Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.
The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARgamma ligand, 15-deoxy-(12,14)-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARgamma dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARgamma in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARgamma antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARgamma-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARgamma. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases.
Plasma hepatitis C virus (HCV) originates from hepatocytes. However, in certain subjects, B cells may harbour both plasma strains and occult HCV strains tha t are not detected in the plasma. The internal ribosome entry site (IRES) of these latter strains is mutated, suggesting that the efficiency of viral translation could drive the cellular tropism of HCV.
Crigler-Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin-uridine 5-diphosphate-glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver.
This report describes the development, optimization and implementation of a persistent cell-based system to test inhibitors of hepatitis C (HCV) translation. The assay is based on a heterologous human immunodeficiency virus-1/simian immunodeficiency virus (HIV-1/SIV) lentiviral vector expressing the bicistronic cassette containing the firefly and renilla luciferase genes, respectively, as reporters, and the HCV internal ribosome entry site (IRES) inserted in between, under the control of the cytomegalovirus (CMV) promoter. The drug target in this assay is the HCV IRES, the activity of which leads to modulation of the renilla luciferase gene expression under its control, which is monitored by luminometry. The system has been validated using interferon (IFN), which is still the only consensual antiviral agent against HCV infection, associated with ribavirin. This bicistronic vector, extended to other viral IRESs and assayed in different cell lines, exhibited weak cell tropism, allowing its broad use in gene therapy, which frequently needs a multicistronic transfer vector to follow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicide gene, expressed from the same transcript.
Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.
Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.
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