RESUMEN

BACKGROUND: Gene therapy is designed to provide people with hemophilia B with a steady and elevated factor (F)IX activity, thereby strengthening protection and relieving the burden of frequent replacement therapy infusions. The European Medicines Agency has approved gene therapy for the severe and moderately severe forms of hemophilia B that uses the FIX-Padua variant (etranacogene dezaparvovec). OBJECTIVES: The aim was to provide a document dedicated to hemophilia B gene therapy and give a comprehensive overview of the topic. METHODS: An Italian group of experts in hemophilia carried out a narrative review of the literature and discussed during a virtual meeting several key aspects of the delivery of this treatment in Italy. The discussion covered the organizational model, the role of the multidisciplinary team, the laboratory surveillance, and the patient's journey, from the follow-up to the identification of safety issues and outcome measures. RESULTS: This article highlights the need to follow the Hub and Spoke organizational model and sheds light on the role of each professional figure within the multidisciplinary teams to favor patient engagement, management, and retention. Moreover, this article stresses the need to perform laboratory tests for patient screening and follow-up and proposes a checklist to help patient identification. Finally, the needs of Italian hemophilia centers have been considered to ensure an efficient implementation of the care delivery model. CONCLUSION: It is crucial to ensure that centers are appropriately organized, equipped, and trained to adequately select patients, deliver the gene therapy, and perform follow-up.

RESUMEN

BACKGROUND: The X-linked bleeding disorder hemophilia B, caused by mutation(s) in the coagulation factor (F)IX gene, leads to partial or total loss of its function, requiring lifelong FIX replacement therapy. Although new recombinant FIX (rIX) therapeutics like albumin fusion proteins (rIX-FP) enable longer plasma half-life and thus less frequent administration, the complexity of intravenous (i.v.) injection remains. OBJECTIVES: The study aimed to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic profile with beneficial characteristics for subcutaneous (s.c.) administration. METHODS: Two rIX-FP variants, R338L ("Padua variant") and R338L/E410K, were characterized in vitro. Pharmacokinetic profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after i.v. and s.c. administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after i.v. and s.c. administration (dosing based on activity) in a tail clip bleeding model. A marketed wild-type (WT) rIX-FP product served as the comparator. RESULTS: Both rIX-FP variants showed a 4- to 5-fold increase in specific activity in vitro compared with rIX(WT)-FP, while FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared with rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after i.v. and s.c. administration and demonstrated comparable efficacy with rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring ∼4 times lower protein amount. CONCLUSION: rIX(R338L)-FP was shown to be a promising candidate for s.c. administration, exhibiting increased specific activity combined with higher activity-based exposure and indicating efficacy at a lower protein dose.

Asunto(s)

Factor IX , Hemofilia B , Hemorragia , Proteínas Recombinantes de Fusión , Animales , Factor IX/farmacocinética , Factor IX/genética , Factor IX/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/administración & dosificación , Inyecciones Subcutáneas , Hemofilia B/tratamiento farmacológico , Hemofilia B/sangre , Humanos , Modelos Animales de Enfermedad , Ratones Noqueados , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Semivida , Mutación , Coagulantes/farmacocinética , Coagulantes/administración & dosificación , Masculino

RESUMEN

The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD ) 0·5 nM] over wild-type FIX-HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.

Asunto(s)

Coagulación Sanguínea/efectos de los fármacos , Factor IX/farmacología , Proteínas Recombinantes de Fusión/farmacología , Albúmina Sérica Humana/farmacología , Animales , Factor IX/genética , Femenino , Semivida , Hemofilia B/sangre , Hemofilia B/tratamiento farmacológico , Humanos , Masculino , Ratones Transgénicos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica Humana/genética

RESUMEN

Targeting the coagulation factor IX (FIX) expression in platelets has been shown to be effective in ameliorating bleeding in hemophilia B (HB) mice. To improve the therapeutic effects and evaluate the safety of this gene therapy strategy, we generated a transgenic mouse model on an HB background with FIX Padua target expressed in platelets. The transgenic mice exhibited stable expression and storage of FIX Padua in platelets. The platelet-stored FIX Padua could be released with the activation of platelets, and the proportion of platelet-stored FIX Padua in whole blood was the same as that of platelet-stored wild-type human FIX. The platelet-derived FIX Padua showed substantially increased specific activity compared with wild-type FIX. Reduced bleeding volume in the FIX Padua transgenic mice demonstrated that bleeding in the mice was improved. Levels of thrombin-antithrombin complex, fibrinogen, D-Dimer, and blood cell counts were normal in the transgenic mice, suggesting that thrombotic risk was not increased in this mouse model. However, the leakage and failure to overcome the presence of inhibitor to wild-type FIX is also observed with FIX Padua, as expected. Taken together, our results support the conclusion that targeting FIX Padua expression in platelets may be an effective and safe gene therapy strategy for HB, and could provide an ideal model to evaluate the safety of platelet-targeted gene therapy for treating hemophilia.

Asunto(s)

Factor IX , Hemofilia B , Animales , Plaquetas , Factor IX/genética , Hemofilia B/genética , Hemofilia B/terapia , Ratones , Ratones Transgénicos

RESUMEN

Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other engineered proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease.

RESUMEN

Haemophilia is an attractive disease target for gene therapy that fostered the development of the field at large. The delivery of the clotting factor genes into the patients' cells could be accomplished using different types of gene delivery vehicles or vectors. Adeno-associated viral vectors (AAV) and lentiviral vectors represent some of the most promising gene delivery technologies that allow for a relatively efficient delivery of the therapeutic FVIII and FIX transgenes into the relevant target cells. To reduce the risks associated with insertional mutagenesis due to random vector integration, gene-editing approaches have also been considered based primarily on zinc finger nuclease (ZFN) and CRISPR/Cas. However, comprehensive analysis of off-target effects is still required. It is particularly encouraging that relatively stable therapeutic FVIII or FIX expression levels were reached in severe haemophilia patients in recent clinical trials after liver-directed AAV gene therapy. This success could be ascribed in part to improvements in vector design. In particular, clotting factor levels could be increased by codon optimization of coagulation factor transgenes. Alternatively, incorporation of a hyperactive gain-of-function R338L mutation (FIX Padua) in the FIX gene improved the overall efficacy. However, some patients still show transient liver toxicity, especially at high vector doses, possibly due to inflammatory immune responses, requiring the need for transient immunosuppression. The exact immune mechanisms are not fully understood, but may at least in some patients involve an AAV-capsid specific T cell response. Moreover, there is a need to identify the key factors that contribute to the interpatient variability in therapeutic efficacy and safety after gene therapy.

Asunto(s)

Terapia Genética/métodos , Hemofilia A/genética , Hemofilia A/terapia , Animales , Factor IX/genética , Factor VIII/genética , Vectores Genéticos/genética , Humanos

RESUMEN

Hemophilia A and B are congenital, X-linked bleeding disorders caused by mutations in the genes encoding for the blood clotting factor VIII (FVIII) or factor IX (FIX), respectively. Since the beginning of gene therapy, hemophilia has been considered an attractive disease target that served as a trailblazer for the field at large. Different technologies have been explored to efficiently and safely deliver the therapeutic FVIII and FIX genes into the patients' cells. Currently, the most promising vectors for hemophilia gene therapy are adeno-associated viral vectors (AAVs) and lentiviral vectors. More recently, gene editing approaches based on designer nucleases or CRISPR/Cas, have also been considered to minimize risks associated with random vector integration and insertional mutagenesis though off-target issues would have to be carefully and comprehensively assessed. In the past two decades, several phase 1 hemophilia gene therapy clinical trials have been initiated with varying success. In particular, the early gene therapy clinical trials in hemophilia B patients based on AAV showed either transient or subtherapeutic clotting factor expression levels. This could be ascribed, at least in part, to suboptimal vector design and/or inadvertent immune consequences triggering hepatic inflammation. Hence, there was an unmet need to further increase vector safety and efficacy in future trials, preferably by using lower vector doses. It is particularly encouraging that sustained therapeutic FVIII and FIX expression levels have recently been attained after gene therapy in patients with severe hemophilia paving the way towards pivotal trials and commercialization. Nevertheless, transient liver toxicity still occurs and the use of transient immunosuppression was still required to curtail inadvertent immune responses, especially at high vector doses. To further boost clotting factor expression levels, codon-usage optimized synthetic FVIII or FIX transgenes have been employed. Alternatively, we and others have shown that the incorporation of hyperactive gain-of-function R338L mutation in the FIX gene substantially increased the overall efficacy. It is inevitable that the continued improvements in vector engineering and new insights in the vector-patient interactions will further benefit the development of a safe and effective cure for hemophilia A and B.

Asunto(s)

Terapia Genética/tendencias , Vectores Genéticos/uso terapéutico , Hemofilia A/terapia , Hemofilia B/terapia , Sistemas CRISPR-Cas , Factor IX/genética , Factor IX/uso terapéutico , Factor VIII/genética , Factor VIII/uso terapéutico , Edición Génica/tendencias , Vectores Genéticos/genética , Hemofilia A/genética , Hemofilia B/genética , Humanos , Lentivirus