Journal: Frontiers in Immunology

Article Title: Intravitreal AAV vector delivery induces integrin-dependent ocular inflammation, complement activation, and antiviral and DNA damage responses

doi: 10.3389/fimmu.2026.1799327

Figure Lengend Snippet: Preliminary assessment of intravitreal gene transfer in the pig shows early inflammation post-AAV injection. (A) Animals received intravitreal (IVT) injections of AAV2.7m8/CAG-hFIX (n = 3) at 5x1011 vg/eye or vehicle (n = 2) on day 0. Prophylactic methylprednisolone was administered intramuscularly (I.M.) from day -2 and weekly thereafter. Animals were sacrificed on day 28. (B) Total uveitis scores in AAV2.7m8/CAG-hFIX-injected eyes (n = 6) overtime, compared to vehicle control eyes (n = 4) at terminal timepoint. Bars represent mean uveitis score. Symbols show individual scores in each eye. Different symbols represent pairs of eyes belonging to the same animal. Open circles identify eyes from the animal that received corticosteroids on Day 8. Statistical significance was determined by two-way ANOVA with Bonferroni correction. *P = 0.0367. Analysis of (C) complement C3a, (D) cytokines and chemokines MCP-1, IP-10, IL-6 and MIP-1α and (E) anti-AAV2.7m8 IgG in aqueous (AH) and vitreous (VH) humors at terminal timepoint. For C-E, data are represented as mean between 2 vehicle- and 4 AAV-injected eyes. (F) Flow cytometry analysis of the frequencies of CD45+ cells, T cells, antigen presenting cells (APC), monocytes and dendritic cells (DCs) in total ocular homogenates. Data are shown as mean ± SD between 4 vehicle- and 4 AAV-injected eyes. Statistical significance was determined by Mann-Whitney t-test. In all cases, *p = 0.0286.

Article Snippet: IVT dosing of AAV2.7m8 has also been linked to severe, dose-limiting toxicities and vision loss at the high dose of 6×10 11 vector genomes (vg)/eye, leading to discontinuation of Adverum’s INFINITY clinical trial for retinal macular edema ( ).

Techniques: Injection, Control, Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Intravitreal AAV vector delivery induces integrin-dependent ocular inflammation, complement activation, and antiviral and DNA damage responses

doi: 10.3389/fimmu.2026.1799327

Figure Lengend Snippet: Intravitreally dosed AAV2.7m8 distributed across the pig retina and induced the highest transgene expression in GCL. (A) Animals received bilateral intravitreal (IVT) injections of AAV2.7m8/CAG-GFP (n = 7) at 5x1011 vg/eye or vehicle (n = 5) on day 0. Prophylactic methylprednisolone was administered intramuscularly (I.M.) from day -2 and weekly thereafter. Animals were sacrificed on days 15 (week 2), 24 (week 4 cohort 1) or 28 (week 4 cohort 2) as indicated. (B) Representative fundus images of GFP fluorescence (top) and confocal scanning laser ophthalmoscopy with blue autofluorescence (BAF) (bottom) in vehicle- and AAV-injected eyes at different timepoints post-injection. (C) UMAP of GFP mRNA distribution. (D) Stacked barplot showing the cell type frequencies among GFP+ nuclei. (E) Quantification of the percentage of GFP mRNA+ cells and mean GFP reads per cell population in AAV-injected retinas on week 4 presented as mean ± SD per cell population. (F) Representative IF images of GFP protein expression (yellow) and AAV vector genomes by RNAscope using probes against the CAG promoter (white), in retina sections from two different vehicle- and AAV-injected animals, 4 weeks post-injection. Sale bar 100μM.

Article Snippet: IVT dosing of AAV2.7m8 has also been linked to severe, dose-limiting toxicities and vision loss at the high dose of 6×10 11 vector genomes (vg)/eye, leading to discontinuation of Adverum’s INFINITY clinical trial for retinal macular edema ( ).

Techniques: Expressing, Fluorescence, Injection, Plasmid Preparation, RNAscope

Journal: Frontiers in Immunology

Article Title: Intravitreal AAV vector delivery induces integrin-dependent ocular inflammation, complement activation, and antiviral and DNA damage responses

doi: 10.3389/fimmu.2026.1799327

Figure Lengend Snippet: Intravitreally dosed AAV2.7m8 activates DNA damage and robust antiviral responses in pig retinas. (A) Heatmaps showing enriched GSEA terms in retinas from AAV-injected animals collected 2- and 4-weeks post-injection, against the Hallmark gene set (Molecular Signatures Database). Each column represents the average between 2 eyes. GSEA was performed on logFC fold changes in gene expression compared to vehicle controls (NES, normalized enrichment score; *, adjusted p <0.05). (B) Heatmaps showing top upregulated and downregulated genes in retinas from AAV-injected animals at the indicated timepoints and their contribution to IFN signaling according to the Hallmark gene set. ISG, interferon stimulated gene. (C) Representative IF images of p-ɣH2AX+ foci and GFP expression in retina sections from different vehicle and AAV-injected animals, 4 weeks post-injection. Scale bar 200μM. (D) Quantification of nuclear p-ɣH2AX mean fluorescence intensity (MFI) per cell. Dots represent MFI values per individual DAPI+ nucleus, pooled from 3 vehicle- and 5 AAV-injected animals, 2 ROIs per animal. Statistical significance was determined by Welch’s t-test. (E) Representative scatter plot of GFP and p-ɣH2AX colocalization analysis from one representative ROI, generated with MACS IQ View Software. **** symbol corresponds to the P < 0.0001.

Article Snippet: IVT dosing of AAV2.7m8 has also been linked to severe, dose-limiting toxicities and vision loss at the high dose of 6×10 11 vector genomes (vg)/eye, leading to discontinuation of Adverum’s INFINITY clinical trial for retinal macular edema ( ).

Techniques: Injection, Gene Expression, Expressing, Fluorescence, Generated, Software

Journal: Frontiers in Immunology

Article Title: Intravitreal AAV vector delivery induces integrin-dependent ocular inflammation, complement activation, and antiviral and DNA damage responses

doi: 10.3389/fimmu.2026.1799327

Figure Lengend Snippet: Kinetic assessment shows early inflammation accompanied by complement activation, chemokine accumulation and anti-AAV IgG development in ocular humors. (A) Total uveitis scores in AAV/GFP-injected animals over time. Data correspond to the mean between both eyes from the same animal from 7 AAV-injected animals. Statistical significance against vehicle week 4 was determined by one-way ANOVA with Tukey correction. **** P < 0.0001 vs. baseline (week 0). (B) Representative OCT showing horizontal scans from vehicle- and AAV-injected retinas at terminal timepoint week 4. (C) Quantification of total retinal thickness (TRT) derived from horizontal retinal scans over time. Data correspond to individual eyes from 4 vehicle controls (week 4) and 7 AAV-injected animals over time, and are represented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey correction. *P = 0.0383. Analysis of (D) complement factor C3a, (E) MCP-1, (F) IP-10, (G) IL-6 and (H) MIP-1α and (I) anti-AAV2.7m8 IgG in AH and VH at the indicated timepoints. For C-I, eyes with a uveitis score of 0 belong to cohort 2 and are highlighted in light purple. For C-H, data are represented as mean ± SD between individual eyes from 5 vehicle- and 7 AAV/GFP-injected animals. For detailed N refer to supplementary table 5. For AH samples, statistical significance between all timepoints vs. vehicle was determined by one-way ANOVA with Tukey correction. For VH samples, statistical significance between week 4 group and vehicle was determined by Mann-Whitney t-test. *P < 0.05, **p < 0.01, p < 0.001, *** p < 0.0001 vs. vehicle.

Article Snippet: IVT dosing of AAV2.7m8 has also been linked to severe, dose-limiting toxicities and vision loss at the high dose of 6×10 11 vector genomes (vg)/eye, leading to discontinuation of Adverum’s INFINITY clinical trial for retinal macular edema ( ).

Techniques: Activation Assay, Injection, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Intravitreal AAV vector delivery induces integrin-dependent ocular inflammation, complement activation, and antiviral and DNA damage responses

doi: 10.3389/fimmu.2026.1799327

Figure Lengend Snippet: LFA-1/VLA-4 neutralization prevents IOI after intravitreal AAV vector dosing in mice. (A) Study design. Female C57Bl/6J mice received intravitreal (IVT) bilateral injections of AAV2.7m8/CAG-GFP (1x10 9 vg/eye) or vehicle control on day 0 and sacrificed at different timepoints as indicated in the scheme. One cohort of AAV-injected mice received concomitant intraperitoneal (I.P.) injections of anti-LFA-1/VLA-4 antibodies on day 0, and every 3 days until termination (week 4). (B) FACS analysis of ocular infiltrates in ocular homogenates. Both eyes from from the same animal were pooled for analysis. Data are shown as mean ± SD between n = 5 week 1, n = 5 week 2, n = 9 week 4 and n = 10 αLFA-1/VLA-4-treated week 4 animals, and represented as fold change between AAV-injected animals relative to vehicle controls. The dashed line represents values in vehicle controls, set at 1. Statistical significance for each timepoint was determined respect to its own vehicle control by one-way ANOVA with Fisher’s LSD test (n = 5 for vehicles week 1 and 2, n = 10 vehicle week 4). *P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. vehicle; ### p < 0.001, #### p < 0.0001 vs. AAV week 4. (C) Quantification of cytokines and chemokines in vitreous humors. Each dot corresponds to a pool between 4 eyes from 2 animals from the same treatment group. Data are shown as ± SD between pools (n = 5 for vehicle and αLFA-1/VLA-4-treated animals, n = 4 for AAV-treated only). Statistical significance was determined by two-way ANOVA with Tukey’s correction. ****P < 0.0001. (D) Representative fundus images of GFP expression over time. (E) Quantification of GFP mean fluorescence intensity (MFI) in mice injected with AAV only (n = 8) or with AAV + anti-LFA-1/anti-VLA-4 (n = 9). Data are shown as mean ± SD between individual eyes. Statistical significance was determined by two-way ANOVA with Bonferroni correction. *** P = 0.0005.

Article Snippet: IVT dosing of AAV2.7m8 has also been linked to severe, dose-limiting toxicities and vision loss at the high dose of 6×10 11 vector genomes (vg)/eye, leading to discontinuation of Adverum’s INFINITY clinical trial for retinal macular edema ( ).

Techniques: Neutralization, Plasmid Preparation, Control, Injection, Expressing, Fluorescence