stuffer DNA on the Suppression of Choroidal Neovascularization by a rAAV Expressing a mTOR-Inhibiting shRNA  Steven Hyun Seung Lee, HeeSoon Chang, Hee Jong Kim, Jun-Sub Choi, Jin Kim, Ji Hyun Kim, Ha-Na Woo, Seung Kwan Nah, Sang Joon Jung, Joo Yong Lee, Keerang Park, Tae Kwann Park, Heuiran Lee  Molecular Therapy - Methods & Clinical Development  Volume 14, Pages 171-179 (September 2019) DOI: 10.1016/j.omtm.2019.06.004 Copyright © 2019 The Authors Terms and Conditions

Figure 1 Characterization of rAAV2-shmTOR-SD In Vitro Schematic representation of the various virus vectors (A). The entire GFP expression cassette of rAAV2-shmTOR-GFP has been replaced with a stuffer DNA derived from the 3′ UTR of the human UBE3A gene to produce rAAV2-shmTOR-SD. Normalized relative to mock-treated ARPE-19 cells, quantitative analysis conducted via qRT-PCR showed that both the GFP- and stuffer DNA-containing virus vectors yielded significantly reduced mTOR mRNA expression when compared to their counterparts containing a control shRNA (B). At 48 h after treatment with the various virus vectors, mTOR expression was determined via western blotting from ARPE-19 cells (C), with β-actin used as a protein loading control, and the results were quantified thereafter (D). Molecular Therapy - Methods & Clinical Development 2019 14, 171-179DOI: (10.1016/j.omtm.2019.06.004) Copyright © 2019 The Authors Terms and Conditions

Figure 2 In Vivo Activity of rAAV2-shmTOR-SD and Retinal Transduction of the Mouse Model Western blots of retinal tissue samples, again using β-actin as a loading control (A), demonstrated that rAAV2-shCon-SD treatment had little effect on mTOR expression, while it was significantly downregulated by rAAV2-shmTOR-SD (B) (n = 3). Immunohistochemistry performed on frozen sections showed that the various virus vectors were able to successfully transduce the retinas of the laser-induced mouse model and exert their desired effects on the target tissue (C). Molecular Therapy - Methods & Clinical Development 2019 14, 171-179DOI: (10.1016/j.omtm.2019.06.004) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Fundus Fluorescein Angiography FFA performed 1 day prior to sacrifice showed extensive new vessel development resulting from laser photocoagulation in the retinas of mock-treated mice and in those treated with control shRNA-containing virus vectors. This activity was significantly abrogated upon rAAV2-shmTOR-SD and rAAV2-shmTOR-GFP transduction (A), which was then quantified (B) (n = 3). Molecular Therapy - Methods & Clinical Development 2019 14, 171-179DOI: (10.1016/j.omtm.2019.06.004) Copyright © 2019 The Authors Terms and Conditions

Figure 4 Visualization of CNV Extensiveness Resulting from Laser Photocoagulation Whole-mount immunostaining using anti-CD31 to observe endothelial cells revealed that CNV was widespread in control mice and those treated with rAAV2-shCon-SD and rAAV2-shCon-GFP. In contrast, CNV extensiveness was markedly reduced in mice treated with rAAV2-shmTOR-SD and rAAV2-shmTOR-GFP (A), with the difference being especially pronounced between the virus vectors containing the stuffer DNA (B) (n = 6). Molecular Therapy - Methods & Clinical Development 2019 14, 171-179DOI: (10.1016/j.omtm.2019.06.004) Copyright © 2019 The Authors Terms and Conditions

Figure 5 Anti-apoptotic Effect of rAAV2-shmTOR-SD, as Determined by TUNEL Assay TUNEL-positive cells were observed in retinal sections of mock-treated mice and mice treated with virus vectors containing control shRNA. Significantly fewer apoptotic cells were detected in the retinas of mice treated with rAAV2-shmTOR-SD and rAAV2-shmTOR-GFP (A), showing that the mTOR shRNA has anti-apoptotic activity, with the results then quantified (B) (n = 3). Molecular Therapy - Methods & Clinical Development 2019 14, 171-179DOI: (10.1016/j.omtm.2019.06.004) Copyright © 2019 The Authors Terms and Conditions