Figure 5.

AAV-trap-mediated expression of hFIX and therapeutics effect in APOC3-humanized hemophilia model

(A) Brief information for AAV transduction in dual mutant mice. (B) The indels in AAV-transduced livers were confirmed by T7E1 analysis (left) and targeted deep sequencing (right). Red arrowhead indicates DNA fragments cleaved by T7E1. (C) Confirmation of the existence of KI by PCR and analysis of KI patterns by sequencing. For KI pattern analysis, 15 clones from forward and reverse sites were subjected to sequencing and compared with the expected KI map. Yellow box, unexpected indel. (D) AAV-trap-mediated in vivo expression of hFIX (n = 4 for each group). (E) Cross-reactivity of hFIX and mFIX was evaluated by transduction of AAV-CMV-hF9 into F9^Mut mice. Plasma was collected at 6 weeks after transduction of 4 × 10¹³ vg/kg AAV into mice. The hFIX concentration was approximately 2,930 ng/mL in the AAV-CMV-hF9 group (detailed data are not shown). Next, aPTT was conducted to evaluate coagulation activity and cross-reactivity between hFIX and mFIX (n = 4 for each group). (F) hF9 gene KI-mediated therapeutic effects were evaluated by aPTT (n = 3–4 per group). Each dot indicates data from an individual mouse. Statistical analysis was performed using the Student t test. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.