HUMAN GENE THERAPY 26:186–192 (April 2015) ª Mary Ann Liebert, Inc. DOI: 10.1089/hum.2015.029

Brief Reviews

Gene Therapy for Inherited Diseases of Liver Metabolism Pasquale Piccolo1 and Nicola Brunetti-Pierri1,2


Gene therapy is entering the stage of initial clinical development to treat a growing number of inherited metabolic diseases. This review outlines the development of liver-directed gene therapy for diseases caused by deficiencies of enzymes that are primarily expressed in the liver and discusses the disorders that appear most promising for clinical translation.



nherited metabolic diseases have historically played an important role in the development of gene therapy. In the prerecombinant DNA era, patients diagnosed with argininemia, a urea cycle disorder (UCD) caused by arginase deficiency, were unsuccessfully injected with the papilloma Shope virus based on the incorrect assumption that the viral genome encoded the arginase gene.1 Several years later, a genetically modified retroviral vector encoding the human hypoxanthine phosphoribosyltransferase was used to infect mouse bone marrow cells ex vivo and transduced cells were subsequently transplanted into a Lesch–Nyhan disease mouse model, one of the very first examples of preclinical gene therapy.2 Inborn errors of metabolism continue to be attractive targets for gene therapy because of their prevalence, severity, and lack of effective treatments. Although individually rare, these diseases are relatively frequent as a group as their collective incidence may approach 1 in 800–2500 births.3,4 Expanded newborn screening is allowing early detection of several of these disorders.5 However, we are still lacking effective and safe therapies for most of them. The conventional approach to treating inborn errors of metabolism involves the following basic principles: (1) restriction of upstream essential nutrients to prevent intoxication, (2) supplementation of downstream nutrients to prevent secondary deficiency, and (3) stimulation of alternative routes for disposal of precursor metabolites. Inborn errors of metabolism are currently being treated based on these principles, using nutritional and pharmacological therapies, such as vitamin cofactors, end-product replacement, and drugs that induce specific enzymes or alternative pathways. Moreover, enzyme replacement therapies have been developed for lysosomal storage disorders (LSD) based on the concept that lysosomal enzymes are distributed distally to other tissues via secretion and uptake mediated by the mannose-6-phosphate receptor. 1 2

Despite treatment efforts, the prognosis of several inherited metabolic disorders is poor, especially for conditions at increased risk of decompensations during the course of intercurrent illness. The availability of gene therapy to be performed at the time of newborn screening, before the onset of symptoms and before development of irreversible damages, has the potential to radically improve the natural history of these diseases. The liver is a key organ for most metabolic pathways, and therefore numerous metabolic inherited diseases are caused by defects in this organ. This has led to the consideration of liver transplantation for the treatment of various metabolic inherited defects (Table 1). Patients who have undergone solid organ transplantation have benefited from innovative surgical techniques and novel, less toxic nonsteroidal immunosuppressive regimens.6 However, several patients (approximately 15%) succumb while waiting for a donor organ, and shortterm peritransplant and long-term morbidities continue to be significant issues.6–10 Liver transplantation is not a ‘‘cure’’ but rather a disorder in itself with a defined acute mortality and chronic morbidity, which is mostly related to lifelong immunosuppression. Patients with inherited metabolic diseases have generally normal liver, except for a defect in a single gene, suggesting that alternative approaches to treating them would involve correcting the genetic defect or replacing it with a normal version by gene therapy. However, the path toward clinical gene therapy has been difficult and early trials have shown that the immune system is a major barrier to clinical applications. Clinical Trials and Vectors

General skepticism toward gene therapy was raised by the death of one patient in the clinical trial for ornithine transcarbamylase (OTC) deficiency11,12 and by the development of leukemia in patients with severe combined immunodeficiencies (SCID) and Wiskott–Aldrich syndrome who were treated

Telethon Institute of Genetics and Medicine, Pozzuoli, Naples 80078, Italy. Department of Translational Medicine, Federico II University of Naples, Naples 80131, Italy.



Table 1. Most Common Inherited Liver Diseases in Which Hepatic Transplantation Has Been Performed Diseases with liver damage Wilson disease Progressive familial intrahepatic cholestasis Alpha1-antitrypsin deficiency Tyrosinemia type I Glycogen storage disease types I, III, and IV Diseases without liver damage Urea cycle disordersa Crigler–Najjar syndrome Familial hypercholesterolemia Primary hyperoxaluria Maple syrup urine disease Propionic acidemia Methylmalonic acidemia Acute intermittent porphyria Diseases with liver and extra-hepatic damage Lysosomal storage disorders Fatty acid oxidation defects Mitochondrial cytopathies a

Except argininosuccinate lyase deficiency.

with retroviral ex vivo gene therapy.13,14 The trial for OTC deficiency, the first with direct administration of a gene transfer vector (an earlier adenoviral vector) in patients with a genetic disease, resulted in lethal toxicity and death of one of the two subjects injected with the highest dose, whereas all 17 remaining subjects in the trial, including a subject injected with the highest dose, experienced only mild and transient toxicity.15 The patient who did die developed coagulopathy and hyperammonemia, followed by respiratory distress syndrome and multiple organ failure within 24 hr from vector infusion.11 It remains unclear whether the underlying more severe OTC deficiency, genetic susceptibility to enhanced innate immune response, or previous exposure to the adenovirus contributed to the fatal outcome. Nevertheless, it became clear that systemic injection of adenoviral vectors could be associated with dose-dependent toxicity, mediated by the vector capsid proteins that activate a potent inflammatory response.11,16,17 In the case of severe forms of SCID, while they have obviously raised concerns about the safety of the gene transfer, the development of leukemia is arguably an acceptable risk given the clear demonstration of sustained clinical benefit by gene therapy. After these drawbacks, encouraging results from a gene therapy trial using serotype 8 adeno-associated virus (AAV8) in hemophilia B patients18 sparked new enthusiasm for development of clinical trials for liver-directed gene therapy in inborn errors of metabolism. A single intravenous infusion of vector in 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX (FIX) to therapeutic levels that were sustained over a period of about 3 years. Importantly, clinical efficacy was achieved in the high-dose group, as shown by reduction in the use of prophylactic FIX concentrates and decreased bleeding episodes. However, an increase in alanine aminotransferase (ALT) because of a cytotoxic T lymphocyte (CTL) immune response occurred between 7 and 10 weeks after vector administration in 4 of the 6 patients in the high-dose group but was resolved after prednisolone treatment.18,19 Although the


increase in ALT was mild and transient, this trial once again highlights the obstacles posed by the immune response against viral gene transfer vectors directly administered in the human organism. Nevertheless, based on this promising study, liver-directed gene therapy, mostly utilizing AAV vectors, has been investigated for application in several inborn errors of metabolism, and it is currently under evaluation in patients with intermittent acute porphyria.20 Although AAV vectors are at this time the most promising for clinical applications, other vectors, such as helperdependent adenoviral (HDAd) vectors, have also generated encouraging results in terms of high levels of hepatic transduction, long-term expression, and safety when administered at low vector doses through localized hepatic delivery.21,22 Therefore, investigation of other vector platforms for liver-directed gene therapy should not been dismissed. In all animal models studied so far, HDAd-transduced hepatocytes (as well as all other examined target cell types) were not destroyed by an adaptive cellular immune response, thus leading to multiyear transgene expression.22,23 Whether or not this holds true for humans is unknown. Similar to AAV, HDAd capsid proteins derived from the administered particles may be a source of immunogens24,25 and following administration into mice may also provoke a CTL response directed against viral proteins derived from the capsid. However, whether or not these adenoviral-specific CTLs eliminate HDAd-transduced cells in vivo remains to be shown and animal modeling may not be adequate enough to address this important issue, given that it was not predictive of the human outcomes by AAV vectors.26–28 Nonviral vectors offer a number of advantages over viralbased strategies, including minimal toxicity from the vector, long-term transgene expression, lack of a humoral response to the vector and the consequent ability to repeat dose, and simple, cost-effective production. They could replace many viral vector approaches if they can be delivered with higher efficacy. Clinically relevant methods for regional hydrodynamic delivery of nonviral vectors demonstrate the feasibility of intravascular delivery to the liver using minimally invasive approaches,29,30 and are a step in the direction of human clinical trials. However, further improvements in formulations or carriers of the gene transfer material resulting in larger percentage of transfected hepatocytes are needed to make this approach attractive for therapy of inherited metabolic diseases. The purpose of this review is to highlight examples of liver-directed gene therapy for inherited metabolic diseases caused by deficiencies of enzymes that are primarily expressed in the liver and to address approaches and diseases that appear to be most promising for clinical translation. Approaches and Disease Targets

Ex vivo gene transfer has been shown to be effective in a selected number of immunological disorders in which expression of the therapeutic gene provides a selective growth advantage over untransduced cells, such as adenosine deaminase deficiency.31,32 Ex vivo gene therapy directed at hematopoietic stem cells by retroviral and lentiviral vectors has also generated encouraging results for treatment of multisystemic LSD. Vector-transduced bone marrow-derived cells overexpressing lysosomal enzymes can migrate into the central nervous system and mediate cross-correction of


neighboring brain cells. This approach has resulted in excellent outcomes, preventing the development of clinical manifestations in metachromatic leukodystrophy.33 Patients with peroxisomal disorder X-linked adrenoleukodystrophy treated with ex vivo lentiviral vector-mediated gene therapy have also exhibited evidence of clinical benefits.34 Ex vivo approaches are desirable as an alternative to hepatic transplantation in inborn errors of liver metabolism because they are less invasive and do not require life-long immunosuppression. However, for liver-directed approaches, ex vivo gene therapy has been less effective because of the limited engraftment of genetically modified hepatocytes.35 Liver repopulation by corrected hepatocytes occurs only if recipient hepatocytes are affected by a cytotoxic disease process or are removed, and if corrected cells have a selective growth advantage over uncorrected cells. In tyrosinemia type 1, hepatocytes corrected for fumarylacetoacetate hydrolase deficiency have a selective growth advantage and proliferate enough to efficiently repopulate mutant livers. However, strong selection for corrected cells in tyrosinemia type 1 is not replicated in the majority of inherited metabolic liver diseases. Moreover, ex vivo approaches to liver gene therapy are complicated: surgery is needed to remove hepatic tissue to obtain hepatocytes, cultured primary hepatocytes undergo a few rounds of cell division and cannot be substantially expanded, genetically modified cells must be re-infused surgically via the portal vein into the patient’s liver, and engraftment of transplanted hepatocyte is inefficient and short-term. Therefore, gene therapy for inherited metabolic liver diseases is mostly based on in vivo approaches. Liver sinusoidal endothelial cells have a fenestrated structure, allowing vector particles to be injected intravenously to access and infect hepatocytes. These fenestrations are about *107 nm in size in humans36 and are a major barrier for larger viral particles, like adenoviral vectors.37–39 In contrast, smaller particles such as the AAV that are *20 nm in size can easily cross the fenestrations to infect hepatocytes. However, Kupffer cells that are attached to endothelial and parenchymal cells remove foreign and damaged materials, including viral vector particles, from the circulation. This limits the efficiency of hepatocyte gene transfer.40,41 Inborn errors of metabolism result in disease through various mechanisms: (1) direct toxicity of accumulating metabolites upstream of the enzyme defect, (2) deficiency of downstream metabolites, (3) feedback inhibition/activation by the metabolite on the same or different pathway, and (4) diversion of metabolic flux to secondary pathways causing buildup of toxic metabolites. Ideal disease candidates for in vivo gene therapy are disorders that can be entirely corrected by delivery of therapeutic genes to hepatocytes. In contrast, metabolic diseases presenting with multisystemic involvement are more challenging to treat because they require transduction of multiple tissues for disease correction. A thorough understanding of disease pathogenesis permits prediction of disorders amenable to correction by liverdirected gene therapy, which is critical for clinical development of gene therapy. However, for several disorders, such as glycogen storage disease type I, organic acidemias and fatty acid oxidation defects, it is still unclear whether or not liver-directed gene therapy can correct all disease manifestations.42–44


Clinical experience with liver transplantation is extremely useful to predict the outcomes of liver-directed gene therapy. In many ways, liver transplantation is comparable to gene therapy, although a wasteful version of it, given that tens of thousands of normal genes are replaced in order to substitute one defective gene. Every disease treated by liver transplantation is amenable to correction by gene therapy.45–47 Some UCD, Crigler–Najjar syndrome, familiar hypercholesterolemia (FH), and primary hyperoxaluria type 1 (PH1) are examples of diseases that can be completely corrected by liver transplantation.45,46 However, there is a major difference between organ transplantation and gene therapy: in its current early stages, gene transfer will affect a limited percentage of cells, while the remaining cells in the target tissue remain uncorrected. Therefore, diseases requiring smaller percentages of hepatocyte transduction to achieve clinically relevant benefits are more likely to be corrected by gene therapy, and thus are attractive candidates. Diseases caused by enzymatic deficiencies generally require higher percentages of correction compared with diseases such as hemophilia. The amount of correction needed for a given inborn error of metabolism depends ultimately on the magnitude of the metabolic flux in the biochemical pathway at the cellular and organ levels.48 Tissue integrity is another important issue to consider, and conditions characterized by liver parenchymal damage, such as Wilson disease and cholestatic genetic diseases, are less attractive candidates for gene therapy because altered liver microarchitecture lessens the efficiency of liver gene transfer (Table 1). Therefore, to treat these disorders, liver gene therapy needs to be performed in stages before the development of tissue damage. Although LSD affect multiple tissues and organs as a result of lysosomal accumulation of undegraded products in cells, the defective enzyme can be transferred from one cell to another through the mannose-6-phosphate pathway. As a consequence, the enzyme can be produced in a single target tissue, secreted into circulation, and taken up by other diseased tissues. Therefore, in several studies, hepatic gene transfer has been performed with the goal of converting the liver into a factory organ for production and secretion of lysosomal enzymes, which can be then distributed to multiple affected tissues via blood circulation.49,50 However, this approach is ineffective for several LSD with brain involvement because the enzymes secreted by vectortransduced hepatocytes do not efficiently cross the blood– brain barrier. Urea cycle disorders

UCD have been studied for gene replacement therapy because risk/benefit assessments deem them appropriate as early clinical targets. They have high prevalence and a mortality rate of up to 24% in neonatal onset cases and 11% in late onset cases.51 The most common precipitant of clinical hyperammonemic episodes in the postneonatal period is intercurrent infection.51 Several studies, including the clinical trial discussed previously, have focused on OTC deficiency because of its severity and higher prevalence. Progress toward gene therapy for UCD has been underpinned by recent developments in clinical AAV gene therapy, and AAV vectors have been found to be efficient for correction of mouse OTC deficiency.52,53 Nevertheless,


while results in mice are encouraging, translating these data into the clinic is a major challenge. First, the cell-autonomous nature of the enzymatic defects requires a high percentage of hepatocytes to be transduced for phenotypic correction. The metabolic defect results in toxicity within each affected cell, and in order for the gene therapy to be effective, the functional gene must be delivered to a large percentage of cells. Second, successful treatment of severe neonatal disease is considerably difficult: rapid onset of hyperammonemia and consequent death within 24 hr of birth makes it difficult to achieve adequate levels of transgene expression with sufficient rapidity, despite vector delivery soon after birth. Third, loss of vector genomes as a consequence of liver growth requires vector re-administration, which in turn is hampered by the induction of immune responses against the vector. Several studies have repeatedly shown loss of episomal AAV vector genomes as a consequence of liver growth.54,55 The decline in transgene expression, particularly during the early rapid growth phase of dividing tissues, is a substantial problem that compromises long-term phenotypic correction. The human liver doubles in weight between the newborn period and the first 3 months of a life, doubles again by 10 months, and then once more by the age of 5 years.56 Loss of episomal vectors is a major challenge for UCD and other inborn errors metabolism requiring treatment early in life. Moreover, early timing of gene delivery appears to be a critical factor for genotoxicity and risks of hepatocellular carcinoma after AAV gene delivery, at least in mice,57,58 whereas no evidence of insertional mutagenesis and cancer was observed in adult mice for 18 months,59 in dogs for a period of 8 years,60 and in nonhuman primates for up to 5 years.61 It should also be noted that in mice that developed cancer the AAV integrated in the Mir341 within the Rian locus that has no orthologues in the human genome.56 Crigler–Najjar syndrome

Crigler–Najjar syndrome is an autosomal recessive disease caused by deficiency of bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1) and presents with nonhemolytic unconjugated hyperbilirubinemia. Patients with Crigler–Najjar syndrome type I are refractory to phenobarbital treatment, experience life-threatening elevations of bilirubin, and are generally managed with phototherapy throughout childhood and adolescence. Although effective, phototherapy is cumbersome, is inconvenient, significantly impairs the quality of life, and diminishes its efficacy with time as a consequence of increased skin thickness and decreased surface/ mass ratio. Moreover, despite treatment, patients remain at risk for brain damage when intercurrent infections increase levels of bilirubin above those that are controllable by phototherapy.62 Therefore, patients with Crigler–Najjar type I are often advised to consider liver transplantation, most often between 18 and 25 years of age. Crigler–Najjar syndrome has long been considered a paradigm for developing gene therapies for metabolic liver diseases because (1) the fraction of corrected hepatocytes required for clinical benefit is small, as deduced from hepatocyte transplantation studies,63 and even limited liver transduction is sufficient for correction of hyperbilirubinemia64; (2) UGT1A1 does not require strict gene regulation for normal activity; (3) animal models, the Gunn rat


and a mouse model,65,66 recapitulating the human disease are available; and (4) the outcome of experimental therapies can be easily determined by measuring bilirubin levels in serum and bile. For these reasons, Crigler–Najjar syndrome type I has been a disease target in several studies using a wide variety of vector systems and multiple routes of administration.67 The AAV serotype 8, which was found to be efficient in correcting hyperbilirubinemia in the Gunn rat and in the mouse model of disease,68,69 appears to be the most promising vector for clinical development. Familial hypercholesterolemia

Homozygous FH caused by mutations of the LDL receptor (LDLR) gene is a lethal disorder that causes affected patients to die of premature coronary artery disease. Affected children as young as 4 years old experience sudden death because of acute myocardial infarction, with nearly complete occlusion of the coronary artery.70 Until recently, the only valid treatment available was hepatic transplantation since the liver is the primary organ responsible for regulating cholesterol homeostasis.71 When liver transplantation was found effectively to lower cholesterol levels in FH patients, the concept of hepatic gene transfer of the LDL receptor was fostered and a clinical trial of gene therapy was developed.35 This trial is the only example of ex vivo liver-directed gene therapy in humans. Hepatocytes isolated from an FH patient were transduced with the LDLR gene using a retroviral vector and were subsequently re-implanted in the patients.35 Only a marginal response to the treatment was observed because of aforementioned reasons. Therefore, it became clear that only in vivo gene therapy has potential to effectively correct FH. Since high levels of hepatocyte transduction are required for correction of FH, initial attempts with AAV2 vectors failed in preclinical models and it was not until AAV8 vectors were developed that a substantial longterm reduction of serum cholesterol levels was obtained.72,73 Based on these promising data, current efforts are focused on developing an AAV8-mediated clinical trial for FH.74 Recently, two pharmacologic treatments have been approved for FH, the microsomal triglyceride transfer protein inhibitor lomitapide and the apolipoprotein B antisense oligonucleotide mipomersen.75,76 Although these drugs appear promising, further clinical investigations are needed to determine their efficacy and to address concerns about their potential hepatotoxicities. Nevertheless, gene therapy remains attractive for long-term correction of the disease. Primary hyperoxaluria type I

PH1 is caused by deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT), which results in a severe disease characterized by overproduction of oxalate. Oxalate insoluble calcium salts accumulate in the kidney, resulting in nephrocalcinosis and urolithiasis, as well as in other organs causing myocarditis, arrhythmias, stroke, and peripheral vascular occlusions. PH1 is estimated to account for about 1% of pediatric cases of end-stage renal failure.77,78 Combined liver–kidney transplantation is currently the only available treatment for most patients with PH1. Before the use of liver–kidney transplantation, 80% of patients died by the age of 20 years.79,80 Following liver transplantation, the rate of endogenous oxalate synthesis


drops to normal levels as expected based on pattern of AGT expression.81,82 PH1 behaves like a cell-autonomous defect in which one corrected hepatocyte cannot compensate for overproduction of toxic metabolites in its neighboring cells. In the context of gene therapy, it is expected that a large proportion of hepatocytes has to be transduced by gene therapy to minimize the deleterious effect of uncorrected hepatocytes, which continue to synthesize oxalate. Combined liver and kidney transplantation has sufficient risk to make the attempt of hepatocyte gene therapy justifiable from the perspective of a risk/benefit ratio. Preclinical proof of concept of efficacy of AAV8 in PH1 mice has been provided: AAV-mediated gene transfer has resulted in sufficiently robust liver expression of AGT and normalization of urinary oxalate excretion at doses that will be acceptable in humans.83 Future Considerations

Gene therapy for treatment of inborn errors of metabolism is appealing researchers and clinicians. In the past few years gene therapy has become widely accepted and has emerged as a novel therapeutic modality for several human disorders. However, these studies have also revealed several problems, such as unexpected toxicity and insertional oncogenesis. Despite these problems, clinical trials for SCID,31 retinal diseases,84 hemophilia B,18 and metachromatic leukodytrophy33 have met success and clearly support further development of gene therapies. Nevertheless, a careful risk/benefit assessment must be made for each condition, incorporating the underlying pathophysiology, long-term prognosis, and inherent and potential unforeseen risks of the different gene transfer approaches. Gene therapy for liver metabolic diseases is a logical objective given the problems and limitations of currently available treatments. Disorders such as Crigler–Najjar syndrome, UCD, FH, and PH1 are examples of excellent disease candidates because of their poor prognosis. Based on the significant progress made to date, in spite of the expected setbacks of all drug development efforts, gene therapy for liver metabolic disorders is becoming a viable option for treatment in future clinical trials. However, safety issues, such as immunogenicity of the vector and/or transgene product, prevalence of preexisting immunity against the vector, and loss of expression because of liver growth, remain important concerns and need to be further investigated. The therapeutic levels of hepatocyte transduction required for disease correction also need clarification, and it is currently unknown whether or not higher levels of hepatocyte correction can be achieved in humans using AAV vectors for disorders like UCD, FH, and PH1. While AAV-based gene replacement strategies are entering the phases of clinical development, novel technologies (zinc finger, TALEN, and CRISPR/Cas9) have recently been developed to obtain hepatic gene correction in vivo85 and have the potential for being the next generation of drugs for gene therapy of inherited metabolic diseases. Although these approaches are still far from being used in clinical applications because of significant issues related to safety and delivery, they have the potential to overcome the issue of loss of transgene expression because of hepatocyte division and might permit gene expression within its physiologic genomic context.


In conclusion, clinical gene replacement therapy has achieved important successes, and based on the steady progress to date, we are expecting several inherited metabolic diseases to be investigated in clinical trials in the immediate future. Acknowledgments

N.B.-P. is supported by the Italian Telethon Foundation (TCBMT3TELD), the European Research Council (IEMTx), and the Italian Ministry of Health (GR-2009-1594913). Author Disclosure Statement

The authors have no conflicts of interest to disclose. References

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Address correspondence to: Dr. Nicola Brunetti-Pierri Telethon Institute of Genetics and Medicine Via Campi Flegrei 34 80078 Pozzuoli, Napoli Italy E-mail: [email protected] Received for publication February 27, 2015; accepted after revision March 2, 2015. Published online: March 11, 2015.

Gene therapy for inherited diseases of liver metabolism.

Gene therapy is entering the stage of initial clinical development to treat a growing number of inherited metabolic diseases. This review outlines the...
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