1. Volume 25, Issue 3, Pages 765-779 (March 2017)
AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress 
Katharine J. Liang, Kenton T. Woodard, Mark A. Weaver, John Paul Gaylor, Ellen R. Weiss, R. Jude Samulski 
Molecular Therapy 
Volume 25, Issue 3, Pages (March 2017)
DOI: /j.ymthe
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2. Molecular Therapy 2017 25, 765-779DOI: (10.1016/j.ymthe.2016.12.016)
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3. Figure 1
AAV-Nrf2 Transduction Drives Nrf2 and Downstream Gene Expression and Decreases ROS In Vitro
(A) Western blot verifying NRF2 expression and corresponding densitometric analysis of total γ-glutamylcysteine synthase (γ-GCSm) downstream gene activation following pAAV-Nrf2 transfection compared to no-virus control in 293T cells. Western blot shown is representative of several westerns quantified in corresponding densitometric analysis (n = 4; *p < 0.1, paired t test). (B) Western blot and corresponding densitometric quantitation of pAAV-Nrf2-induced heme oxygenase-1 (HO-1) gene activation and Nrf2-siRNA repression compared to no-virus and pAAV-AAT transfection controls are shown. Lanes 1 and 4, none; lanes 2 and 4, pAAV-CBA-AAT; lanes 3 and 6, pAAV-CBh-Nrf2. (C) Fluorescent ROS assay was used to measure H2O2 and arsenic-induced oxidative stress levels in either untransduced HEK293T cells or cells transduced with 50,000 MOI AAV-Nrf2. Plotted values represent the ratio of fluorescence measured in cells stressed with H2O2 and arsenic to reference cells treated with vehicle (n = 12 paired biological replicates from 12 independent experiments, each n representing the mean value of a triplicate; **p < 0.001, linear mixed model with random plate effects).
Molecular Therapy  , DOI: ( /j.ymthe )
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4. Figure 2 AAV-Nrf2 Protects Mice from Acute Acetaminophen Toxicity
(A) Nrf2 was cloned into AAV2 ITR backbones under the control of a CBA promoter. Nrf2−/− and wild-type mice were injected with AAV2-CBA-Nrf2 prior to the administration of acetaminophen by oral gavage, and they were observed for survival at 24 and 48 hr following oral gavage. (B) AAV-Nrf2 injection of Nrf2−/− mice resulted in protection from acetaminophen-induced lethality (600 mg/kg; exact log-rank test p < for Nrf2−/− mice versus Nrf2−/− mice + AAV-Nrf2 at 48 hr). (C) H&E staining of liver sections revealed severe centrilobular necrosis in Nrf2−/− mice following 600 mg/kg acetaminophen stress, which was not present in Nrf2−/− mice that had received AAV-Nrf2 prior to acetaminophen stress (D). (E) AAV-Nrf2 injection of wild-type mice resulted in protection from acetaminophen-induced lethality (1,000 mg/kg; exact log-rank test p = for injected versus uninjected mice at 48 hr). H&E staining of liver sections revealed centrilobular necrosis in wild-type mice following 1,000 mg/kg acetaminophen stress (F), which was not present in mice that had received AAV-Nrf2 prior to acetaminophen stress (G). Scale bars, 100 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
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5. Figure 3 AAV Transduction in the Retina via Intravitreal Delivery
(A) eGFP and Nrf2 were cloned into AAV2 ITR backbones under the control of a CBh promoter. (B) Western blot analysis of mouse retinal lysates from uninjected eyes compared to eyes injected with AAV-Nrf2 is shown. The expression of the ARE-controlled gene (HO-1) in Nrf2−/− mouse retina lysates was compared to that from uninjected wild-type mice, as well as to 293T cells that were either untreated or transfected with pAAV-Nrf2. α-tubulin and calnexin were used as loading controls for Nrf2 and HO-1 blots, respectively. (C) AAV2- (upper) and AAV2.5-CBh-GFP- (lower) mediated GFP expression is visualized by in vivo fluorescent fundus in intravitreally injected mouse eyes. (D) Corresponding 10-μm cryosections were immunostained for GFP and the Müller glia marker glutamine synthetase (GS). Scale bars, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
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6. Figure 4 Retinal Pathology following Light Damage
(A) Retinal layers are labeled on in vivo OCT scans and H&E-stained sections from naive adult BALB/c mice. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment. (B) Mice were intravitreally injected with AAV-Nrf2 in one eye and the fellow eye was left uninjected. OCT scans from 1 week and 3 months following light damage were blinded and scored for pathological features (n = 5 mice per group, four scans per eye, 20 scans per group). Retinal pathology scores were plotted as paired values of injected and uninjected eyes from the same animal (detailed scoring table, Table S1). AAV-Nrf2-injected eyes exhibited significantly decreased pathological features compared to uninjected eyes at 3 months following light damage (n = 5; *p = 0.02, paired t test). (C) Representative OCT scans and H&E-stained sections of AAV-Nrf2-injected and uninjected eyes, 1 week and 3 months following exposure to bright white light, are shown. OCT scale bars, 100 μm; H&E scale bars, 60 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
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7. Figure 5 AAV-Nrf2 Protects Retinal Thickness following Light Damage
Total retinal thickness (A) and ONL thickness (B) were evaluated using segmentation of OCT images taken of two separate groups of mouse eyes in parallel in vivo experiments at 1 week and 3 months post-light stress. Prior to light stress, mice (n = 5) were injected in one eye with AAV-Nrf2 (cyan, n = 5 eyes) and the fellow eye was left uninjected (black, n = 5 eyes). Age-matched naive mice (yellow, n = 5 mice, ten eyes) also were compared to light-stressed mice. Individual raw traces of retinal thickness and smoothed mean lines are shown, with gray areas representing ±SD (smoothed) values for each group (complete list of p values, Table S2).
Molecular Therapy  , DOI: ( /j.ymthe )
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8. Figure 6 Dark-Adapted Retinal Function following Severe Light Damage
ERG recordings and amplitudes showing rod-dominated retinal function at 1 week (A and B) and 3 months (C and D) following 3 hr of light stress. (A and C) Representative ERG traces are shown for injected and uninjected eyes. (B and D) Average ERG amplitudes for a-waves (upper) and b-waves (lower) were plotted for mice exposed to light damage following AAV-Nrf2 intravitreal injection (closed black circles) or left uninjected (open circles), compared to age-matched naive mice (gray circles). Error bars represent SEM (n = 5/group; complete list of p values, Tables S3 and S4).
Molecular Therapy  , DOI: ( /j.ymthe )
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9. Figure 7 Nrf2-ETGE Exhibits Increased ARE Activity In Vitro
Nrf2 activity was quantified as a ratio of Firefly to Renilla luciferase activity following transfection of HEK293T cells with ARE-Firefly luciferase, CMV-Renilla luciferase, and Keap1 expression plasmids, along with AAV packaging plasmids CBA-AAT, CBh-Nrf2-WT, or CBh-Nrf2-ETGE. Assay was performed in triplicate and data are reported for three separate biological replicates (black, gray, and open circles). Lines represent geometric means (p < for all comparisons except Nrf2-WT versus Nrf2-ETGE in the absence of Keap1; complete list of p values, Table S5).
Molecular Therapy  , DOI: ( /j.ymthe )
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10. Figure 8 Dark-Adapted Retinal Function following Moderate Light Damage
ERG recordings and amplitudes showing rod-dominated retinal function 1 week (A and B) and 1 month (C and D) following 2 hr of light stress. (A and C) Representative traces are shown for injected and uninjected eyes. (B and D) Average ERG amplitudes for a-waves (upper) and b-waves (lower) were plotted for mice exposed to light damage following AAV-Nrf2 intravitreal injection (closed black circles) or left uninjected (open circles), compared to age-matched naive mice (gray circles). Error bars represent SEM (n = 5 per group; complete list of p values, Tables S3 and S4).
Molecular Therapy  , DOI: ( /j.ymthe )
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