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Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

ABSTRACT: Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1.FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

SUBMITTER: Wang X

PROVIDER: S-EPMC4174336 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

Wang Xia X Sarkar Debi P DP Mani Prashant P Steer Clifford J CJ Chen Yong Y Guha Chandan C Chandrasekhar Voshavar V Chaudhuri Arabinda A Roy-Chowdhury Namita N Kren Betsy T BT Roy-Chowdhury Jayanta J

Hepatology (Baltimore, Md.) 20090901 3

<h4>Unlabelled</h4>Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes ...[more]

PMID: 19585550

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Project description:A Sleeping Beauty (SB) transposon system is made of a transposon plasmid (containing gene encoding a desired functional or therapeutic protein) and a transposase plasmid (encoding an enzyme capable of cutting and pasting the gene into the host cell genome). It is a kind of natural, nonviral gene delivery vehicle, which can achieve efficient genomic insertion, providing long-term transgenic expression. However, before the SB transposon system could play a role in promoting gene expression, it has to be delivered efficiently first across cell membrane and then into cell nuclei. Towards this end, we used a nanoparticle-like lipid-based protocell, a closed bilayer of the neutral lipids with the DNA encapsulated inside, to deliver the SB transposon system to cancer cells. The SB transposon system was amplified in situ inside the protocells by a polymerase chain reaction (PCR) process, realizing more efficient loading and delivery of the target gene. To reach a high transfection efficiency, we introduced two targeting moieties, folic acid (FA) as a cancer cell-targeting motif and Dexamethasone (DEX) as a nuclear localization signaling molecule, into the protocells. As a result, the FA enabled the modified targeting protocells to deliver the DNA into the cancer cells with an increased efficiency and the DEX promoted the DNA to translocate to cell nuclei, eventually leading to the increased chromosome insertion efficiency of the SB transposon. In vivo study strongly suggested that the transfection efficiency of FA-modified protocells in the tumor tissue was much higher than that in other tissues, which was consistent with the in vitro results. Our studies implied that with the targeting ligand modification, the protocells could be utilized as an efficient targeting gene carrier. Since the protocells were made of neutral lipids without cationic charges, the cytotoxicity of protocells was significantly lower than that of traditional cationic gene carriers such as cationic liposomes and polyethylenimine, enabling the protocells to be employed in a wider dosage range in gene therapy. Our work shows that the protocells are a promising gene carrier for future clinical applications.

Dataset Information

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

Publications

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

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