Current Gene Therapy - Volume 9, Issue 2, 2009

In 1992, the group of Jim Wilson in Philadelphia conducted the first gene therapy trial aimed at treating a liver inherited disease [1]. This seminal study involved hypercholesterolemic patients suffering from a complete hepatic deficiency of LDL receptor. It was based on an ex vivo strategy which entailed the harvest of hepatocytes from a surgical biopsy followed by transduction of cultured cells with MoMULV retroviral vectors. The corrected cells were then reintroduced via the portal vein. Although no substantial therapeutic effect was observed, this trial demonstrated the feasibility and safety of liver directed gene therapy. Almost ten years thereafter, another liver directed gene therapy protocol was carried out by the group of Kathy High [2]. In that case, recombinant AAV vectors ferrying the human factor IX cDNA were injected directly in the hepatic artery of hemophilia B patients. A significant increase in serum FIX was recorded in some patients receiving the highest vector dose. However, this increase was only transient and disappeared in the following weeks. It was subsequently demonstrated that an immune response against the viral capsid proteins was responsible for the elimination of corrected cells by the immune system. In the meantime, the field of gene therapy was devastated in 1998 by the death of a young patient following administration of adenoviral vectors to treat ornithine carbamyl transferase deficiency [3]. These historical cases epitomize the progress of knowledge in the field of liver directed gene therapy, which has been gained from preclinical as well as clinical studies during the past twenty years. They also point out to the caveats that deserve further attention to improve the therapeutic efficiency of future clinical applications of gene therapy for liver diseases. The liver is an attractive target for gene therapy applications because it is accessible to both ex vivo and in vivo gene therapy strategies. Although the ex vivo approach used in the LDL receptor deficiency trial was limited initially by the low transduction rate of the hepatocytes as well as the poor efficiency of engraftment of the cells, important progress makes this approach still worth of consideration. The particular anatomy of hepatic vascularization makes the liver also readily accessible to in vivo gene transfer strategies based on delivery of vectors to the blood stream. Bloodborn viral particles may easily cross the fenestrated endothelium lining the sinusoidal plates and enter the space of Disse which separates the hepatocyte sinusoidal membrane from the blood vessels. This simplified description may be actually be more complicated. The size of the fenestrations may differ according to the species. Also, non parenchymal cells are present in the vicinity of the space of Diss (Kupffer cells, endothelial cels etc….). All these cells are also the target of gene transfer vectors and contribute, usually negatively, to the final outcome of gene transfer. Such off target infection may result in deleterious responses, as in the case of adenoviral vectors. Furthermore, when expression of the transgene is specifically restricted to hepatocytes, tolerance to the transgene product may ensue with an as yet not clearly defined role of regulatory T cells. Therefore many efforts are devoted to circumvent infection of non parenchymal liver cells.

Interleukin-12 (IL-12) is a multifunctional cytokine that stimulates both innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses. The immunomodulating and antiangiogenic functions of IL-12 have provided the rationale for exploiting this cytokine as an anticancer and antiviral agent. The promising data obtained by the administration of IL-12 recombinant protein in preclinical animal models of cancer and chronic viral hepatitis raised hopes that recombinant IL-12 could be a powerful therapeutic agent against both pathologies. However, clinical trials revealed a modest clinical response that was limited by the development of an adaptive response that down-regulated IL-12 activity and by severe toxicity when high doses of this cytokine were used. Gene therapy can significantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efficacy/toxicity ratio. Early clinical trials with short-term IL-12 expression vectors have set the proof-of-concept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells, sometimes followed by tumor regression. Recent advances in long-term expression vectors for the delivery of IL-12 or lytic viruses armed with this cytokine may be key to unlocking the therapeutic potential of IL-12. However, the new generation of IL- 12 gene therapy protocols should cope with two major limitations. First, promoter silencing induced by IL-12 may abrogate long-term production of this cytokine. Second, regulatory immune systems induced by IL-12 should be blocked to maximize antitumor and antiviral activity.

Crigler-Najjar (CN) syndrome is a recessive inherited disorder caused by deficiency of uridine diphosphoglucuronosyl transferase 1A1. This hepatic enzyme catalyzes the glucuronidation of bilirubin, an essential step in excretion into bile of this neurotoxic compound. As a result, CN patients suffer from severe unconjugated hyperbilirubinemia and are at risk of bilirubin encephalopathy. Over the last decades ex vivo and in vivo gene therapy using viral and nonviral vectors has been used to correct hyperbilirubinemia in the relevant animal model for CN syndrome, the Gunn rat. Several of these approaches did result in long-term correction of serum bilirubin levels in this animal model. However, none have been translated into a clinical trial. In this review we will recapitulate the strategies used and discuss their suitability for clinical application in the near future. We will also address specific safety measures in the gene therapy protocol needed to prevent adverse effects such as bilirubin toxicity. Since CN seems an ideal model for other monogenetic inherited metabolic liver disorders, development of liver-directed gene-therapy has relevance beyond this rare disease.

The liver is a key organ in numerous metabolic pathways, in cholesterol metabolism, and in production of coagulation factors. Therefore, gene transfer to hepatocytes has been extensively pursued. There are numerous biological parameters that may affect the outcome of hepatocyte-directed gene transfer. Species or strain variation of any of these multiple determinants hinders the process of clinical translation. This review specifically focuses on functional aspects of liver histology that are pertinent for gene transfer to parenchymal liver cells. We discuss the reticulo-endothelial cells of the liver and the spleen, and their impact on innate immune responses after adenoviral transfer and on vector clearance. Liver sinusoidal endothelial cells contain pores, called fenestrae, and have no basal lamina. Fenestrae are clustered in sieve plates and may provide direct access for circulating gene transfer vectors to the space of Disse, in which microvilli of parenchymal liver cells protrude. We present multiple lines of evidence that the species differences in the diameter of sinusoidal fenestrae are a critical determinant of transgene expression after adenoviral transfer. The small diameter of fenestrae in humans should be considered in any rational design of gene transfer technologies for hepatocyte-directed transfer. Hydrodynamic gene transfer is highly successful in rodents. The significantly lower efficacy in higher species may also partially be due to species differences in liver architecture. Finally, we discuss species differences in adaptive immune responses against the transgene product that may constitute one of the most significant hurdles for clinical translation.

Globally, hepatic diseases are an important cause of mortality and morbidity. Harnessing RNA interference (RNAi) to silence pathology-causing genes specifically offers exciting possibilities for improvement of treatment. Nevertheless achieving efficient and safe delivery of RNAi activators remains an important objective before this gene silencing approach realizes its full therapeutic potential. Several viral and non viral vectors (NVVs) are being developed for hepatotropic delivery of synthetic and expressed RNAi activators. Each has advantages and disadvantages that are suited to particular disease conditions. Amongst the viral vectors, recombinant adeno-associated viruses and PEG-modified helper dependent adenoviruses show promise for situations that require intermediate to long term expression of RNAi activators. Recombinant lentiviruses have not been used extensively as hepatotropic RNAi vectors, but are likely to find application where lasting therapeutic silencing is required. NVVs are a particularly important class of vector and are effective for delivery of synthetic RNAi activators to the liver. Preclinical investigations using RNAi-mediated gene silencing to counter persistent hepatitis B virus, hepatitis C virus, hepatocellular carcinoma, hypercholesterolemia and cirrhosis are discussed in this review. Although obstacles remain, vigorous research has given impetus to the field and RNAi-based treatment of liver diseases is likely to become a reality in the near future.

The liver is a preferred target organ for gene therapy not only for liver-specific diseases but also for disorders that require systemic delivery of a protein. Diseases that could benefit from hepatic gene transfer include hemophilia, metabolic disorders, lysosomal storage disorders, and others. For a successful delivery of the transgene and sustained expression, the protocol must avoid immune responses in order to be efficacious. A growing number of studies have demonstrated that liver-directed transfer can induce transgene product-specific immune tolerance. Tolerance obtained via this route requires optimal engineering of the vector to eliminate transgene expression in antigen presenting cells while restricting high levels of therapeutic expression to hepatocytes. Innate immune responses may prevent tolerance induction, cause toxicity, and have to be minimized. Discussed in our review is the crucial role of CD4+CD25+ regulatory T cells in tolerance to the hepatocyte-derived gene product, the immunobiology of the liver and our current understanding of its tolerogenic properties, current and proposed research as to the mechanisms behind the liver's unique cellular environment, as well as development of the tools for tolerance induction such as advanced vector systems.

Adenovirus (Ad) are valuable vectors for liver gene therapy because of their intrinsic ability to transduce hepatocytes following intravenous administration. However, the effective application of these vectors, including helperdependent Ad unable to trigger viral gene expression, for liver gene therapy in humans has been limited due to several obstacles. First, their high immunogenicity triggers a complex immune response, both innate and adaptive, that leads to hepatocyte destruction, reducing the duration of transgene expression. This high immunogenicity also induces a long lasting cellular and humoral immunity that impairs subsequent re-administration. Second, Ad vectors transduce not only hepatocytes but also other cell types from the liver or other organs. This Ad vector dissemination contributes to their toxicity and immunogenicity, further reducing the effective period of transgene expression. A better understanding of the interactions between Ad vectors and their host underlying the acute liver toxicity and hepatocyte transduction is required to improve the efficacy and duration of gene delivery in vivo. The aim of this review is to discuss insights into the cellular and molecular mechanisms involved in Ad vector-mediated innate immune responses. Current advances in the knowledge of Ad liver tropism and the influence of blood components on Ad vector uptake by the liver will be discussed. Finally, different approaches developed to minimize Ad vector toxicity, optimize delivery and increase transgene expression will be summarized. The full potential of Ad vectors will only be reached when their immunogenicity is abolished and hepatocytespecific transduction achieved.

Hydrodynamic gene delivery to the liver has potential as a safe and effective approach for clinical liver gene therapy. However, the simplicity of the technique in rodents - an intravenous injection - belies the theoretical and practical complexity for clinical application. A key issue is that outflow obstruction of the DNA solution from the liver is a critical factor for raising intrahepatic vascular pressure, which in turn provides the force to swell the liver and effect gene delivery. For conventional hydrodynamic gene delivery via tail vein injection, this outflow obstruction is provided naturally by the vascular resistance of the gut, spleen and pancreas. For regional hydrodynamic gene delivery to the liver, outflow obstruction to create a closed system requires surgical intervention, making it unlikely that minimally invasive techniques will be possible in the clinic. Intrinsic factors, in particular compliance (elasticity) of the liver are likely to be crucial in determining the degree of swelling for a given level of intrahepatic vascular pressure. Liver compliance is likely to be the major reason for the low level of hydrodynamic gene delivery in the pig model, and will influence the effectiveness of the approach in man, both in general and in different disease states.

Transplantation of hepatocytes, whether genetically modified or not, has become an alternative to orthotopic liver transplantation for the treatment of patients with metabolic disease. However, more than ten years after the first clinical trial of ex vivo gene therapy to treat patients with Familial Hypercholesterolemia, there are still a number of impediments to these approaches. Numerous animal models are still being developed on the one hand to improve hepatocyte integration within hepatic parenchyma and function, and on the other hand to develop vectors that drive long-term transgene expression in situ. These include large animal models such as non-human primates, which have recently led to significant progress in hepatocyte transplantation. Simultaneous development of lentiviral vectors from different lentivirus species has permitted the transfer of genes into mitotically-quiescent primary cells including differentiated hepatocytes. Particularly third generation vectors derived from HIV-1 lentivirus are the most widely used and have significantly improved the safety and efficiency of these vectors. Given the shortage of organs and problems related to immunosuppression on one hand, and recent progresses in hepatocyte transduction and transplantation on the other hand, ex vivo approach is becoming a real alternative to allogeneic hepatocyte transplantation. We review the present progresses and limits of the ex vivo liver gene therapy approach in different animal models, emphasizing clinically relevant procedures.