Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice
Betsy T. Kren, Gretchen M. Unger, Lucas Sjeklocha, Alycia A. Trossen, Vicci Korman, Brenda M. Diethelm-Okita, Mark T. Reding, Clifford J. Steer
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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Technical Advance Genetics

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

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 2

Delivery of nanoencapsulated DsRed2 SB-Tns in vivo to either hepatocytes or LSECs.

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Delivery of nanoencapsulated DsRed2 SB-Tns in vivo to either hepatocytes...
Eight-week-old mice were administered 100 μg of the encapsulated cis pT2/DsRed2 Tns via tail vein injection and sacrificed 1 week after injection. Expression of DsRed2 targeted to hepatocytes with ASOR or to LSECs with HA was visualized by confocal microscopy. LSECs were identified by anti-CD14 Ab, a marker specific for the discontinuous endothelial cells in the liver, and a Cy5-labeled secondary Ab. The confocal micrographs (A) show Cy5-labeled LSECs (green) with DsRed2 fluorescence (red). The targeting ligand for the nanocapsules and the relevant protein are indicated at left and above, respectively. The merged images (right) of the DsRed2 and CD14 micrographs demonstrate colocalization (yellow) of fluorescence when HA was used as the targeting ligand. The inset in the top left panel shows SYTOX green–stained nuclei (green) of hepatocytes expressing DsRed2; original magnification, ×60. Scale bar, 50 μm. (B) Western blot analysis of 100 μg total liver protein extracts from control mice and mice treated with the ASOR or HA nanocapsules shown in A. The proteins were detected by ECL as described in Methods. The treatment group is indicated above the lanes. (C) Western blot analysis of 100 μg total protein extracts from kidney, spleen, and lung to determine nonspecific uptake of nanocapsules. The β-actin lanes for loading controls are shown below. Control, mice treated with HA nanocapsules not containing DsRed2-expressing SB-Tns; DsRed2, purified recombinant DsRed2 protein.