Volume 23, Issue 8, Pages 1308-1319 (August 2015) CNTF Gene Therapy Confers Lifelong Neuroprotection in a Mouse Model of Human Retinitis Pigmentosa  Daniel M Lipinski, Alun R Barnard, Mandeep S Singh, Chris Martin, Edward J Lee, Wayne I L Davies, Robert E MacLaren  Molecular Therapy  Volume 23, Issue 8, Pages 1308-1319 (August 2015) DOI: 10.1038/mt.2015.68 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Long-term quantification of intrinsically fluorescent cone photoreceptors through repetitive in vivo imaging following rAAV2/2.hCNTF treatment. (a) Autofluorescence (AF) image of Rho-/- Tg(OPN1LW-EGFP)+/0 mouse fundus showing cone photoreceptors represented as punctate white dots. (a’) Increase magnification AF image demonstrating a distinctive group of cone photoreceptors (aligned β to γ) and individual cones (white arrows) at the nasal vascular bifurcation (α, dotted line). (b) Near infrared reflectance (NIR) fundus image detailing the retina vasculature and (b’) magnified NIR image detailing the nasal vascular bifurcation (α, dotted line). (c) flat mounted post-mortem retina with retinal vasculature counterstained with lectin. (c’) magnified image of the retinal flat mount demonstrating the distribution of cone photoreceptors (green dots) at the nasal bifurcation (α, dotted line). Note that the pattern of cones in panel (c’) correlates precisely with that observed by in vivo AF imaging (a’); including a distinctive group of cone photoreceptors (aligned β to γ) and individual cones (white arrows). (d–g) Repetitive AF fundus imaging of a Rho-/-, Tg(OPN1LW-EGFP)+/0 mouse treated with high dose rAAV2/2.hCNTF from postnatal week (PW) 8 to PW30. (d’–g’) Magnified AF images showing how retinal vascular l lifetime of each animal. Cone photoreceptors (white dots) can be quantified (red dot overlay) within the regions of interest allowing highly reproducible longitudinal assessment of cone survival. Survival is expressed as a function (%) of cone photoreceptor numbers at baseline (PW8). All scale bars ~100 μm. (h) Survival of cone photoreceptors expressed as a function (%) of cone photoreceptor numbers at baseline (PW8) for each treatment group: high dose (dark blue), medium dose (bright blue), low dose (pale blue) or PBS sham (grey). hCNTF treatment has a significant effect on cone photorecptor survival: *P < 0.05; **P < 0.01; ***P < 0.001, two-way repeated measures analysis of variance with dose and time as factors. High-dose group n = 5, medium-dose group n = 6, low-dose group n = 8, at PW30. Molecular Therapy 2015 23, 1308-1319DOI: (10.1038/mt.2015.68) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Near infrared reflectance (NIR) and autofluorescence (AF) fundus imaging of high dose rAAV2/2.hCNTF-treated and PBS sham-treated mice at PW8 and PW30. Sham-treated controls demonstrate areas of increased reflectance indicative of retinal pigment epithelium (RPE) atrophy (a,c) and loss of EGFP-expressing cone photoreceptors (b,d) by PW30. High-dose rAAV2/2.hCNTF-treated eyes show no signs of RPE atrophy (e,g) and minimal loss of cone photoreceptors (f,h) over the same period. Representative histology of phosphate-buffered saline (PBS) sham treated (i) retina at PW30 demonstrates and absence of both rod and cone photoreceptors. Histology of high dose rAAV2/2.hCNTF treated (j–o) retina at PW30 demonstrating preservation of cone (GFP-expressing) and rod (non-GFP-expressing) photoreceptors in the outer nuclear layer (ONL). (j,k) Cone photoreceptors (l–o) express Mws-opsin (arrow head) in the correct cellular compartment (outer segment (OS)) with no evidence of opsin mis-localization in the cell body (*). Cone photoreceptor morphology indicates compression between the inner nuclear layer (INL) and RPE due to the loss of multiple nuclear rows following rod degeneration. Molecular Therapy 2015 23, 1308-1319DOI: (10.1038/mt.2015.68) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Assessment of cone photoreceptor function in PW30 rAAV2/2.hCNTF treated mice through laser speckle imaging of the visual cortex. (a,b) change in cortical blood flow (CBF) in untreated Rho-/-TgOPN1LW-EGFP+/- mice at PW6 demonstrating that laser speckle imaging (LSI) detect changes in micro-vascular blood flow primarily in the visual cortex following stimulation with a 510 nm flicker stimulus. (c) Schematic representation of the visual pathway in mice demonstrating that visual input is primarily processed in the visual cortex located in the hemisphere contralateral to the eye stimulated. White arrow = superior sagital sinus; arrow head = lambda; OS = oculus sinister (left); OD = oculus dexter (right). Changes in CBF were examined in (d,e) medium and (g–i) high dose treated Rho-/-, Tg(OPN1LW-EGFP)+/0 mice at PW30 following independent stimulation of PBS-treated (d,g) or rAAV2/2.hCNTF-treated (e,h) treated eyes, revealing significant differences in CBF from high-dose-treated eyes (i) compared to sham controls. ns, not significant. *P < 0.05, Wilcoxon matched-paired signed rank test. LSI: high dose n = 3; medium dose n = 6. Molecular Therapy 2015 23, 1308-1319DOI: (10.1038/mt.2015.68) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Cone-photoreceptor mediated vision is preserved only in rAAV2/2.hCNTF treated eyes at PW30. (a) schematic representation of the optomotor response (OMR) in relation to the direction of the drums rotation. (b) Number of head-tracking responses (three tests per animal) from individual mice at PW30. Error = SEM; X = zero responses; L = low dose, M = medium dose, H = high dose. (c) Mean group responses to OMR testing at PW30 demonstrating significantly greater number of head-tracks from medium (bright blue) and high dose (dark blue) compared to paired PBS sham-treated controls. ns, not significant. ***P < 0.001, one-way analysis of variance. Low dose n = 5, medium dose n = 6, high dose n = 4. Molecular Therapy 2015 23, 1308-1319DOI: (10.1038/mt.2015.68) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 RNAseq analysis comparing the transcriptional profile of rAAV2/2.hCNTF eyes with contralateral PBS sham treated controls revealed upregulation of proteolysis inhibitors and complement. (a) Hierarchical clustering of transcriptome output from low-, medium-, and high-dose rAAV2/2.hCNTF-treated mice showing altered gene expression relative to paired PBS sham-treated controls (n = 4 mice per group) at PW30, where the solid blue line in each column represents fold-change compared to baseline (dotted blue line). Two clusters relating to genes with the highest transcriptional changes are enlarged: the upper panel shows downregulation of genes involved in ion transport and visual cycle; the lower panel shows widespread upregulation of genes encoding serine-, cysteine-, and metallopeptidase inhibitors. (b) Clustering of genes based function using genome search meta analysis ontology visualized using the enrichment map plugin for cytoscape. Molecular Therapy 2015 23, 1308-1319DOI: (10.1038/mt.2015.68) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions