Volume 8, Issue 2, Pages 188-195 (August 2003) AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa  Alexander J Smith, Frank C Schlichtenbrede, Marion Tschernutter, James W Bainbridge, Adrian J Thrasher, Robin R Ali  Molecular Therapy  Volume 8, Issue 2, Pages 188-195 (August 2003) DOI: 10.1016/S1525-0016(03)00144-8 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Expression of Gfp 6 weeks after subretinal injection of AAV-CMV-GFP or AAV-RPE-GFP in the RCS rat. Vectors were injected subretinally into 10-day-old RCS rats. (A) Six weeks after injection of AAV-CMV-GFP transgene expression was observed in the RPE and in the photoreceptor cells. (B) Following subretinal injection of AAV-RPE-GFP, the reporter gene is expressed mainly in the RPE. Due to the degeneration there are few photoreceptors remaining in the ONL and there is a large amount of debris (labeled D) in the subretinal space, which results in a high level of autofluorescence. Images were taken using green and red filters and overlaid to distinguish between GFP fluorescence (green) and autofluorescence (yellow). Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 RT-PCR amplification of Mertk from injected mouse eyes. RNA was isolated from eyes injected with AAV-CMV-Mertk (lanes 1 and 7) and AAV-RPE65-Mertk (lanes 3 and 9) and from the uninjected control eyes (lanes 2 and 8 and lanes 4 and 10, respectively). Samples were treated with DNase to remove genomic and viral DNA before reverse transcription and amplification of the transgenic Mertk mRNA. A band of the correct size was amplified from the eyes treated with either vector (lanes 1 and 3); whereas there was no transgenic mRNA detectable in the uninjected eyes (lanes 2 and 4). Lane 5 shows a cDNA positive control for the amplification of Mertk, lane 6 is a 250 bp ladder. Omission of the reverse transcriptase results in the absence of signal (lanes 7 to 10). Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 ERG recordings following gene delivery. A representative intensity series of right and left eyes is shown 6 weeks after various treatments. After treatment with (A) AAV-CMV-Mertk or (B) AAV-RPE65-Mertk an increased b-wave amplitude was present in the treated eye, indicative of a prolonged photoreceptor function. (C) Injection of control vector AAV-CMV-GFP does not lead to altered traces at any time point. (D) When AAV-CMV-Mertk was injected into normal (nondystrophic) rat eyes, no differences were found in b-wave amplitude. Please note the different y-axis scale compared to the other graphs. Flash intensities (in mcds/m2) are shown next to the traces in D. Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 ERG recordings from a single animal at various time points following gene delivery. Averaged responses at a single stimulus intensity (100 mcds/m2) from one individual RCS rat are shown over 12 weeks following treatment with AAV-CMV-Mertk. The time course clearly reveals the improvement in b-wave amplitude and waveform. These effects remain prominent for up to 9 weeks, when the b-wave amplitude gradually declines on the treated side. Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Mean ERG b-wave amplitudes after injection of recombinant AAV. The median of the b-wave amplitudes and the range between the 25 and 75% quartiles of the treated and untreated eyes are shown at various time points after treatment with (A) AAV-CMV-Mertk, (B) AAV-RPE65-Mertk, and (C) AAV-CMV-GFP. All measurements were performed at a flash intensity of 100 mcds/m2. Statistical significance of the differences between the treated and the untreated eyes was determined using a paired nonparametric test (*P < 0.05, **P < 0.01). After treatment with virus containing Mertk a significantly improved ERG response is present up to 9 weeks after injection, whereas injection of AAV-CMV-GFP did not increase the b-wave amplitude. Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Histological analysis of AAV-CMV-Mertk-treated eyes. Semithin sections of (A) AAV-CMV-Mertk-treated and (B) the contralateral untreated eye were stained with hematoxylin and eosin 9 weeks after injection. The amount of debris deposited between the outer nuclear layer and the RPE is lower in the treated eyes than in the untreated eyes, indicative of a restoration of the phagocytic function of the RPE. The number of photoreceptor cell nuclei is higher after injection with therapeutic virus, confirming that fewer photoreceptor cells were lost. Molecular Therapy 2003 8, 188-195DOI: (10.1016/S1525-0016(03)00144-8) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions