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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 3

Cell and promoter specificity of nanocapsule targeting in vivo.

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Cell and promoter specificity of nanocapsule targeting in vivo.
Eight-we...
Eight-week-old male and female mice were administered 100 μg of the encapsulated plasmid targeted to liver using ASOR or HA via tail vein injection and sacrificed 1 week after injection. (A–D) β-gal expression in 6-μm liver cryosections was visualized by immunohistochemical staining using rabbit anti–β-gal and a mouse cocktail specific for microvessels (78). The micrographs show the red color of the secondary anti-rabbit Qdot 565 conjugate in hepatocytes expressing β-gal using either the hepatocyte-specific SV40:Alb promoter (B) or the constitutive SV40:Ear (C) delivered to hepatocytes via ASOR. In contrast, when LSECs were targeted by HA nanocapsules, no detectable expression of β-gal using the SV40:Alb promoter occurred (A). (D) Background staining observed in control liver from vehicle-injected mice. The green FITC-labeled microvessels are shown to provide a reference for β-gal expression. The targeting ligand and plasmid are indicated above and below the respective panels. (E–J) Expression of β-gal in animals receiving pcDNA3.1 delivered using ASOR (E and H), HA (F and I), or vehicle control (G and J) determined by IHC against the Xpress epitope tag. The positive Xpress signal (green) gives an obvious hepatocyte (E) and LSEC (F) expression pattern, while no staining is observed in the negative vehicle control (G). The merged panels below show the β-gal expression with the blue nuclei counterstain TO-PRO-3. The targeting ligand is indicated above and the Ab and nuclear stain on the right.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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