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Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

ABSTRACT: Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose-dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas.This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases.

SUBMITTER: Viecelli HM

PROVIDER: S-EPMC4449723 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

Viecelli Hiu Man HM Harbottle Richard P RP Wong Suet Ping SP Schlegel Andrea A Chuah Marinee K MK VandenDriessche Thierry T Harding Cary O CO Thöny Beat B

Hepatology (Baltimore, Md.) 20140729 3

<h4>Unlabelled</h4>Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a live ...[more]

PMID: 24585515

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Project description:Non-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses.Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFR?, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction.Minicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFR? and FGFR2, respectively.A novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

Dataset Information

Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

Publications

Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

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