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Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice
Betsy T. Kren, … , Mark T. Reding, Clifford J. Steer
Betsy T. Kren, … , Mark T. Reding, Clifford J. Steer
Published June 8, 2009
Citation Information: J Clin Invest. 2009;119(7):2086-2099. https://doi.org/10.1172/JCI34332.
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Technical Advance Genetics

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

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Abstract

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

Authors

Betsy T. Kren, Gretchen M. Unger, Lucas Sjeklocha, Alycia A. Trossen, Vicci Korman, Brenda M. Diethelm-Okita, Mark T. Reding, Clifford J. Steer

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Figure 6

Targeting of cis CAGGS-BΔcFVIII SB-Tns to LSECs and correction of the bleeding diathesis in knockout hemophilia A mice.

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Targeting of cis CAGGS-BΔcFVIII SB-Tns to LSECs and correction of the bl...
(A) Mice treated with 25 μg of cisSB-Tn in HA s50 nanocapsules were bled 5, 12, 16, 19, and 50 weeks after injection and aPTTs determined in duplicate as described in Methods. Treated mice (n = 6) had aPTTs of 25.5 ± 3.1 seconds at 5 weeks and 28.8 ± 3.7 seconds at 50 weeks; these were not significantly different from those of age-matched wild-type (n = 3) aPTTs of 23.5 ± 1.3 seconds (5 weeks; light gray) and 27.9 ± 1.6 seconds (50 weeks, dark gray). In contrast, untreated mice (n = 3) had aPTTs ranging from 46.7 ± 3.5 to 65.7 ± 9.6 seconds. The data show the mean ± 1 SD. *P < 0.001 compared with untreated hemophilia A mice. Far-right dark gray bar, plasma from a separate group of untreated hemophilia A mice collected and assayed with the other 50-week samples. (B) Coamatic determination of FVIII activity in mice. FVIII activity in plasma samples was also determined using the Coamatic assay, which measures the conversion of FX to FXa mediated by FIXa and its cofactor FVIII. The graph shows the mean ± 1 SD of values obtained using the procedure outlined in Methods and wild-type mouse plasma as positive control. By 5 weeks after injection, the treated animals showed greater than 95% wild-type mouse plasma activity; and this increased to greater than 100% by 50 weeks. In contrast, the plasma from untreated hemophilia A mice exhibited no detectable activity. ANOVA analysis indicated no significant difference between the plasma from treated hemophilia A mice and that from wild-type animals.
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