Fig 1: Collagen VII (C7) expression in primary RDEB patient cells, RDEB mice, and human RDEB xenografts on immunodeficient mice following B-VEC therapy.a, RDEB keratinocytes and fibroblasts were infected in vitro with B-VEC vector at various ratios of cell to viral particle (MOI). Cells were fixed 48 h after infection and analyzed by indirect immunofluorescence microscopy (IDIF) to validate C7 expression in keratinocytes (kc; yellow) and fibroblasts (fb; green). Scale bar, 100 µm. b,c, Infection efficiency and live cell number in cultures after infection (n = 3 for each condition). d, Keratinocytes and fibroblasts were collected after various MOI (n = 3 for each condition) and analyzed by Western blot. C, control vector. e, Mice were injected intradermally at four separate sites on the back (one vehicle control site and three B-VEC treatment sites, one injection per site). Some mice received a second injection on day 3 at the same four sites. At day 3 after B-VEC treatment (dose of 4.6 x 107 p.f.u. per 50 µl per injection), RDEB mice injected intradermally with B-VEC vector had linear C7 basement membrane zone (BMZ) expression (yellow, dotted line) including in hair follicle basement membranes (arrows). Scale bar, 100 µm. f, At day 7, C7 expression (yellow) was analyzed using IDIF after one or two injections in RDEB mouse skin. Scale bar, 200 µm. g,h, COL7A1 DNA (g) and C7 transcript expression levels (h) were analyzed using qPCR or RT–qPCR, after one or two injections (dose of 4.6 x 107 p.f.u. per 50 µl per injection) in RDEB mouse skin, on day 3 and 7 after single or repeated injections (n = 9 injections, triplicate for each condition, per timepoint). i, Demonstration of C7 expression dose dependency in RDEB mouse skin for low-dose injections (top row) and high-dose injections (bottom row) at days 3, 5, and 7 after injection. The far right panels show C7 expression at high and low B-VEC doses 7 days after both injections. j, Heterozygous RDEB mice were treated with daily topical B-VEC or vehicle (PBS) applications on wounded skin for 5 days. Fourteen days after the first application, linear C7 expression using human-specific antibody is shown in yellow at the epidermal–dermal junction and hair follicle epithelial–dermal junctions, and co-localization with the BMZ marker a6 integrin staining is shown in red. The inserts in the right panels demonstrate higher magnification of the sample region. Four areas on the back were treated per mouse (1 control and 3 B-VEC) and four mice in total were tested. Scale bar, 100 µm. k, Xenografts comprising human RDEB fibroblasts and keratinocytes were treated by topical B-VEC and imaged 5 days later. The grafts were analyzed by light (first row) and immunofluorescence (rows 2–5) microscopy. Light microscopy demonstrated areas of dermal–epidermal blistering (left panel, arrow), which was not seen after B-VEC treatment (right panel). In the treated region, expression of the NC1 (red) and NC2 (green) domains of C7 co-localized with the control BMZ marker laminin-332 (yellow). Row 5 demonstrates the persistence and location of human fibroblasts in the dermis of the graft. Scale bar, 100 µm. Representative of eight grafts treated with B-VEC (mice) and 2 with placebo. l, Human RDEB skin xenografts were treated with topical B-VEC and analyzed after 3 and 12 days using immunoelectron microscopy with C7 NC1-directed antibodies (NP185), followed by gold nanoparticles (first, third rows; white arrows), and NC2-directed primary antibodies (LH24), followed by gold nanoparticles (second, fourth rows; black arrows). Error bars on all panels represent standard error of the mean of all replicates. Representative of eight grafts treated with B-VEC and two grafts treated with placebo. Scale bars, 300 nm. Data plots, including error bars and P values, were generated using GraphPad Prism v8.3.0.
Supplier Page from MilliporeSigma for Anti-COL7A1 antibody produced in rabbit