Journal: Molecular Therapy Oncology
Article Title: AAV immuno-gene therapy platform delivering vectorized cytokines defines a new modality for high-grade glioma treatment
doi: 10.1016/j.omton.2026.201183
Figure Lengend Snippet: Vectorized IFNβ drives durable signaling and complete tumor regression in human glioblastoma models in vivo (A) Sustained hIFNβ secretion in human GBM6 cells treated with AAV9-hIFNβ (red, MOI = 4E5 vg/cell) or recombinant hIFNβ cytokine (r-hIFNβ, purple, 47 IU/mL, equivalent to 114 pg/mL), measured by ELISA at indicated time points. 50% media washouts every 5 h for the first 20 h in the r-hIFNβ condition mimic in vivo cytokine clearance (half-life = 4–5 h). Full media exchanges were performed at 24, 48, 72, and 96 h post-treatment. (B) Number of differentially expressed genes (DEGs, p -Adj<0.01) in GBM6 cells 24–96 h post-treatment with AAV9-hIFNβ or r-hIFNβ vs. media controls. (C) Enrichment scores for type I IFN and TNFα response pathways across treatments and time points. (D) Heatmap of the top 10 IFN and TNFα response genes (Log2FC vs. media controls) in GBM6 cells treated as in (A). (E) Schematic of orthotopic PDX (SF11411) and cell line-derived xenograft ([CDX], GBM6-FLuc) studies in athymic nu/nu mice treated intratumorally with saline, AAV9-GFP, or AAV9-hIFNβ via CED. (F) Kaplan-Meier survival curves for PDX mice treated as in (E). Saline = black, AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ (2E11 vg/brain) = red. Vertical dashed line = day of treatment (day 9). p < 0.04 by log-rank (Mantel-Cox) test. n = 30 (10 per treatment arm). (G) Longitudinal BLI of GBM6-FLuc tumor growth in CDX mice treated as in (E). Saline = black, AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ (2E11 vg/brain) = red. Thin lines = individual mice, thick lines = geometric mean. Vertical dashed line = day of treatment (day 9). ∗ p < 0.04 by Kruskal-Wallis test with Dunn’s multiple comparisons correction on day 22. n = 30 (10 per treatment arm). (G′) Representative BLI images from each treatment group 11 days post-treatment. (H) Kaplan-Meier survival curves for CDX mice. p < 0.001 by log-rank (Mantel-Cox) test. (I) Distribution of treatment responses in CDX by BLI flux (photons/second) at day 27. Tumor free = BLI flux <2.5 × 10 5 p/s, tumor reduction = ≥30% decrease from assignment on day 9, no change = between 30% decrease and 20% increase from assignment on day 9, tumor growth = ≥20% increase from assignment on day 9, death = mice that died before day 27. (J) Dose-response analysis of AAV9-hIFNβ efficacy in CDX mice. AAV9-GFP (2E11 vg/brain) = blue, AAV9-hIFNβ hi (2E11 vg/brain) = solid red, and AAV9-hIFNβ lo (1E11 vg/brain) = dashed red. Thin lines = individual mice, thick lines = geometric mean. Vertical dashed line = day of treatment (day 9). ∗∗ p < 0.02 by Kruskal-Wallis test with Dunn’s multiple comparisons correction on day 20. n = 45 (15 per treatment arm). For data interpretation, tumor burden threshold = 2.5 × 10 5 . (J′) Representative BLI images of tumors 11 days post-treatment. (K) Kaplan-Meier survival curves from (J). p < 0.002 (AAV9-hIFNβ hi), p < 0.005 (AAV9-hIFNβ lo) by log-rank (Mantel-Cox) test compared to AAV9-GFP. (I) Distribution of treatment responses in CDX mice at day 27 by BLI flux as in (I).
Article Snippet: 24, 48, 72, and 96 h after treatment, cell supernatants were collected and IFN variant levels were measured using IFN ELISA kits following the manufacturer’s instructions (human IFNα [PBL Cat# 41135-1], human IFNβ [PBL Cat#41410], and human IFNγ [R&D Systems Cat#: DIF50C]).
Techniques: In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay, Saline