Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice

Figures

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  Figure 1.. Generation of engineered activated hPC (human protein C). A, Schematic presentation of the engineered hPC. An indicated a furin-cleaving peptide sequence was inserted into the thrombin cleavage site of hPC. B, hPC:C levels (mean±SEM [n=3–4]) in the supernatant obtained from the HEK293 cells stably expressing an indicated engineered hPC. C, hPC:C levels (mean±SEM [n=3–4]) measured without snake venom. The P values, 1-way ANOVA with Dunnett multiple comparison test compared with wild-type (WT) hPC. D and E, Normal human plasma was incubated with an indicated concentration of WT hPC, the previously reported engineered hPC (QGNSEDY), or the engineered activated hPC (hPC-2RKR) for 15 minutes. Activated partial thromboplastin time (APTT; D) and prothrombin time (PT; E; mean±SEM [n=3]) were measured using an automated coagulation analyzer. F, Protein S–deficient plasma was incubated with an indicated concentration of hPC or hPC-2RKR for 15 minutes. APTT (mean±SEM [n=3]) was measured using an automated coagulation analyzer. The P values, 2-way ANOVA with Bonferroni multiple comparison test compared with WT hPC. hPC:C: hPC activity expressed as the percentage of pooled human plasma.…

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  Figure 2.. Prevention of pathogenic thrombus formation by the engineered activated PC (protein C) in mice. Adeno-associated virus (AAV)-8 vectors expressing wild-type mPC (mouse PC) or the engineered activated mPC (mPC-2RKR) under the control of HCRhAAT promoter were intravenously administered into 7-week-old C57BL/6J male mice (low, medium, and high doses: 4.0×1010, 1.2×1011, and 4.0×1011 vg/mouse, respectively). A, Plasma mPC antigen (mPC:Ag) levels (mean±SEM [n=4]) at 4 weeks after vector injection were measured using ELISA. Activated partial thromboplastin time (APTT; B), FV:C (C), and FVIII:C (D) were measured using an automated coagulation analyzer. Values represent mean±SEM (n=4, except FVIII:C in mPC Medium [n=2] and mPC-2RKR High [n=3]). E, AAV genome (mean±SEM [n=4]) in the liver tissue at 8 to 12 weeks post-injection was measured using quantitative polymerase chain reaction. The P values, 2-way ANOVA with Bonferroni multiple comparison test comparing between mPC and mPC-2RKR at the same vector dose. F, Thrombus formation in testicular veins induced by laser-induced reactive oxygen species (ROS) production was observed using intravital confocal microscopy. The green signal…

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  Figure 3.. Insertion of cDNA sequence by genome editing in neonatal mice. A, Schematic presentation of genome editing experiment. Self-cleaving P2A (2A peptide derived from porcine teschorivus-1) peptide sequence followed by EGFP (enhanced green fluorescent protein) cDNA was inserted into double-strand break (DSB) induced by SaCas9 (Staphylococcus aureus Cas9) with homology-directed repair (HDR) in vivo. B through F, Neonatal C57BL/6J mice received an adeno-associated virus (AAV) donor vector harboring EGFP (2.0×1011 vg/mouse) without (EGFP donor) or with an AAV vector expressing SaCas9 and single guide RNA for intron 14 of Alb locus (6.0×1010 vg/mouse; SaCas9 and EGFP donor). B, mRNA expression of EGFP fused with Alb in the liver was detected by reverse transcription polymerase chain reaction. Representative data from 3 mice are shown. Liver tissues obtained from C57BL/6J were used as negative control. C, Immunoblotting with anti-GFP (green fluorescent protein) antibody in liver tissues obtained from 3 mice. Representative data from 3 mice are shown. Liver tissues obtained from C57BL/6J were used as negative control. D, EGFP expression in liver specimens assessed by EGFP immunostaining was…

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  Figure 4.. Phenotypic correction of PC (protein C)-deficient homozygotic mice treated with genome editing to insert the engineered activated PC. A, Proc+/− female mice were treated with an FVIII (factor VIII) antibody (No. GMA8015; 20 µg/mouse) and then mated with Proc+/− male mice. The antibody administration was repeated every 7 days until delivery. All neonatal mice were treated via genome editing with adeno-associated virus (AAV)-8 vector harboring mPC-2RKR (2.0×1011 vg/mouse [high dose] or 2.0×1010 vg/mouse [low dose]) and AAV8 vector expressing SaCas9 (Staphylococcus aureus Cas9) and single guide RNA for intron 14 of Alb locus (6.0×1010 vg/mouse [high dose] or 6.0×109 vg/mouse [low dose]), together with 1 µg of the FVIII antibody. B, Kaplan-Meier survival curves for Proc−/− mice (n=13), Proc−/− mice treated with genome editing (high dose, n=9; low dose, n=10), and Proc−/− F8−/− mice (n=10). The P values, log-rank test, compared with Proc−/− mice. C through F, Plasma mouse PC antigen (mPC:Ag; C), FV:C (D), and FVIII:C (E), and coagulation time (activated partial thromboplastin time [APTT]; F). Values represent mean±SEM (n=5–10, except Proc−/− Treated [low dose, n=2]). The P values,…

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  Figure 5.. Thrombus formation and bleeding phenotype of PC (protein C)-deficient homozygotic mice treated with genome editing to insert the engineered activated PC. Proc+/− female mice were treated with an FVIII (factor VIII) antibody (No. GMA8015; 20 µg/mouse) and then mated with Proc+/− male mice. The antibody administration was repeated every 7 days until delivery. All neonatal mice were treated via genome editing with adeno-associated virus (AAV)-8 vector harboring mPC-2RKR (2.0×1011 vg/mouse [high dose] or 2.0×1010 vg/mouse [low dose]) and AAV8 vector expressing SaCas9 (Staphylococcus aureus Cas9) and single guide RNA for intron 14 of Alb locus (6.0×1010 vg/mouse [high dose] or 6.0×109 vg/mouse [low dose]), together with 1 µg of the FVIII antibody. A, Representative photographs of hematoxylin-eosin staining of liver and lung tissues in an indicated mouse. Scale bars=100 µm. B, Representative photograph of filter paper to assess bleeding after tail clipping in an indicated mouse. C and D, Bleeding time and volume after the tail clip assay. Values represent mean±SEM (n=3–7, except Proc−/− mice treated with genome editing [Proc−/− Treated, n=2]). The P values, 1-way ANOVA with Dunnett…