Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40-60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti-CD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.
Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.
Retroviral vectors have been used for several decades for the transfer of therapeutic genes to various cells or organs including the liver. Initial studies aimed at treating inherited liver deficiencies were carried out with murine oncoretroviral vectors either delivered directly to the organ or using an ex vivo strategy that entailed harvest of the hepatocytes, transduction during a culture phase and further reinfusion to the patient. However, although a clinical trial was performed in the early 1990s, a complete cure of animal models of metabolic diseases was rarely achieved. The advent of lentiviral vectors derived from HIV1 profoundly changed the field and this vector type now appears to be of the most attractive for liver directed gene therapy. Indeed, lentiviral vectors do not require complete cell division to transduce the target cells. There are however still bottlenecks that limit the clinical development of gene therapy using retroviral vectors. In the present review we will specifically focus on specific aspects such as the risk of insertional mutagenesis, the potential requirement of cell cycle activation to enhance transduction and the major issue of an immune response directed against the transgene as well as some specific aspects of ex vivo gene transfer. Finally we will briefly consider the future developments of these vectors made possible by the availability of new techniques in cell and molecular biology.
Immunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC), especially as secondary treatment to prevent recurrence. It has been shown that a patients survival is directly correlated to the type and number of tumor-infiltrating immune cells, indicating that immune responses have a direct effect on the clinical course of the disease. We have assessed the potential of immunotherapy against HCC in preclinical models of low tumor burden. An antigen-specific strategy targeting ?-fetoprotein, and consisting of immunization with a DNA-based synthetic vector (DNAmAFP/704), was tested on an autochthonous model of chemical hepatocarcinogenesis and led to an important (65%) reduction of the tumor burden. A nonspecific approach of CD25(+) T-cell depletion by injection of PC61 antibody was also tested on an orthotopic HCC model and led to a significant protection against tumor development. Antigen-specific immunotherapy and Treg depletion are promising strategies in physiologically relevant HCC preclinical models. Future clinical trials will demonstrate if a combination of Treg depletion with an antigen-specific immunotherapy will also translate into clinical responses in HCC patients.
Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (i.m.) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2-3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the i.m. or the intravenous (i.v.) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.
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.
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.
In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver.
Metabolic inherited liver diseases are attractive targets for gene therapy. Recombinant lentiviruses are very powerful viral vectors able to infect nonmitotic cells. We wanted to develop a new surgical approach to improve gene transfer in adult liver using low viral doses.
When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.
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.
CASE HISTORY AND PRESENTATION: Two non-human primates (Macaca fascicularis), weight 3.5 kg, enrolled in an experimental protocol received a 25 ?g hour(-1) transdermal fentanyl patch for postoperative analgesia. The following day both animals were clinically normal, but after a new induction of anaesthesia with ketamine, they developed severe and prolonged respiratory distress, profound coma and myosis. MANAGEMENT AND FOLLOW-UP: Attempted reversal with naloxone was ineffective. After several hours of ventilation, both primates eventually died, 7 and 15 hours after ketamine injection, respectively. In both cases, the patch was discovered in the animals cheek pouch. Subsequent fentanyl serum concentration measurements (8.29 and 14.80 ?g L(-1) ) confirmed fentanyl overdose.
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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