Figure 1.
Targeting codon-optimized FIX-Padua expression to platelets in FIXnullmice (the noninhibitor model). Two strains of FIX-deficient (FIXnull) mice were used to test 2bCoF9R338L expression. FIX expression was introduced by 2bCoF9R338L LV transduction of Sca-1+ cells isolated from donors and transplanted into littermate recipients under a sublethal 6.6 Gy TBI. After HSCT and at least 4 weeks of BM reconstitution, blood samples were collected from recipients at various time points for assays. (A) Diagrams of experimental design. (Ai) Mice in Model 1 had a pure C57BL/6 background. (Aii) Mice in Model 2 had a C57BL/6-B6-129S mixed background. (B) Platelet-FIX expression in 2bCoF9R338L-transduced recipients in Model 1. Platelets were isolated and lysed in 0.5% CHAPS (the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MP Biomedicals). Functional platelet-FIX activity (Platelet-FIX:C) in platelet lysates was determined by a chromogenic assay. Platelet-FIX antigen (Platelet-FIX:Ag) levels in platelet lysates were quantified by enzyme-linked immunosorbent assay (ELISA). (C) The ratio of platelet-FIX:C to platelet-FIX:Ag in transduced recipients in Model 1. (D) Platelet-FIX expression in 2bCoF9R338L-transduced recipients in Model 2. (E) The ratio of platelet-FIX:C to platelet-FIX:Ag in 2bCoF9R338L-transduced recipients in Model 2. (F) Average platelet-FIX:C and platelet-FIX:Ag levels in 2bCoF9R338L-transduced recipients. Individual mice were analyzed at various time points during the study, and the average platelet-FIX level was calculated. Normal plasma from the International Society of Thrombosis and Hemostasis (ISTH) was used as a reference. (G) The average ratio of platelet-FIX:C to platelet-FIX:Ag in 2bCoF9R338L-transduced recipients. (H) Average copy number of 2bCoF9R338L proviral DNA in 2bCoF9R338L-transduced recipients. DNA was purified from leukocytes isolated from peripheral blood. The proviral DNA copy number was determined by quantitative real-time PCR (qRT-PCR). Individual mice were analyzed more than once during the study, and the average copy number was calculated. (I) Average platelet-FIX:C expression in sequential transplantation recipients from a representative experiment. BM mononuclear cells were isolated from the primary 2bCoF9R338L-tranduced recipient (1°), transplanted into secondary recipients (2°), and subsequently tertiary recipients (3°). Individual mice were analyzed more than once during the study, and the average level was calculated. Data are presented as mean ± standard deviation (SD). ****P < .0001. n.s., not statistically significant; Plt, platelet.

Targeting codon-optimized FIX-Padua expression to platelets in FIXnullmice (the noninhibitor model). Two strains of FIX-deficient (FIXnull) mice were used to test 2bCoF9R338L expression. FIX expression was introduced by 2bCoF9R338L LV transduction of Sca-1+ cells isolated from donors and transplanted into littermate recipients under a sublethal 6.6 Gy TBI. After HSCT and at least 4 weeks of BM reconstitution, blood samples were collected from recipients at various time points for assays. (A) Diagrams of experimental design. (Ai) Mice in Model 1 had a pure C57BL/6 background. (Aii) Mice in Model 2 had a C57BL/6-B6-129S mixed background. (B) Platelet-FIX expression in 2bCoF9R338L-transduced recipients in Model 1. Platelets were isolated and lysed in 0.5% CHAPS (the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MP Biomedicals). Functional platelet-FIX activity (Platelet-FIX:C) in platelet lysates was determined by a chromogenic assay. Platelet-FIX antigen (Platelet-FIX:Ag) levels in platelet lysates were quantified by enzyme-linked immunosorbent assay (ELISA). (C) The ratio of platelet-FIX:C to platelet-FIX:Ag in transduced recipients in Model 1. (D) Platelet-FIX expression in 2bCoF9R338L-transduced recipients in Model 2. (E) The ratio of platelet-FIX:C to platelet-FIX:Ag in 2bCoF9R338L-transduced recipients in Model 2. (F) Average platelet-FIX:C and platelet-FIX:Ag levels in 2bCoF9R338L-transduced recipients. Individual mice were analyzed at various time points during the study, and the average platelet-FIX level was calculated. Normal plasma from the International Society of Thrombosis and Hemostasis (ISTH) was used as a reference. (G) The average ratio of platelet-FIX:C to platelet-FIX:Ag in 2bCoF9R338L-transduced recipients. (H) Average copy number of 2bCoF9R338L proviral DNA in 2bCoF9R338L-transduced recipients. DNA was purified from leukocytes isolated from peripheral blood. The proviral DNA copy number was determined by quantitative real-time PCR (qRT-PCR). Individual mice were analyzed more than once during the study, and the average copy number was calculated. (I) Average platelet-FIX:C expression in sequential transplantation recipients from a representative experiment. BM mononuclear cells were isolated from the primary 2bCoF9R338L-tranduced recipient (1°), transplanted into secondary recipients (2°), and subsequently tertiary recipients (3°). Individual mice were analyzed more than once during the study, and the average level was calculated. Data are presented as mean ± standard deviation (SD). ****P < .0001. n.s., not statistically significant; Plt, platelet.

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