Current Pharmaceutical Design - Volume 17, Issue 24, 2011

Successful liver-directed gene therapy has the potential to revolutionize medicine. Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have shown tremendous potential for liver-direct gene therapy. In small and large animals, hepatic transduction with HDAd has resulted in high level, long-term transgene expression without chronic toxicity in a variety of disease models. Recent advancements in the large-scale manufacture of HDAd have permitted contemplation of clinical application. However, dose-dependent activation of the host innate inflammatory response remains an obstacle for clinical translation. Recent advancements in vector capsid modifications, immune modulation regimes, as well as novel routes of vector administration may yet permit clinical liver-directed gene therapy with HDAd.

Gene therapy holds great promise for the treatment of inherited metabolic disease. Among different vector systems used to date, vectors based on adeno-associated virus (AAV) have shown great potential for systemic expression of therapeutic transgenes. The main advantages of AAV are the beneficial safety profile and the possibility to generate long-term transgene expression without the necessity for chromosomal integration. Successful transduction of hepatocytes in the absence of immunological complications has been achieved in a number of animal species, including mice, dogs and nonhuman primates. Despite this plethora of successful studies, clinical applications have been lagging. Up to date, one clinical trial for liver-directed gene transfer has been performed and results underscored the important role of neutralizing antibodies towards the AAV capsid and the generation of cytotoxic T cell responses against transduced hepatocytes. Recently, a wide variation of novel AAV serotypes has emerged that shows great promise for improved gene transfer efficiency. However, one important factor hampering clinical progress has been the large degree of variability in terms of transduction efficiency and transgene expression levels of different AAV serotypes and the subsequent difficulties in the selection of a single serotype for clinical development. The aim of this review is to critically reevaluate pre-clinical data obtained in animal models of metabolic diseases in light of the progress that has been achieved in liver-directed gene transfer using AAV vectors. Using this evidence-based rationale, we have selected AAV8 as the serotype that combines the most favorable features for clinical applications of hepatic gene transfer.

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.

The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer.

Hepatocytes are a key target for gene transfer directed at correction of inborn errors of metabolism. The theoretical potential of hepatocyte-directed gene transfer contrasts with the hurdles for clinical translation of this technology. Innate immune responses following gene transfer are initiated by recognition of pathogen-associated molecular patterns by pattern recognition receptors like Toll-like receptors. Adaptive immune responses may constitute the most significant hurdle for efficient gene transfer. Besides the challenge imposed by adaptive immune responses against the vector and the potential problem of pre-existing immunity, immune responses against the transgene product may also constitute an obstacle. The liver is a tolerogenic organ. Naive T cells encounter liver antigens initially in the liver, rather than in lymphoid tissue. Lymph nodes and the spleen are anatomical compartments that provide a particular microarchitecture and microenvironment for the induction of immunity. In contrast, antigen presentation in the liver takes place in a completely different microarchitecture and microenvironment. This is a key aspect of the hepatic adaptive immune tolerance induction. Consistent with the tolerogenic nature of the liver microenvironment, the risk of antibody formation against the transgene product may be limited in the setting of hepatocyte-directed gene transfer and specifically by restricting transgene expression to hepatocytes by use of hepatocyte-specific expression cassettes. However, it is unclear to which extent animal experimental data following gene transfer predict immune responses in humans. Extrapolations from animals to humans are required but should be performed with sufficient insight into the dramatic species differences of the immune system.

There are numerous inborn errors of metabolism of the liver, and they are all rare to very rare. To get a clear picture of the indications for gene transfer in these conditions, it is essential to get a clear view on the current (lack of) insight in the pathophysiology of these disorders, the current treatment options and hence on the window of opportunity for new treatments as gene transfer. The aim of this review, is to illustrate the problems related to treatment of inborn errors of metabolism of the liver. General aspects defining the quest for treatments for very rare diseases are touched upon, but for the sake of clarity, this review is restricted to five illustrative examples: Crigler-Najjar type I, the urea cycle defects, phenylketonuria, classic galactosemia and propionic acidemia. These examples reflect the problems that are currently experienced and can be expected, when gene transfer trials for these disorders are undertaken.

Lysosomal Storage Diseases (LSDs) comprise a group of over fifty inherited metabolic disorders, with their hallmark feature being deficient catabolism and accumulation (storage) of macromolecules in the lysosomes due to genetic deficiency of specific lysosomal enzymes. The combined incidence of LSDs is estimated to be ∼1 in 7,000 births. LSD symptoms can vary significantly, primarily due to the nature of the gene defect (null or missense mutations) as well as which cells are affected. Cumulatively, LSDs place a significant burden on patients and their families, causing much in the way of morbidity and mortality. Currently, there is no cure for any LSD. This review will describe currently available treatment options for LSD patients, and then focus upon gene therapy prospects for various LSDs. Worldwide, researchers have accumulated significant data in humans affected by LSDs, as well as several small and large animal models. As a result, various viral and non-viral gene transfer platforms have been developed and specifically optimized to treat LSDs. In this review we will describe advances suggesting that the LSDs may be some of the most amenable diseases to treat by gene therapy based approaches. However, to overcome the several remaining limitations encountered by these approaches, a deep understanding of the biology of the LSDs is required, as well as the host innate and adaptive immune responses to the act of gene transfer.

Familial hypercholesterolemia (FH) is an inherited metabolic disorder characterized by high levels of plasma low density lipoproteins (LDL) and an increased risk of premature atherosclerosis and coronary heart disease. LDL receptor (LDLr) deficiency is the most prevalent cause of FH. Therefore, hepatocyte-directed LDLr gene transfer constitutes an important strategy for the treatment of this monogenetic disease. Nowadays, homozygous FH patients are treated with lipid-lowering drugs complemented by plasma or LDL apheresis. Liver transplantation can restore metabolism of apolipoprotein B containing lipoproteins, but requires lifelong immunosuppression to prevent organ rejection. Recently, significant progress in gene transfer technology has encouraged investigators to further develop LDLr gene transfer approaches for the treatment of FH. In experimental animal models of FH, LDLr overexpression following viral vector-based gene transfer has been shown to be associated with long-term stable correction of hyperlipidemia, with attenuation of atherosclerosis progression, and in certain cases even with lesion regression. The first part of this review provides a thorough overview of familial hypercholesterolemia including its diagnosis, lipoprotein metabolism, and current management. In the second part, we critically review experimental LDLr gene transfer studies demonstrating the progress that has been made from the initial proof of principle studies to recent investigations showing dramatic regression of atherosclerosis in experimental models.