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 β-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 μg/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. Conclusion FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

“…These observations are shared by many others (2,5,9,13,18,24,25); however, there are reports demonstrating a complete removal of the plasmids by degradation, posttransposition (37). We are presently unable to consolidate these contradictory findings; however, it is possible that the extend of this random, nontranspositional insertion of plasmids is cell and tissue specific or dependent on plasmid architecture and methods used in plasmid preparation.…”

Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion

“…This knowledge may help in developing safe and effective antiviral strategies, especially with the emergence of drug resistance due to structural alteration of viral components (19,32,58). Although we have successfully completed a preclinical study on bilirubin gene therapy in jaundiced rats by use of FV (57), an exploration of the present knowledge of modulation of host cell signaling in fusion enhancement in such a model is awaited.…”

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

“…Hyaluronan-and asialoorosomucoidcoated nanocapsules, were found to target SB-based vectors carrying an FVIII transgene to hepatocytes in vivo and to improve the phenotype of hemophilia A mice, after intravenous injection (Kren et al, 2009). Similarly, packaging an SB vector expressing chUGT1A1 into proteoliposomes incorporating a fusogenic glycoprotein that promoted asialoglycoprotein receptor (ASGPR)-mediated endocytosis allowed successful in vivo delivery and sustainable, therapeutic gene expression in the hepatocytes of a rat model of Crigler-Najjar syndrome type 1 (Wang et al, 2009). Importantly, in neither study was a significant host immune response toward the gene delivery vehicle or the transgene product observed (Kren et al, 2009;Wang et al, 2009).…”

“…Similarly, packaging an SB vector expressing chUGT1A1 into proteoliposomes incorporating a fusogenic glycoprotein that promoted asialoglycoprotein receptor (ASGPR)-mediated endocytosis allowed successful in vivo delivery and sustainable, therapeutic gene expression in the hepatocytes of a rat model of Crigler-Najjar syndrome type 1 (Wang et al, 2009). Importantly, in neither study was a significant host immune response toward the gene delivery vehicle or the transgene product observed (Kren et al, 2009;Wang et al, 2009). In summary, when combined with nonviral delivery approaches, the SB transposon system shows considerable efficacy in providing sustained levels of therapeutic gene expression both ex vivo and in vivo.…”