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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 7

PCR identification of DNA flanking the SB-Tn insertion sites.

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PCR identification of DNA flanking the SB-Tn insertion sites.
   
(A) DN...
(A) DNA isolated from the livers of hemophilia A mice injected with HA pT2/CAGGS-BΔcFVIII//IFSB10 nanocapsules and wild-type controls served as template using primer pairs specific for the FVIII hemophilia A knockout (top) or the wild-type allele (bottom). (B) Lack of SB10 CDS persistence in the HA-treated knockout mice. PCR amplification of the SB10 CDS was performed (19) using liver DNA isolated from the DsRed2 animals and treated hemophilia A mice. The size or identity of the predicted amplicons and selected bands of the DNA marker (M) are indicated. The time (after injection) that the livers were harvested is indicated above the gels. (C) Schematic of the inverted-nested PCR strategy used to identify DNA flanking the SB-Tn insertion site. The genomic DNA digested by NcoI or XhoI was subjected to self-ligation and the products used as template for the initial inverted PCR amplification with primer pairs RP1/LP1 and RNP1/FNP1 for XhoI- and NcoI-digested DNA, respectively. The second PCR amplification used the XhoI and NcoI initial reactions as template with internal nested primer pairs LP2/RP2 and RNP2/FNP2, respectively. The PCR products were analyzed by agarose gel electrophoresis and visualized by UV light after ethidium bromide staining. The size of the 3 heavy bands is shown to the left of the NcoI gel. (D) Identification of the insertion sites in hemophilia A mice treated with HA-encapsulated pT2/CAGGS-BΔcFVIII//IFSB10. The region of the ID/DR of the Tn and the requisite duplicated TA (gray) followed by 40 nt of genomic DNA flanking the identified insertion sites are indicated above the sequences. The chromosomal location established by BLAST analysis is shown at right, and the intronic insertion sites are marked with asterisks. The closest adjacent genes for the other insertions are listed, with the distance in kb from the Tn insertion indicated in parentheses if less than 100 kb.
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