Fig 2: Differential gene expression in the cochlea by RNA sequencing and qRT‐PCR Venn diagram showing overlap between Ndp‐KO versus WT DEGs and Ndp‐KO versus P2‐L DEGs.Heatmap showing levels of expression of the 18 pathology‐related DEGs identified between Ndp‐KO (red) versus P2‐L (blue) in WT, Ndp‐KO and P2‐L treated cochleas (green). Note that these genes showed trends of differential expression between Ndp‐KO and WT cochleas that did not reach significance. No genes were found to be significantly differentially expressed between WT and P2‐L cochleas, indicating rescue by the treatment. In heat map, red indicates upregulated and blue indicates downregulated gene expression in Ndp‐KO.Gene set enrichment analyses of differentially expressed genes between WT and Ndp‐KO using gene sets were previously defined by transcriptome profiling of FACS sorted CNS vs peripheral endothelial cells (Daneman et al, 2010) and previously used to assess transcriptomes of WT and Ndp‐KO retinas (Zhou et al, 2014). Gene sets characterising barrier vasculature, BBB1 and 2, BBB endothelial transporters, CNS endothelial and CNS pericyte were significantly positively correlated (FDR < 0.25) with the WT genotype.Dot plot using scRNA seq data of the adult mouse cochlear lateral wall from GEO database: accession numbers GSM5124299, GSM5124300, GSM5124301, and GSM5124302. Gene markers used to distinguish 30 cell type clusters in the UMAP were as previously reported (Gu et al, 2020; Bryant et al, 2022).Dot plot showing expression of the 45 DEGs identified in WT versus Ndp‐KO analysis in the 30 cell type clusters identified in the adult mouse cochlear lateral wall at the single cell level. Blue arrows indicate endothelial cell genes (Cldn5, Abcb1a and Flt1) downregulated in Ndp‐KO, and orange arrows indicate upregulated genes in Ndp‐KO. Box indicates genes significantly different in the P2‐L versus Ndp‐KO comparison. Shaded DEGs are downregulated in the Ndp‐KO cochlea. Note expression of some DEGs across several different cell type clusters.

Fig 3: Effects of AAV9.NDP treatment on the retinal vessel morphology and visual function A, B Schematic of the normal retinal vasculature showing three vascular plexi in (A) cross‐sections and (B) whole mount, colour depth projection scheme indicated for (C–F).C–F Pseudocoloured depth projections of vascular plexi in the central retina of retinal whole mounts at 3 months. Vasculature staining with isolectin B4 (IB4) shows three plexi in the WT and after treatment at P2. C–F images are z‐projections of 50 slices of 0.75 μm. Treatment groups: C, WT; D, Ndp‐KO; E, P2‐L; F, P30‐H.G–J Vasculature: IB4, nuclei: DAPI at 2 months.K–N Localisation of tight junction marker claudin‐5 (CLDN5) and PLVAP at 2 months.O, P qRT‐PCR analysis of pathology‐related genes (O) Cldn5 (P) Plvap in retina at 2 months. Data information: Genotype and treatment ages (WT, Ndp‐KO, P2‐L, P21‐L, P21‐H, P30‐H) indicated on x axes. Mean ± SD; n = biological replicates. Sample numbers: n (WT) = 7, n (Ndp‐KO) = 6, n (P2‐L) = 6, n (P21‐L) = 6, n (P21‐H) = 6, n (P30‐H) = 6. Statistical analysis: analysed with one‐way ANOVA with Sidak's post hoc test, all values compared to WT (blue asterisks) and Ndp‐KO (red asterisks).Q–T Evaluation of visual function recovery with scotopic electroretinography (ERG) at 1.5 months of age. Example of ERG waves at 10 cd/s.m2 flash intensity stimulus in WT, Ndp‐KO, P2‐L and P21‐H groups, n = 1 (average of 10 repeated flashes; a‐wave, b‐wave, op—oscillatory potential) (Q). Oscillatory potentials (R), mean ERG b‐wave at increasing stimulus flash intensities in P2‐L (S) and P21‐H (T) groups compared with Ndp‐KO and WT. Data are shown as individual traces in (Q) and as mean ± SD in (R and T). Data information: Sample numbers for C–N: n = 4 per each group. O, P, R analysed with one‐way ANOVA with Sidak's post hoc test, S‐T analysed with two‐way repeated measures ANOVA with Tukey's post hoc test; all values compared to respective WT (blue asterisks) and Ndp‐KO (red asterisks). Post hoc test values: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, non‐significant. Sample numbers for ERG analysis: n (WT) = 10, n (Ndp‐KO) = 10, n (P2‐L) = 7, n (P21‐H) = 10. n = biological replicates. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Numbers 1, 2 and 3 label the superficial, intermediate and deep vascular plexuses. Arrows point to deep plexus vessels. Arrowheads point to abnormal neovascular tufts in Ndp‐KO and P21‐H. Scale bars: C–J 50 μm, K–N 20 μm. C–F and G–N images are z‐projections of 20 slices of 0.45 μm. Source data are available online for this figure.

Fig 4: AAV9.NDP vector transduction in the cochlea and retina A–E Organ of Corti and lateral wall wholemounts stained with anti‐GFP antibody. (A‐A″) Untreated WT cochlea; (B‐B″) P2‐L dose; (C‐C″) P21‐L dose; (D‐D″) P21‐H dose; (E‐E″) P30‐H dose. Scale bars = 500 μm (A‐E), 500 μm (A′‐E′), enlarged view of boxed region shown in A″‐E″. SGN, spiral ganglia region; arrowheads, fibrocyte shaped cells in the lateral wall.F P30‐H dose; cochlea stained with anti‐MyoVIIa antibody showing that hair cells are not transduced. Scale bar 100 μm.G, H EGFP immunostaining at 2 months in retinal cryosections after AAV9.NDP treatment at P2 (G) and P21 (H); optic nerve: o.n. Arrows indicate transduced region of the retina; arrowheads indicate transduced cells in the RPE. Scale bar 500 μm. Source data are available online for this figure.

Fig 5: Preservation of gene expression in the cochlea by 2 months of age demonstrated by RNA‐seq and qRT‐PCR A Heatmap showing levels of expression of 45 pathology‐related DEGs identified between WT (blue) and Ndp‐KO (red) in WT, Ndp‐KO and P2‐L treated cochleas. Treatment restored expression to levels comparable to the WT (no significant difference in the WT versus P2‐L comparison for all 45 genes). Box indicates 16 genes significantly different in the P2‐L versus Ndp‐KO comparison.B–J qRT‐PCR analysis of the expression of the pathology‐related biomarker genes in all treatment groups. Expression of pathology‐related genes (B) Plvap, (C) Clu, (D) Ceacam16, (E) Nr1h4, (F) Abcb1a, (G) Cldn5, (H) Slc7a1, (I) Slc7a5, (J) Sox17. Data information: Genotype and treatment ages (WT, Ndp‐KO, P2‐L, P21‐L, P21‐H, P30‐H) indicated on x axes. B–E data are shown as mean ± SD; n = biological replicates. Sample numbers: n (WT) = 9, n (Ndp‐KO) = 12, n (P2‐L) = 10, n (P21‐L) = 8, n (P21‐H) = 6, n (P30‐H) = 8. Statistical analysis: B–J analysed with one‐way ANOVA with Sidak's post hoc test, all values compared to WT (blue asterisks) and Ndp‐KO (red asterisks). Post hoc test values: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; ns, non‐significant. Source data are available online for this figure.