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

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: Spatial transcriptomics reveals rapid, localized transcriptional remodeling of the tumor microenvironment following vectorized hIFNβ treatment (A) Coronal brain sections from representative human GBM6-FLuc CDX mice collected pre-treatment (0 h, n = 1) or 48 h ( n = 1) after intratumoral AAV9-hIFNβ infusion (2E11 vg/brain), stained with H&E (left) and subjected to Visium Spatial Gene Expression profiling (right). Annotated clusters were assigned based on anatomical localization and marker gene expression. Dashed lines denote tumor borders. Scale bars, 1 mm. (B) Top 10 marker genes for each spatially resolved cluster identified across 0 h and 48 h datasets. Values are shown as log-normalized expression centered at 0 (Seurat “scale.data”). (C) Spatial expression of canonical human GBM tumor markers ( CD44 , VIM , TOP2A , and NOTCH1 ) delineating tumor and peri-tumor regions before (0 h) (top) and after (bottom) (48 h) AAV9-hIFNβ treatment. (D) Expression maps of the human IFNβ payload and hallmark IFN-response genes ( CXCL10 , IFIT1 , and IFIT2 ), demonstrating tumor-restricted transgene expression and induction of an IFN-specific transcriptional program within 48 h. (E) Spatial expression of host mouse immune-response genes ( Gfap , Ifitm3 , and Irf7 ) showing localized activation of astroglial and innate immune pathways proximal to the tumor. (F) Integrated datasets (0 and 48 h) visualized using canonical correlation analysis (CCA), showing distinct clustering of tumor and peri-tumor regions (left) and enrichment of IFN-response gene module expression (right). (G) Volcano plot depicting differential gene expression between 0- and 48-h tumor clusters. Red, IFN-response genes; gray, other significantly upregulated genes ( p -Adj <0.01); blue, non-significant. (H) Top enriched Gene Ontology (GO) terms among upregulated genes in 48-h tumor cells, highlighting interferon and inflammatory response pathways (∗∗ p -Adj <0.01; ∗ p -Adj <0.05).

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: Spatial Transcriptomics, Staining, Gene Expression, Marker, Expressing, Activation Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: RAW cells were incubated for 1 h without (−) or with (+) 1.0 μM JQ1 or 1.0 μM BI2536 and then stimulated for 8 h without (−) or with (+) poly(I:C) (10 μg/ml) ( A and C ) or for 4 h without (−) or with (+) LPS (100 ng/ml) ( B and D ). At each time point, the total RNA was extracted from the cells and Ifnb mRNA was quantified by qPCR ( A and B ) and the concentration of IFNβ in the cell culture medium was determined by ELISA ( C and D ). Results are means+S.E.M for triplicate determinations. ( A ) and ( B ) show the fold increase in mRNA levels relative to the values measured in cells that had not been stimulated with LPS or poly(I:C).

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: ( A and B ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM) or I-BET151 (1.0 μM) and then stimulated for 8 h with CL097 (1.0 μg/ml) ( A ) or for 12 h with ODN1826 (1.0 μM) ( B ). The concentration of IFNβ in the culture medium was measured by ELISA. Results are means+S.D. from two independent experiments each performed in duplicate. ( C ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM), I-BET151 (1.0 μM) or BI-605906 (5.0 μM) and then stimulated for 1 h with CL097. Staining with anti-IRF5, or DAPI to reveal nuclei, followed by deconvolution microscopy was performed as described in the Materials and methods section. ( D ) Gen2.2 cells were incubated for 1 h with BI-2536 (1 μM) or JQ1 (1 μM), then stimulated for 1 h with the TLR7 agonist CL097 (1 μg/ml), cross-linked and lysed. Chromatin was sheared by sonication and ChIP was performed using anti-IRF5. The enrichment of the Ifnb promoter was measured by qPCR, normalizing to input. Results are means+S.D. similar results were obtained in three independent experiments each performed in duplicate. IP, immunoprecipitation.

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Sonication, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease

doi: 10.1371/journal.ppat.1000667

Figure Lengend Snippet: Freshly isolated PBMC were stimulated with CpG or the non-CpG ODN control. After 22 hours IFN-α was quantified in culture supernatants by ELISA. ( A ) Levels of IFN-α upon CpG or the non-CpG ODN control in healthy, HIV-1 and HIV-2 cohorts. The infected cohorts were further stratified according to CD4 T cell counts ( B ) or the presence or absence of detectable viremia ( C ) and the levels of IFN-α upon CpG stimulation are shown. Results expressed as “Net IFN-α” refer to the amount of IFN-α produced upon CpG stimulation subtracted by the IFN-α measured with medium alone. The “Net IFN-α” was divided by the total number of pDC in the culture and results are shown for the healthy, HIV-1 and HIV-2 cohorts ( D ), as well as for the infected cohorts stratified according to CD4 T cell counts ( E ) or the presence or absence of detectable viremia ( F ). Each dot represents one individual. Bars represent mean±SEM. Numbers under the bars represent the total individuals analyzed. The subgroups of patients analyzed are representative of their respective patient population described in with respect to CD4 counts and viral load. ** p<0.01 and *** p<0.001 as compared to controls. # p<0.05 and ## p<0.01 between the two groups of the same infected cohort. & p<0.05 between HIV-1 and HIV-2 patients with more than 350 CD4 T cells/µl.

Article Snippet: IFN-α was quantified in serum samples and in culture supernatants using the VeriKineTM Human IFN-Alpha Serum Sample ELISA and Human IFN-Alpha ELISA Kit, respectively (PBL InterferonSource, Piscataway, NJ), according to manufacturer's instructions.

Techniques: Isolation, Control, Enzyme-linked Immunosorbent Assay, Infection, Produced

Journal: Retrovirology

Article Title: Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys

doi: 10.1186/s12977-015-0219-8

Figure Lengend Snippet: BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon Alpha A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma IFN-alpha levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r , Spearman’s correlation coefficient; p , p value

Article Snippet: PBMC from three healthy animals were stimulated with 10, 50 and 100 ng Human Interferon Alpha A (Alpha 2a) (PBL Biomedical Laboratories) for 16 h. Fold induction of BST2 mRNA expression was calculated using GAPDH as internal control.

Techniques: Standard Deviation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Infection, Transformation Assay