1. Volume 13, Issue 6, Pages 1074-1084 (June 2006)
Safety of Recombinant Adeno-Associated Virus Type 2–RPE65 Vector Delivered by Ocular Subretinal Injection 
Samuel G. Jacobson, Gregory M. Acland, Gustavo D. Aguirre, Tomas S. Aleman, Sharon B. Schwartz, Artur V. Cideciyan, Caroline J. Zeiss, Andras M. Komaromy, Shalesh Kaushal, Alejandro J. Roman, Elizabeth A.M. Windsor, Alexander Sumaroka, Susan E. Pearce- Kelling, Thomas J. Conlon, Vincent A. Chiodo, Sanford L. Boye, Terence R. Flotte, Albert M. Maguire, Jean Bennett, William W. Hauswirth 
Molecular Therapy 
Volume 13, Issue 6, Pages (June 2006)
DOI: /j.ymthe
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Design of the rAAV-2/2.RPE65 vectors in this study. (A) AAV2-CBo-hRPE65, 4070 bp, and (B) AAV2-CBSB-hRPE65, 3921 bp, differ by 152 bp at the 5′ end of the CMV immediate early enhancer. ITR, AAV2 inverted terminal repeats; CMV ie enhancer, cytomegalovirus immediate early enhancer; β-actin, chicken β-actin promoter; Exon1, chicken β-actin exon 1; Intron, hybrid chicken β-actin and rabbit β-globin intron; Exon2, rabbit β-globin exon 3; hRPE65, human RPE65 cDNA; SV40 poly(A), SV40 polyadenylation signal.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Schematics showing the sites of subretinal injections in the 18 RPE65-mutant dogs of this study. Injection sites of (A) vehicle control and (B) rAAV-2/2.RPE65 vector are shown for right eyes (RE) and left eyes (LE).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
In vivo ocular examination of 15 RPE65 mutant dogs after ocular delivery of rAAV-2/2.RPE65 vector (circles) compared to vehicle control (diamonds). Inflammatory changes (conjunctival hyperemia, chemosis, or discharge; cellularity/precipitates in anterior chamber and/or vitreous) as well as changes in transparency of ocular media (cornea/lens/vitreous) were assessed by slit-lamp biomicroscopy and indirect ophthalmoscopy. Clinical changes were graded at three levels of severity. Results from individual eyes are presented from left to right in order of increasing relative vector dose levels; data points are arbitrarily offset in the vertical direction within each dose level and ocular evaluation category. Eight vector doses (Table 1) are represented by levels of gray in the symbols (scale at bottom right).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
(A–I) Retinal histopathology and (J, K) outer nuclear layer (ONL) thickness analyses in 15 RPE65-mutant dogs at 3 months after intraocular delivery of vehicle or rAAV-2/2.RPE65 vector. Histological sections illustrate injection site traumatic lesions (A–C), inflammatory changes in the vitreous and retina (C–E), rosette formation within a large traumatic lesion (F), and outer retinal thinning at dose 1× (H, I) compared with vehicle (G). Note that the images in G, H, and I are from the same retinal location. Calibration bars (A, D, G) apply to entire rows of micrographs except F, which is at a higher magnification. (J) ONL thickness measurements along the vertical meridian of the eyes of the 15 dogs studied; circles and triangles within each vector-dose graph represent different animals (filled, RE; open, LE). Black lines connect individual measurements in each eye. Thicker red lines define region of subretinal injection (in this meridian). (K) Summary data comparing uninjected (black squares) with injected (red squares) retina for each eye, within each vector-dose level. Data from the same eye are connected by a vertical line. *Statistically significant difference between uninjected and injected data for that eye. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Retinal function after subretinal delivery of different doses of rAAV-2/2.RPE65 vector to 18 RPE65-mutant dogs. (A) Dark-adapted ERGs evoked by increasing intensities of light in RPE65-mutant dogs 2–3 months after subretinal delivery of rAAV-2/2.RPE65 (dose 1×; right) compared to an age-matched vehicle-control-injected RPE65-mutant dog (middle). ERGs from a normal dog are also shown (left). Stimulus onset is at trace onset; stimulus intensity is at the left of key traces; calibration bars, bottom right of responses. (B) Light-adapted flicker (29 Hz) ERGs elicited by white-flash stimuli in the same animals. Vertical gray bars represent stimulus onset; calibration bars, right of responses. (C) ERG amplitudes under dark- and light-adapted conditions in normal (N) dogs (squares) and in different groups of RPE65-mutant dogs: untreated (U) eyes (hexagon; error bar, mean + 3 SD), vehicle control (VC)-injected eyes (diamonds), and eyes injected with increasing dose of rAAV-2/2.RPE65 vector (circles, vector AAV2-CBo-hRPE65; circles with dots, AAV2-CBSB-hRPE65). Eight levels of vector doses are represented by levels of gray fill.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2006 The American Society of Gene Therapy Terms and Conditions