Haemophilia is an X-linked bleeding disorder characterized by spontaneous or provoked, often uncontrollable, bleeding into muscles, joints, and other soft tissues, resulting in significant pain, swelling and permanent damage if not treated. Haemophilia is caused by a genetic defect in specific proteins in the blood called clotting factors. Approximately 80% of affected children are born deficient in factor VIII (haemophilia A) and 20% are deficient in factor IX (haemophilia B). As hemophilia is an X-linked recessive disease, haemophilia occurs almost exclusively in males, but can also occur in some carrier females owing to variances in inactivation of X chromosomes, or females with homozygosity or Turner syndrome.
Gene therapy either by transferring a normal copy or correcting an abnormal gene has the potential of providing continuous, endogenous production of a specific protein and has been an attractive goal for 30 years. Haemophilia is an optimal candidate for gene therapy as a single gene mutation is responsible for the disease phenotype, factor expression is easy to measure by standard clinical assays, the wide range of normal factor levels does not demand tight regulation of expression, and even low factor levels are effective in reducing bleeding. It was first shown in 1989 that human factor IX could be synthesized and secreted into the circulation of experimental animals after transplantation of genetically modified human fibroblasts by using a human factor IX cDNA containing retrovirus. Subsequently, several translational methods have been developed for the clinical application of gene therapy in haemophilia, however most of these only resulted in transient benefits. Until 2011, a new adeno-associated virus (AAV)-derived vector for factor IX gene transduction was developed, which could directly be given to a patient by a peripheral vein infusion. Since then, various AAV and lentiviral vector (LV)-based gene therapy approaches have been developed and are currently in clinical investigation.
AAV-derived vector-mediated transfer of a normal FVIII or FIX gene is most advanced in clinical development. The first human, liver-directed AAV-mediated gene transfer trial for haemophilia used intrahepatic artery delivery of AAV-FIX vector and indicated safety and efficacy, but factor expression failed to last. Immune reactivity against AAV capsid antigens leading to the destruction of transduced liver cells and loss of factor expression was discovered as a major obstacle. Later, the first successful AAV-FIX trial used peripheral vein administration. Immune-mediated, vector-induced transaminitis occurred at higher vector doses, but was a solitary event about 4 to 12 weeks after vector infusion that resolved with corticosteroid therapy. Trial participants have had stable FIX levels of 2%-7% for 8 years with no long-term toxicity. Recent phase I/II trials have used improved vectors and achieved levels of FIX and FVIII expression in the normal or mild haemophilia range associated with a significant reduction in bleeding rate and factor use. No new safety concerns were observed. Multiple phase I/II and III trials are ongoing.
The single observed safety finding in AAV-mediated gene therapy has been immunotoxicity in the form of transient subclinical liver inflammation. Concerns of insertional mutagenesis at integration sites, gene silencing or loss of expression over time, overexpression or ectopic or dysregulated transgene expression, and horizontal and vertical transmission have remained theoretical but are important aspects of the ongoing investigation and long-term monitoring. Although FIX expression in the St. Jude/UCL trial has been stable beyond 8 years, the AAV5-FVIII trial by Biomarin reported a decline of FVIII levels by 43% within year 2 and further 10% over year 3 after infusion. Finally, questions related to circumvention of AAV-NAb-mediated immune reactivity during the first or subsequent vector administration and efficacy of AAV-mediated gene transfer in children with a rapidly growing liver remain unresolved.
HIV-derived (LV) are replication-defective hybrid enveloped viral particles made by a minimal set of capsid and enzymatic proteins of the parental virus, a surface protein of an unrelated virus (known as pseudotype) and a recombinant viral genome, comprising the cis-acting viral genome elements necessary for gene transfer and a transgene expression cassette of choice. The vesicular stomatitis virus surface glycoprotein (VSV.G) is most often used to pseudotype LV, as it confers high stability and wide tropism mediated by the low-density lipoprotein receptor (LDL-R). Upon iv administration, both the filtering action of liver sinusoids and the engagement of LDL-R on hepatocytes by VSV.G on LV particles provide for substantial liver tropism. LV integrates into the target cell chromatin and is maintained as the cells duplicate their genome, a potential advantage for establishing long-term expression especially in pediatric patients, in which the liver undergoes substantial growth.
Figure 1. Schematic representation of third-generation LV designed for liver-directed gene therapy. (Cantore A, Naldini L. 2020)
LVs intended for liver-directed gene therapy are designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. Systemic administration of such LV provided stable multi-year transgene expression in the liver of dogs and mice. Recently, the vector surface has been modified by changing the protein composition of the producer cell plasma membrane to reduce the immunogenicity of LV particles. In order to apply this strategy to haemophilia A, LVs with human B-domain deleted FVIII have been generated. Persistent FVIII expression was observed post-LV treatment of newborn haemophilia A mice by the 6-month study period despite a greater than 10-fold increase in body weight of the treated animals, with coFVIII and coFVIIIXTEN conferring several-fold improvement on plasma FVIII levels. Administration of coFVIII-or coFVIIIXTEN-expressing MHC-free/CD47hi LV to NHP resulted in FVIII levels in the therapeutic range up to 100% in treated animals, under immune suppression.
As a new therapeutic modality, DNA delivery to produce proteins in human bioreactors represents a huge paradigm shift in medicine. Gene-based delivery technologies have made great progress over the past 30 years to bring persons with haemophilias A and B into the subnormal to normal ranges of clotting factor activity. But many questions regarding safety and efficacy have not been adequately answered, and will not be addressed before licensure. QVirusTM Platform, a division of Creative Biogene, can offer a series of AAV and lentivirus services to accelerate your haemophilia gene therapy projects. If you have any special requirements, please feel free to contact us.
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