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. 2023 Aug 29;15(10):e17393. doi: 10.15252/emmm.202317393

Figure 1. Gene therapy construct evaluation and study design.

Figure 1

  • A
    Schematic of the AAV9.NDP expression construct. Ubiquitous CAG promoter (cytomegalovirus (CMV) immediate enhancer fused to the chicken beta‐actin promoter) drives expression of the transgene cassette: enhanced green fluorescent protein, EGFP; P2A self‐cleaving linker, human NDP cDNA with a C‐terminal FLAG tag.
  • B‐B″
    Expression of AAV9.NDP construct in transfected HEK293 cells. EGFP (green), anti‐FLAG (red), colocalisation (yellow). Scale bar: 50 μm.
  • C
    Western blot of transfected (+) and untransfected (−) HEK293 cell lysate. Detection of NDP, GFP and GAPDH proteins (GAPDH provides a loading control; Appendix Fig S1 shows uncropped Western blot images).
  • D
    Schematic of the NDP induced β‐catenin signalling. NDP dimer binds to FZ4 complex with essential co‐receptor LRP5 or LRP6 and signal amplifying co‐receptor TSPAN12 and induces β‐catenin binding to TCF/LEF sites in the promoters of the downstream target genes activating transcription.
  • E
    TopFlash assay shows activity of transgenic NDP in HEK293 cells. AAV9.NDP construct was co‐transfected with FZ4, LRP6, TSPAN12 expression plasmids and TopFlash plasmid encoding firefly luciferase under a promoter containing TCF/LEF binding sites; mCherry plasmid was used as a transfection control. LiCl addition was used as a positive control as it mimics the destruction complex inhibition allowing β‐catenin binding. Luciferase activity was measured as relative luminescence levels; mean ± SD, N = 3 technical replicates.
  • F
    Experimental design of the treatment administration, endpoints and approximately corresponding stages in human development. Intravenous treatment of the AAV9.NDP virus was administered at P2, P21 or P30 with doses indicated in the schematic. Eye and ear histology in all groups was analysed at 2 months of age; a separate set of mice was analysed for visual function (ERG) at 1.5 months and audiology (DPOAE, ABR) at 3 months of age.

Source data are available online for this figure.