Fig. 1: Histopathology and hFVIII-SQ DNA transduction efficiency in liver biopsy samples from five participants 2.6–4.1 years after gene transfer with valoctocogene roxaparvovec.
a, Representative liver histopathology sections stained with H&E (participants 1, 3, 4 and 11) or hematoxylin and Van Gieson (participant 15); histologic sections per participant were reviewed by the local pathologist and central pathologist, with consistent results: three biopsy levels (participant 1), two levels (participants 11 and 15), one level (participant 3) and four levels (participant 4). Images were captured in QuPath v0.2.3 using the export snapshot feature; no subsequent processing or image enhancement was performed. Scale bars, 180 µm (participant 1); 150 µm (participants 11, 15, 3 and 4) b, Representative liver biopsy sections from each participant showing hFVIII-SQ DNA (brown foci) by ISH. Images were captured at 1,600 × 1,200 pixels and output at 300 pixels per inch (ppi). Each focus (brown dot) represents at least one vector DNA molecule; it is possible to have multiple copies of vector genome within a single focus. Scale bars, 50 µm. c, Percentage of hepatocyte nuclei stained positive for hFVIII-SQ DNA by ISH. Data are means across 11 (participants 1 and 11) or 27–28 (participants 3, 4 and 15) images per biopsy section, spanning ≥50% of the tissue area (biopsy tissue area was larger for participants 3, 4 and 15). Error bars represent the s.e.m., dots represent quantification of each individual image, and data labels show mean values. d, Circular genomes (full length or H–T ITR fused) detected in liver biopsy samples via drop-phase ddPCR following DNA sample treatment with PS-DNase and KpnI (with PS-DNase, all linear forms of DNA are hydrolyzed, and only circular forms of DNA remain; KpnI treatment separates out vector genome units within the concatemeric forms, enabling quantification of genome units within concatemeric vector genomes). e, Qualitative Southern blot analysis of circular episomes after PS-DNase treatment of DNA from liver biopsy samples. Biopsy samples from control, participant 1 and participant 11, and for participants 3, 4 and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. kbp, kilobase pairs; SC, supercoiled markers; W, week.
