Abstract
Gene therapy is an exciting therapeutic concept that offers the promise of a cure for an array of inherited and acquired disorders. The liver has always been a key target for gene therapy as it controls essential biological processes including digestion, metabolism, detoxification, immunity, and blood coagulation. Metabolic disorders of hepatic origin number several hundreds, and for many, liver transplantation remains the only cure. Liver-targeted gene therapy is an attractive treatment modality for many of these conditions. After years of failure, substantial progress in this field in the past decade has resulted in promising clinical efficacy and safety in patients with monogenetic disorders with Valoctocogene roxaparvovec (Roctavian), the first gene therapy for treatment for hemophilia A, to be approved in Europe. Another, Etranacogene dezaparvovec (AMT-061) for hemophilia B is also in the final stages of approval. A number of other liver targeted gene therapy products are at an advanced stage of development, thus heralding a new era of potentially curative molecular medicine. This review explores the recent clinical advances in liver targeted gene therapy as well as the challenges that need to be overcome for the widespread adoption of this new treatment paradigm.
INTRODUCTION
Gene therapy aims at restoring, modifying, or enhancing cellular functions through the introduction of genetic material into a target cell to treat genetic disorders, infection, inflammatory diseases, or cancer. The liver is the largest solid organ in the human body and a key target organ for gene therapy as it expresses two-thirds of the 20,000 protein coding genes that are normally expressed in humans, controlling essential biological processes, including digestion, detoxification, metabolism, immunity, and blood clotting.
Consequently, numerous inherited metabolic disorders have their origin in this organ and more precisely in the hepatocytes that constitute 67% of the liver parenchyma. Inborn errors of metabolism may lead to accumulation of toxic products in the liver, leading to irreversible hepatotoxicity as observed in disorders such as α1-antitrypsin deficiency, type I tyrosinemia, or Wilson disease. In other disorders including familial hypercholesterolemia and hemophilia A and B, the clinical manifestations are primarily extrahepatic.
Primary and secondary cancers affecting the liver as well as chronic life-threatening infections such as hepatitis B and C are also amenable to gene therapy. The liver contains 10–15% of the body's total blood volume, making it ideal for use as a factory to synthesize and deliver proteins into the circulatory system for systemic therapy, including those not normally manufactured in the liver. Therefore, liver directed gene therapy can target an array of organ specific and systemic disorders, including those that cannot be effectively treated with drugs, biologics, or transplantation.
Gene therapy has the potential to create ever-expanding therapeutic opportunities through one of two broad approaches: (1) gene addition, the more commonly used technique that adds genetic material into somatic cells to provide the target cell with new functional attributes (e.g., chimeric antigen receptor [CAR]-T cells) or compensate for a missing or faulty gene, or (2) gene editing, which aims at treating diseases by directly modifying DNA in somatic cells through inactivation/disruption (also called gene silencing, knockdown, or knockout), or gene correction.
Most approaches in clinic are based on gene replacement and this will be the focus of this review. Strategies based on genome editing are at an earlier stage of clinical development, some of which are listed in Table 1. The simplicity of the basic concepts of gene therapy, together with exciting early results in animal models, fueled great enthusiasm for this new direction in medicine in the mid-1980s. Gene therapy was expected to transform the treatment of a variety of genetic and acquired disorders.
A brief summary of clinical trials evaluating systemic delivery of adeno-associated virus vectors
These expectations were dashed by a disappointing series of clinical trials in the mid-1990s that failed due to the inadequacies of the therapeutic gene delivery systems (or vectors), poor expression of the recombinant genes in humans. Expectations were further dampened, in 1999, when a patient with ornithine transcarbamylase (OTC) deficiency died following therapy. 1 The turn of this century saw the first tangible clinical success for gene therapy in infants with life-threatening X-linked severe combined immune deficiency (SCID). 2 –4
Just when hopes were being raised for a variety of catastrophic disorders, two SCID treated gene therapy patients developed leukemia, as a consequence of insertional mutagenesis, highlighting the dangers of gene therapy. The gene therapy community responded to these tragedies, with a hive of research activity leading to the development of safer gene transfer technology and a better understanding of the critical interactions of gene transfer on host genes.
VECTORS FOR LIVER GENE THERAPY
The success of liver gene therapy depends on the development of vectors that can efficiently introduce the therapeutic genes into hepatocytes. A number of gene transfer vehicles have been developed that can broadly be divided into two categories—non-viral and viral vectors.
Non-viral vectors
Non-viral vectors include naked DNA, which has been effectively used for liver targeted gene delivery in rodents and in dogs through hydrodynamic injections that entails rapid injection of large volumes of solution containing DNA into the circulation to greatly increase intravascular volume, forcing permeability of liver parenchymal cells, to facilitating delivery and uptake of the DNA by hepatocytes. This method is less efficient in larger animals and results in immune activation and a substantial level of liver toxicity.
In addition, expression following hydrodynamic injections is transient. More recently, nucleic acid complex with lipoplex or polyplex has gained popularity for non-viral delivery to the liver, with ligands that provide hepatocyte specificity such as triantennary N-acetyl galactosamine that bind to the asialoglycoprotein receptor (ASGPR), which enhances the delivery of genes to the liver. This approach has been successful in delivering a variety of small interfering RNA conjugates to the liver with notable clinical successes. These include (1) Givosiran for the treatment of acute hepatic porphyria, 5 caused by mutations in genes associated with haem biosynthesis pathway, and (2) Patisiran, a double-stranded small interfering RNA that reduces transthyretin production in hereditary transthyretin amyloidosis. 6
Viral vector systems
Viruses are effective gene delivery vehicles as they have evolved over time to display tropism for specific human cell types and once within target cells can actively deliver their payload to the nucleus. This is critical, as DNA is non-functional outside the nucleus. Recombinant viral vectors are designed to harness the viral infection pathway into target cells but lack the ability to replicate in the host cell, as coding sequences for viral replication are replaced with the gene of interest. The number of different viruses that are under development as gene-therapy vectors is steadily increasing but can be divided into two general categories—integrating and non-integrating.
At present, retroviral vectors based on oncoretro-, foamy-, or lenti (LV)-viruses are the only gene transfer systems that can mediate efficient integration of the transgene into recipient cells. Lentiviral vector (LV) shows most promise for liver targeted gene therapy approaches, because these human immunodeficiency virus-based retroviral vectors have the advantage of being able to transduce dividing and nondividing cells, including hepatocytes. They facilitate integration of transgene into hepatocyte chromosomal DNA, offering the potential advantage for establishing long-term expression, which may be advantageous in the pediatric setting because the liver undergoes substantial growth before reaching maximal adult size.
Further, the lower prevalence of HIV antibodies in the population makes lentivirus attractive vectors for a broader patient population. Lentiviruses have been safely used ex vivo to correct autologous hematopoietic stem cells (HSCs) in a growing number of immune hematological disorders such as SCID 7 and Wiskott-Aldrich syndrome. 8 Lentivirus-based gene therapy also shows promise for globin disorders. 9 Another ex vivo application of LVs is the generation of CAR T cells for cancer immunotherapy (Kymriah). 10
Success with HSC and T cell transduction has supported systemic in vivo evaluation of LV, with initial studies showing low but persistent transgene expression in the liver of mice and dogs. 11 More recently developed phagocytosis-shielded LV show selective targeting of liver and spleen and enhanced hepatocyte gene transfer following systemic delivery in non-human primates, laying the foundation for proof-of-concept clinical trials.
Vectors based on adeno-associated virus (AAV), a nonenveloped, parvovirus virus, show most promise for liver-targeted gene therapy and will be the main focus of this review. 12 They have a remarkable tropism for the liver, enabling administration of AAV vectors into the peripheral vein in humans to mediate preferential and efficient gene transfer into post-mitotic hepatocytes, thus dispensing with the need for invasive procedures previously deployed for organ-specific gene transfer. AAV's 4.7 kb, single-stranded DNA genome contains coding sequences for viral replication machinery (rep) and capsid (cap) proteins, flanked by palindromic inverted terminal repeats (ITR) containing the origin of replication and packaging signals.
Naturally occurring AAV are replication-defective and establish a latent asymptomatic infection when coinfected with a helper virus such as adeno- or herpes viruses. AAV is thought to be non-pathogenic in humans and weakly immunogenic. In addition, recombinant AAV vectors are entirely devoid of wild-type viral coding sequences, thus reducing the potential for invoking cell-mediated immune response to foreign viral proteins.
Following AAV uncoating in the nucleus, the ITRs flanking the vector genome drive recombination to form episomal constructs. Genomic integration occurs but at a low frequency, thus reducing the potential for genotoxicity. Animal studies and accruing data in humans suggest that AAV-mediated expression of transgenic protein can be stably maintained for periods exceeding 5 years in large animal models and humans. 12 AAV vectors, therefore, have the best safety profile among vectors of viral origin. Although most of the early gene therapy studies focused on AAV2, a common human isolate that was the first to be characterized, there are now over 100 naturally occurring AAV serotypes with differing tropism and immune-biological properties available for gene therapy approaches.
Additional approaches based on capsid engineering, phylogenetic reconstitution of AAV, and directed evolution have been used to generate a vast repertoire of AAV vectors with unique attributes, including tissue tropism, biodistribution, and immunogenicity.
The AAV transgene is relatively easy to manipulate, enabling the addition of stronger tissue-specific promoters and codon-optimization of the transgene complementary DNA (cDNA) to improve levels of expression. One limitation of AAV vectors is their small packaging size (∼5.0 kb, including ITRs) compared with other viral vectors. This limited packaging capacity is a hurdle for a variety of disorders, including Hemophilia A, Wilson's disease, and carbamoyl phosphate synthethase 1 (CPS1), disorders that are due to defects in large genes. In Hemophilia A and Wilson's disease, smaller versions of the genes encoding functionally active proteins have been developed to overcome this obstacle. 13
Alternatively, dual AAV vectors containing fragments of the large transgenes split into two separate portions are used to transduce target cells with the aim of reconstituting a full-length gene via intramolecular recombination. 14 Infection with wild-type AAV does result in long-term persistence of neutralizing anti-AAV antibodies, with AAV2 showing the highest seroprevalence, with antibody titers varying geographically and with age. Systemic delivery of AAV vectors in the presence of such neutralizing antibodies fails to result in clinically relevant transduction in most cases.
Therefore, most study subjects that are positive for neutralizing antibodies are excluded. Moreover, AAV gene therapy leads to high titer, multi-serotype cross-reactive, neutralizing AAV antibodies limiting vector re-administration that might be eventually needed if transgene expression is lost. Multiple strategies have been investigated to overcome neutralizing anti-AAV antibodies, which include the use of alternative serotypes, plasmapheresis, pharmacological modulation of B and T cell function, and treatment with IgG degrading endopeptidases such as imlifidase. 15
Although morphologically similar, hepatocytes differ in their metabolic function along the porto-central axis. These differences may explain why in non-human primates AAV transduction is limited mainly to the periportal region. It is not known whether the same holds true for humans, but should this be the case then transduction bias may influence outcomes of liver directed gene therapy for in-born errors of metabolism. Changes in liver microarchitecture induced by liver diseases resulting in fibrosis may also hamper AAV gene transfer. Therefore, diseases affecting the liver and its cellular composition may significantly affect AAV-mediated hepatocyte transduction in humans.
LIVER-DIRECTED, AAV GENE THERAPY: CLINICAL TRIALS IN HEMOPHILIA
Valoctocogene roxaparvovec (Roctavian), an AAV5-based gene therapy for treatment of adults with severe hemophilia A, is the world's first liver targeted gene therapy to be approved. Market approval for other hemophilia gene therapy approaches beckons on the back of decades of research and clinical data that suggest superiority over standard-of-care treatment. These gene therapy treatments herald a new era in the treatment of congenital bleeding disorders. Hemophilia A and B are caused by a dysfunction or deficiency of coagulation Factor (F) VIII or IX, respectively.
Despite the genetic and biochemical differences, these disorders are clinically indistinguishable with the symptoms dependent on the residual plasma coagulation factor levels. Patients who produce <1% (<1 IU/dL) of normal level of FVIII or FIX have a severe phenotype that is characterized by recurrent spontaneous musculoskeletal, soft tissue, and other life-threatening bleeds such as intracranial hemorrhage, as well as excessive bleeding during and following surgery or trauma. 16 Repeated episodes of intra-articular bleeding cause severe, progressive, destructive arthropathy with deformity leading to complete loss of joint function and attendant disability.
Patients with moderate hemophilia have residual clotting factor levels of 1–5% of normal associated with infrequent spontaneous bleeds but are still at risk from trauma-induced bleeds. In patients with mild hemophilia (clotting factor levels of 5–50%), bleeding is usually restricted to traumatic events.
The hemophilias have always been considered good candidates for gene therapy because their clinical manifestations are due to lack of a single protein that circulates in minute amounts in the blood stream. Years of clinical experience and natural history studies show that a small increase in circulating levels of the deficient clotting factor to ≥5% of normal significantly modifies the bleeding diathesis. Thus, the therapeutic goal for gene therapy of hemophilia is modest in comparison to the majority of monogenetic disorders. Further, tight regulation of transgene expression is not necessary since a wide range of FIX or FVIII is expected to be beneficial and nontoxic.
Early AAV-mediated gene therapy trials focused on hemophilia B in part because the FIX cDNA is relatively small (1.5 kb) and its expression pathway is significantly less complex than that of FVIII. The first study to show circulating therapeutic levels of FIX following AAV2 vector-mediated gene transfer was pioneered by Katherine High's group at CHOP, Philadelphia, PA (NCT00515710). This study entailed the administration of AAV2 vectors into the hepatic artery, following selective catheterization, to mediate efficient gene transfer of the liver of severe hemophilia B patients.
Expression of transgenic FIX was short-lived after gene therapy, being eliminated by an unexpected immunological response toward the transduced cells, a phenomenon not observed in animal models, including nonhuman primates exposed to 10-fold higher doses. 17
The first report of sustained FIX expression at therapeutic levels in severe hemophilia B patients came from our group in a trial sponsored by St. Jude Children's Research Hospital and University College, London (UCL NCT00979238). 18,19 The key aspect that contributed to the success of this study was the use of vector pseudotyped with AAV serotype 8 capsid. This offered two advantages over AAV2 vectors: (1) remarkable tropism of AAV8 for liver leading to efficient transduction of hepatocytes following administration of the vector in the peripheral circulation, thus facilitating a simple non-invasive route of vector administration that dispensed with the need for invasive procedures for catheterization of the hepatic artery in patients with a bleeding diathesis 20,21 and (2) lower seroprevalence of AAV8 in humans (∼25% compared with over 70% for AAV2), affording the luxury of excluding a smaller proportion of patients with neutralizing anti-AAV8 antibodies from the clinical trial. 22
A dose-dependent increase in FIX levels was observed in the 10 subjects sequentially treated at a dose of either 2e11, 6e11, or 2e12 vg/kg. Stable, long-term FIX expression at 1–8% of normal was established in all ten subjects. Asymptomatic, transient elevation of ALT accompanied by a fall in steady-state FIX was observed in four out of the six subjects recruited to the 2e12 vg/kg dose level between 6 and 10 weeks after gene transfer. Each of the four patients with raised liver enzymes received a short tapering course of prednisolone, leading to normalization of ALT and AST levels with preservation of FIX transgene expression in the range of 2–5% of normal levels. Cessation of corticosteroids was not associated with a subsequent rise in ALT levels. 18,19
Transgenic FIX activity levels have remained stable in all 10 subjects over a period of follow-up that extends 10 years, associated with a significant reduction in the annual FIX concentrate usage and frequency of spontaneous bleeding. Importantly, the quality of life of these individuals has improved dramatically as they are now able to undertake activities that previously provoked bleeds without suffering from bleeding episodes. No late toxicity was observed, and neutralizing antibodies to FIX were not detected in any patient. Ongoing monitoring of the liver does not show any evidence of long-lasting damage. 23
The gene therapy studies that followed (Table 1) differed in their selection of AAV capsid, configuration of the vector genome, design of the expression cassette, and method of vectors manufacture (mammalian system vs. insect cell-baculovirus method). In general, higher vector doses were required for therapeutic transgene expression when the vector preparations were made using the insect cell-baculovirus method. For instance AAV5 serotype pseudotyped vectors (AMT-060; UniQure Therapeutics) made using the insect cell-baculovirus method but containing the same FIX gene cassette as that used in the St Jude-UCL trial resulted in mean FIX activity levels of 6.9% despite using a log higher vector dose of 2e13 vg/kg. 24
Raised ALT levels were observed in 3 out of 10 patients recruited to AMT-060, requiring treatment with corticosteroids. In three other Phase 1/2 clinical trials (DTX101 [NCT02618915], AAV8-hFIX19 [NCT01620801], and BAX 335 [NCT01687608]), transaminitis leads to loss of transgenic FIX despite administration of prednisolone at an AAV dose of 1e12 vg/kg or higher. 25,26 This suggests that corticosteroids may not work in all circumstances.
The next generation of hemophilia B trials used a FIX cDNA containing a naturally occurring gain-of-function mutation in humans characterized by leucine (R338L) instead of arginine at position 338 in the catalytic domain (FIX Padua mutation). This mutation enhances FIX activity (FIX:C) by five to eightfold for a given amount of FIX antigen. Therefore, a small increase in plasma FIX antigen levels would lead to a substantial increase in plasma FIX clotting activity. In clinic, the FIX Padua mutation has made it possible to achieve plasma FIX activity levels in the mild range (5–50% of normal) on a consistent basis and with some AAV vector formulation even in the normal and supraphysiological range (∼250% of normal), a feat thought to be impossible almost a decade ago.
Vector-mediated transaminitis remains a problem despite use of prophylactic corticosteroid in some of these studies. 27 –31 AMT-061 (NCT03489291, Etranacogene dezaparvovec,) is the front runner to market for gene therapy of hemophilia B. Data from a 54 patient, open-label Phase 3 study (HOPE-B) is currently under regulatory review. At 1.5 years after a 2e13 vg/kg dose of AMT-61, mean plasma FIX activity levels were 36.9% of normal, resulting in a 64% reduction in bleeding rates and a 97% drop in annual FIX usage. Notably, these benefits were seen regardless of pre-existing antibodies against AAV5. 27
Gene therapy of hemophilia A has been hindered by the limited packaging capacity of AAV vectors (4,680 kb) and the poor expression profile of FVIII. These FVIII cDNA is ∼7 kb, which is beyond the packaging capacity of AAV. This obstacle was overcome by removing the coding sequence for the B domain of FVIII as it is not required for co-factor activity. In additionally, Factor VIII expression was improved 10-fold by re-organization of the wild-type cDNA of human FVIII according to the codon usage of highly expressed human genes. 13
BioMarin who licensed this construct commenced the first clinical trial for hemophilia A using AAV5 pseudotyped vectors made using the Baculovirus-insect cell manufacturing method. This vector (AAV5-hFVIII-SQ, BMN 270) was initially tested in a Phase I trial (NCT02576795, BMN 270-201). 32,33 This Phase I trial supported the conduct of the largest Phase III (NCT03370913, GENEr8-1) gene therapy trial in hemophilia patients. In this single-arm, open-label, multicenter, phase III trial, BioMarin assessed BMN 270 in 134 severe hemophilia A patients at a dose of 6e13 vg/kg.
The safety and efficacy results have recently been published 34 and show mean FVIII activity levels (using a chromogenic assay) between 49 and 52 weeks of 41.9 IU/dL in the 132 human participants evaluated. This was associated with 98.6% decline in annualized bleed rate and 83.8% reduction in FVIII concentrate usage. Transaminitis was observed in 85.8% of the participants and was managed with transient immunosuppression. None of the participants developed inhibitors to FVIII, malignancy, or thromboembolic events. Longer follow-up of the seven patients treated at the 6e13 vg/kg dose level in the BMN 270-210 Phase I trial shows a gradual decline in FVIII activity levels over time from 64% of normal at 1 year to a mean FVIII activity of 11.6% at 5 years (unpublished report).
This contrasts with the data in hemophilia B gene therapy trials. Nevertheless, BMN 270 has received a favorable opinion from the European Medicines Agency (EMA) and will likely be the first gene therapy for treatment for hemophilia A to get market approval.
Several other Hemophilia A gene therapy trials using different capsids, promoters, doses, and manufacturing process are currently in various different phases of clinical trials. 35 –39 Others have also observed a decline in FVIII activity levels over time. For instance, in the Pfizer/Sangamo Therapeutics trial (SB-525, NCT04370054, Giroctocogene fitelparvovec), mean FVIII activity level (chromogenic assay) in the 3e13 vg/kg dose cohort (N = 5) at 1 year was ∼43%, dropping to 25.7% in the second year. Transaminitis was detected in 5 out of the 11 participants enrolled in the study, including 3 participants in 3e13 vg/kg cohort. 37
Thus, the differing AAV hemophilia gene therapy trials offer substantial choices to the hemophilia community and provide an opportunity for patients with pre-existing antibodies to one serotype to explore treatment with alternative serotypes. The key differences between hemophilia A and B gene therapy trials are: (1) higher vector doses appear to be required for hemophilia A when compared with hemophilia B and (2) FVIII transgenic activity appears to be declining over time in contrast to stable levels of FIX observed in hemophilia B subjects.
This may be a reflection of the fact that an oversized transgene is used in most hemophilia A studies, which may influence durability. The decline in transgene expression in the absence of transaminitis appears to be most pronounced in patients with the highest level of FVIII expression. Potential explanations for this ongoing decline in FVIII activity remain unclear but may include: (1) loss of the episomally retained, oversized, FVIII AAV transgene from the transduced hepatocytes and (2) silencing of FVIII transgene.
LIVER-DIRECTED, AAV GENE THERAPY CLINICAL TRIALS IN OTHER MONOGENETIC DISORDERS
The hemophilia gene therapy trials have established a platform for the development of other liver-directed gene therapy approaches for other monogenetic disorders, including OTC deficiency, phenylketonuria, Glycogen storage disease 1a (GSD1a), and Crigler-Najjar, mucopolysaccharidosis type 5, Pompe's disease, Fabry's disease and Gaucher's disease, among others (Table 1). The first liver-directed AAV gene therapy trial for an inherited metabolic disorder of the liver was for the treatment of acute intermittent porphyria. 40
Phase I data showed a good safety profile, but no therapeutic benefit consistent with poor gene transfer efficiency. Since then, the pace of advance in novel liver directed gene therapy approaches has been so rapid that data from many new trials are only available from meeting presentations and/or company news releases. Exceptionally, therefore, we are using those sources of information to bring readers of this review the most current available information for two exciting trials in an advanced stage of development, with the understanding that further experience may change our expectations of the safety and efficacy in these studies.
Gene therapy for GSD1a
Glycogen storage disease type 1a (GSD1a) affects ∼6,000 individuals in the Western World and is caused by a defective gene for the enzyme glucose-6-phosphatase (G6Pase). As a consequence, the body is unable to break down glycogen into glucose required for blood glucose regulation. Severely affected infants present in the neonatal period or early childhood with severe hypoglycemia with or without seizures, lactic acidosis, hyperuricemia, and hypertriglyceridemia.
Glycogen and fat accumulate in the liver and kidneys, resulting in hepatomegaly and nephromegaly. Cognitive impairment occurs in children with prolonged periods of hypoglycemia. GSD1a was almost always fatal until 1971, when it was discovered that continuous glucose therapy could prevent hypoglycemia. In 1982, corn starch therapy was introduced as a slow-release form of glucose, enabling children to reach adulthood. No pharmacologic therapies are approved for GSD1a currently.
In a Phase I/II study of DTX401 (NCT03517085), an AAV8-based gene therapy for the treatment of GSD1a designed to mediate stable expression of G6Pase-α following a single intravenous infusion in all nine patients showed a clinical response, with significant reductions in the need for corn starch and improvements in glucose control and other metabolic parameters compared with baseline. A mean 73% reduction in corn starch requirement was observed in Cohort 1 and 2, with four out of the six patients discontinuing daytime corn starch therapy and one patient completely discontinuing corn starch. Patients in Cohort 3 received a reactive steroid treatment as soon as ALT levels began to rise above baseline, at approximately week 4.
This earlier steroid together with decreasing the cornstarch at the beginning of the controlled fasting challenge resulted in meaningful increases (between 23% and 167%) in time to hypoglycemia. These data have supported the initiation of a Phase III study, which began early in 2022 (NCT05139316).
AAV-based gene therapy for OTC deficiency
The OTC is a critical enzyme in the urea cycle required for the conversion of ammonia to urea in the liver. The OTC deficiency is a rare X-linked disorder of the urea cycle that affects ∼10,000 individuals in the Western World. Severely affected males and heterozygous females experience life-threatening elevations of ammonia in blood and brain, leading to irreversible cognitive impairment, coma, and death. The treatment of OTC deficiency combines dietary restrictions aimed at limiting the amount of protein intake to avoid the development of excess ammonia and the stimulation of alternative methods of converting and excreting nitrogen from the body (alternative pathways therapy).
Currently, the only curative approach for OTC is liver transplantation. DTX301 (NCT02991144) is an investigational AAV8 gene therapy designed to deliver stable expression and activity of the OTC gene using a single intravenous infusion in adults with late-onset OTC deficiency. In total, 11 subjects have been dosed over a dose range of 3.4e12–1.7e13 vg/kg with or without steroids. Eight out of eleven subjects had a rise in ALT that was managed with oral corticosteroids. Evidence of clinical response was observed in 7 out of the 11 dosed patients.
Ultragenyx plans to conduct a Phase 3 clinical study (NCT05345171) to evaluate the effect of DTX301 on ammonia and its ability to reduce patients' need for ammonia scavenger medication and a protein-restricted diet. Halting Ornithine Transcarbamylase Deficiency With Recombinant AAV in ChildrEn (HORACE) is a new Phase I/II clinical trial evaluating a synthetic, AAV-LK03 capsid that is better suited to transduction of humans. 41
CHALLENGES AND FUTURE DIRECTION
Safety considerations
Thus far, the risk of liver toxicity accompanied by loss or reduction of transgene expression appears to be the most worrying toxicity associated with liver-targeted delivery of AAV, as described earlier. Transaminitis appears to occur with all AAV capsids regardless of the AAV genome configuration, transgene promoter, or method of manufacture. It is a self-limiting phenomenon that may be controlled with corticosteroids alone or in combination with other immune suppressive agents in some patients.
The precise pathophysiological basis for transaminitis remains unclear, in part because it has not been possible to recapitulate this toxicity in animal models. 38,42 Clinical data suggest that the number of CpG motifs in the transgene may play a role as previously discussed. It remains unclear in that instance, why the transaminitis is limited to the initial period after gene transfer as there have been no reports of late recurrence of transaminitis beyond a 2-year period. Of note, in this respect, is the recent report of a link between wild-type AAV2 infection and an outbreak of acute hepatitis in children who were co-infected with adenovirus, or less commonly herpes virus HHV6. It is unclear whether AAV2 is pathogenic in these children or just a useful persistent biomarker of recent adenoviral infection. 39
As expected, all subjects in these trials develop long-lasting AAV capsid-specific humoral immunity. Although the rise in anti-AAV IgG does not have direct clinical consequences, its persistence at high titers precludes subsequent successful gene transfer with a vector of the same serotype, in the event that transgene expression should fall below therapeutic levels.
The risk of insertional mutagenesis following AAV-mediated gene transfer has been judged to be low because proviral DNA is maintained predominantly in an episomal form. This is consistent with the fact that wild-type AAV infection in humans, though common, is not associated with oncogenesis. However, deep sequencing studies show that integration of the AAV genome can occur in the liver. 43,44 Indeed, a recent publication has found wild-type AAV2 genome fragments integrated in the proximity of known proto-oncogenes in a small percentage of human hepatocellular carcinoma (HCC) specimens. 45
However, the pathogenic role of AAV2 in this setting is not certain. In December 2020, HCC was reported in a patient enrolled in the HOPE-B gene therapy trial for hemophilia B. The patient had multiple risk factors for HCC, including a history of hepatitis B and C, evidence of nonalcoholic fatty liver disease, smoking history, family history of cancer, and advanced age. An investigation including whole genome sequencing of the tumor and adjacent tissue showed that Etranacogene dezaparvovec was unlikely to have caused the HCC in this subject.
An increased incidence of HCC has, however, been reported in the mucopolysaccharidoses type VII (MPSVII) mouse model following perinatal gene transfer of AAV potentially through integration and disruption of an imprinted region rich in microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) on mouse chromosome 12. 46 Biomarin reported tumors on necropsy of mice at 52 weeks after administration of 2e14 vg/kg of AAV5-phenylalanine hydroxylase (PAH) gene therapy in a tumor prone mouse model that harbored two germ line mutations: one to eliminate the PAH gene and a second to render the mice immunodeficient. Further investigations are ongoing.
Other studies in mice have failed to recapitulate this finding and collectively the available data in mice as well as larger animal models suggest that AAV has a relatively low risk of tumorigenesis. 47
Genotoxicity of AAV may potentially be higher for monogenetic disorders with increased cancer risk (e.g., Fabry disease, Gaucher's disease, Wilson's disease, GSD1, citrulline deficiency). In some of these disorders, there is a higher risk of HCC due to potentially three mechanisms: (1) direct toxicity of accumulating metabolites, (2) metabolites channeling, and (3) mitochondrial dysfunction predisposing to inflammation. In the context of these disorders, the additional risk of insertion mutagenesis and inflammation induced by AAV vector remains a concern. However, correction of metabolic defect in at least a subset of liver cells is also expected to improve the metabolic defect, thus reducing the burden of toxic metabolites associated with increased risk of cancer.
In summary, safety consideration remains paramount and will require careful long-term monitoring of patients, likely beyond the 5 years of follow-up mandated by the FDA.
Stability of transgene expression after gene transfer
Absolute duration of gene expression in transduced liver following intravenous AAV administration is not known. Although FIX expression following AAV gene transfer of the liver has remained stable beyond 10 years in Hemophilia B patients, this does not appear to be the case in patients with Hemophilia A. This may in part be due to the fact that in Hemophilia A an oversized genome is used and is packaged in a truncated form. In addition, AAV doses required for efficient gene transfer and the manufacturing process may influence stability of expression.
Affordability of AAV gene therapy
It is likely that gene therapy will command a high price, at least initially, to recoup the development cost. However, successful gene therapy offers the advantage of continuous endogenous expression of transgenic protein with potentially continuous, 24/7 amelioration of the disease, thereby reducing morbidities and the need for frequent medical interventions while improving quality of life. Thus, gene therapy has the potential to yield significant savings for the health care system and society in general but may still prove to be unaffordable for patients living in developing or emerging economies.
Pre-existing neutralizing anti-AAV antibody
As previously discussed, between 20% and 70% of patients have pre-existing neutralizing anti-AAV antibody (NAbs) to specific AAV serotypes, which can block efficient gene transfer. These patients are currently excluded form gene therapy trials, but NAbs represent a major limitation to the broader applicability of gene therapy. One strategy to overcome NAbs that works well in animal models is to switch AAV serotype 20 but may not be applicable in humans due to cross-reactivity of NAbs. Alternatively, NAbs could be overcome by using immunosuppression or plasmapheresis or by simply increasing vector dose or adding empty capsids. 48
Scale-up of vector production
Continued progression toward flexible, scalable production and purification methodologies is ongoing to support the commercialization of AAV bio-therapeutics. The most widely used method for the generation of AAV entails the transient transfection of HEK-293 cells with plasmids encoding the necessary vector, helper, and packaging genes. This method is cumbersome, but progress has been made on market ready methods of production that can also support Phase III trials. 49 Another method being used by several Bio-Pharma companies because of its scalability is one based on baculovirus grown in SF9 insect cells. 50 However, infectivity of AAV made using the baculovirus system is low due in part to lower levels of VP1 incorporation into the AAV capsid. Attention is currently focused on the downstream purification process so that the purity of clinical grade AAV preparation can be improved.
CONCLUSION
The success of gene therapy for the hemophilias with long-lasting clinical benefits has catalyzed a growing interest in gene therapy for other disorders affecting the liver, including the many inborn errors of metabolism with no or limited treatment opportunities. A number of these liver targeted gene therapy products are advancing through clinical development. Several obstacles still remain, but the field is cautiously moving forward through the development of innovative approaches.
Footnotes
AUTHOR DISCLOSURE
(1) Advisor to Freeline (ACN, JM); (2) Founder of Freeline Therapeutics and Non-Executive Director of Freeline Therapeutics (ACN); (3) Inventor on patents licenced to Freeline Therapeutics and BioMarin (ACN, JM); (4) Equity holder in Freeline Therapeutics (ACN, RS, JM); (5) Employee of Freeline Therapeutics (RS).
FUNDING INFORMATION
No funding was received for this review.