Figure 1
Construction, preparation, and expression of AAV8 vectors. (A) The schematic diagram of AAV constructs. As shown, full-length murine ADAMTS13 protein consists of a signal peptide (S), prodomain (P), a metalloprotease domain (M), a disintegrin domain (D), the first thrombospondin type 1 repeat, a Cys-rich (C), and spacer domain (S) (ie, mdtcs). More distal C-terminus of murine ADAMTS13 contains an additional 7 thrombospondin type 1 repeats (2-8) and CUB domains (for complement C1r/C1s, Uegf, Bmp1). The fragment (∼2.4 kb) encoding amino acid residues 1 to 2055 of murine ADAMTS13, a hAAT promoter, and a bovine growth hormone poly adenylation (BGH-polyA) were cloned into an AAV vector (hAAT-mdtcs). The expression cassette was flanked by 2 inverted terminal repeats (ITR). In addition, a lacZ gene and a hAAT promoter were inserted into the same vector as a control (hAAT-lacZ). (B) The purified recombinant vectors, AAV8-hAAT-mdtcs (lane 1) and AAV8-hAAT-lacZ (lane 2), revealed by Coomassie blue staining. Only 3 viral envelop proteins (VP1, VP2, and VP3) are detected in the final preparations, with the VP3 as the predominant band. Two asterisks indicate 2 minor contaminated proteins or degradation products in lane 1. (C) The amplification of murine ADAMTS13 fragment (∼0.25 kb, closed arrowheads) and β-actin (∼0.5 kb, open arrowheads) mRNA in the brain, lung, heart, liver, spleen, and kidneys in mice treated with AAV8-hAAT-mdtcs (2.6 × 1011 vg/kg) or AAV8-hAAT-lacZ or PBS, as indicated in the figure. The therapeutic transgene product was detected only in the liver of Adamts13−/− mice treated with AAV8-hAAT-mdtcs but not in the controls. (D-E) The positive (arrowheads) and negative staining with anti-murine ADAMTS13 IgG in the hepatocytes 2 weeks after intravenous administration (2.6 × 1011 vg/kg) of AAV8-hAAT-mdtcs and AAV8-hAAT-lacZ, respectively. The staining was performed on the frozen sections after being fixed with ethanol/acetic acid (9/1) for 10 minutes at −20°C. An AlexaFluor568 donkey anti-rabbit IgG (Invitrogen) was used (1:500) for detection (red).

Construction, preparation, and expression of AAV8 vectors. (A) The schematic diagram of AAV constructs. As shown, full-length murine ADAMTS13 protein consists of a signal peptide (S), prodomain (P), a metalloprotease domain (M), a disintegrin domain (D), the first thrombospondin type 1 repeat, a Cys-rich (C), and spacer domain (S) (ie, mdtcs). More distal C-terminus of murine ADAMTS13 contains an additional 7 thrombospondin type 1 repeats (2-8) and CUB domains (for complement C1r/C1s, Uegf, Bmp1). The fragment (∼2.4 kb) encoding amino acid residues 1 to 2055 of murine ADAMTS13, a hAAT promoter, and a bovine growth hormone poly adenylation (BGH-polyA) were cloned into an AAV vector (hAAT-mdtcs). The expression cassette was flanked by 2 inverted terminal repeats (ITR). In addition, a lacZ gene and a hAAT promoter were inserted into the same vector as a control (hAAT-lacZ). (B) The purified recombinant vectors, AAV8-hAAT-mdtcs (lane 1) and AAV8-hAAT-lacZ (lane 2), revealed by Coomassie blue staining. Only 3 viral envelop proteins (VP1, VP2, and VP3) are detected in the final preparations, with the VP3 as the predominant band. Two asterisks indicate 2 minor contaminated proteins or degradation products in lane 1. (C) The amplification of murine ADAMTS13 fragment (∼0.25 kb, closed arrowheads) and β-actin (∼0.5 kb, open arrowheads) mRNA in the brain, lung, heart, liver, spleen, and kidneys in mice treated with AAV8-hAAT-mdtcs (2.6 × 1011 vg/kg) or AAV8-hAAT-lacZ or PBS, as indicated in the figure. The therapeutic transgene product was detected only in the liver of Adamts13−/− mice treated with AAV8-hAAT-mdtcs but not in the controls. (D-E) The positive (arrowheads) and negative staining with anti-murine ADAMTS13 IgG in the hepatocytes 2 weeks after intravenous administration (2.6 × 1011 vg/kg) of AAV8-hAAT-mdtcs and AAV8-hAAT-lacZ, respectively. The staining was performed on the frozen sections after being fixed with ethanol/acetic acid (9/1) for 10 minutes at −20°C. An AlexaFluor568 donkey anti-rabbit IgG (Invitrogen) was used (1:500) for detection (red).

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