3.4. Emerging Advanced Therapeutic Medicinal Products: Gene therapy for haemophilia
Gene replacement therapies represent a frequently submitted category of therapeutic for orphan designation [19]. In this form of ATMPs, a functional copy of a defective gene in the target condition, which is either absent or expressed as a non-functional protein is substituted by a fully functional gene which is inserted using a viral vector thereby offering a highly effective means for overcoming diseases, such as haemophilia. The gene correction in haemophilia is well suited because it is associated with a well understood mutated gene sequence which can be substituted by a corrected DNA sequence leading to the endogenous production of a functional factor VIII or IX [5]. This would therefore alleviate the need for exogenous sources of the defective clotting factors.
An important characteristic of haemophilia treatment is that there is no need to normalize circulating clotting factor levels to obtain a therapeutic effect since a slight increase in plasma clotting factor levels (above 1%) is sufficient to decrease the risk of morbidity and mortality. As a consequence, a small increase in clotting factor levels by gene therapy can greatly improve the clinical symptoms. In addition, therapeutic efficacy of GTMPs can be determined by clear-cut clinical endpoints such as circulating clotting factor levels and bleeding frequency [20]. Thus, gene therapy could provide a continuous source of clotting factor from a single treatment.
Initial clinical gene therapy studies using integrating retroviral, adenoviral, and non-viral (ex vivo ) approaches were associated with transient low-level factor expression [21-22].
This led to a shift toward usage of ‘non-integrating’ recombinant adeno-associated viral (AAV) vectors. AAV vectors are derived from wild-type AAV [23], a member of the parvovirus family. Wild-type AAV is non-pathogenic, weakly immunogenic, and replication-deficient, requiring a helper virus for replication. AAV vectors can deliver a therapeutic transgene cassette up to 5 kb into both dividing and nondividing cells. The DNA sequences carried by recombinant AAV vectors are stabilised predominantly in an episomal form so that long-term expression can occur only with delivery into long-lived, post mitotic cell types; the vector DNA integrates at a very low frequency and is typically lost from replicating cells. The recombinant vector has tropism for a range of target tissues including the liver, cell types in the retina and the central nervous system, skeletal muscle, and cardiac muscle, among others. AAV vectors are the most frequently used viral vectors for gene therapy for Haemophilia (90%), followed by lentiviral vectors (10%).
Table 4 provides a detailed list of GTMPs for Haemophilia, which were granted an orphan designation by European Commission.
FVIII and FIX synthesis primarily takes place in the liver, from where the proteins can easily enter the bloodstream. This makes hepatocytes a suitable target for gene therapy in haemophilia.
Over the years, hepatic in vivo gene transfer using adeno-associated viral (AAV) vectors has shown the best success in preclinical and clinical studies, with several clinical studies for both hemophilia A and B enrolling patients for phase 3 trials. We analyzed the studies present in the Clinical trial.gov and in the EU Clinical Trials Register repositories [24-25].
Many AAV vectors have progressed into clinical studies for both haemophilia A and B. The ClinicalTrials.gov database presently lists 29 active gene therapy clinical trials for haemophilia, 12 clinical trials to evaluate different AAV-based GTMPs for haemophilia B and 11 trials to evaluate AAV-FVIII gene therapy for haemophilia A (Table 5), with some overlap, as the same vector is being evaluated in both phase 1/2 and phase 3 studies.
Of the ongoing Haemophilia A and B AAV clinical trials, there are remarkable successes. The trial sponsored by St. Jude Children’s Research Hospital haemophilia B patients, using an scAAV2/8-LP1-hFIXco vector, was the first long-term success story for haemophilia gene therapy [26].
However, an important limitation to successful AAV-base gene therapy is the pre-existing humoral capsid immunity. Immune recognition by cytotoxic CD8+ T cells or antibody responses to the vector capsid, the transgene product, or both can compromise the therapeutic expression of the transgene. Wild-type AAV infection occurs during childhood, and thus patients may develop neutralising antibiotics (nAb) that prevent gene transfer with AAV vectors. Cross-reactivity with multiple AAV serotypes has also been reported. As a result, many patients have been excluded from recent studies. The main toxicity observed in AAV-based clinical studies has been dose-related elevation of liver transaminase following vector infusion. Within some studies, this coincides with the demonstration of cell-mediated AAV capsid immunity. Most studies have used either early intervention or prophylaxis with corticosteroids in order to protect transduced hepatocytes. Although the majority of episodes have been managed effectively with this approach, some episodes have been associated with partial or complete loss of transgene expression, despite intervention [26].
Due to the pre-existing AAV humoral immunity and the potential loss of vector transduction with time, there is for the need of other viral and non-viral-based vectors for transgene delivery. Lentiviral vectors may complement the therapeutic reach of AAV vectors due to lower incidence of pre-existing humoral immunity and greater packaging capacity. Pre-clinical studies using in-vivo or ex-vivo stem cell (haematopoietic or induced pluripotent) transduction or blood outgrowth endothelial cells have been recently reviewed [27-28]. Two phase 1 studies are registered with plans to evaluate ex-vivo lentiviral stem cell transduction for FVIII (NCT03818763) or FVIII/FIX (NCT03217032/NCT03961243).