1. 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 (September 2019)
DOI: /j.omtm
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2. 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  , DOI: ( /j.omtm )
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3. 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  , DOI: ( /j.omtm )
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4. 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  , DOI: ( /j.omtm )
Copyright © 2019 The Authors Terms and Conditions

5. 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  , DOI: ( /j.omtm )
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6. 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  , DOI: ( /j.omtm )
Copyright © 2019 The Authors Terms and Conditions