A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels.

ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Long-term safety and efficacy outcomes of valoctocogene roxaparvovec gene transfer up to 6 years post-treatment.

INTRODUCTION: Valoctocogene roxaparvovec uses an adeno-associated virus serotype 5 (AAV5) vector to transfer a factor VIII (FVIII) coding sequence to individuals with severe haemophilia A, providing bleeding protection. AIM: To assess safety and efficacy of valoctocogene roxaparvovec 5-6 years post-treatment. METHODS: In a phase 1/2 trial, adult male participants with severe haemophilia A (FVIII ≤1 IU/dL) without FVIII inhibitors or anti-AAV5 antibodies received valoctocogene roxaparvovec and were followed for 6 (6 × 1013 vg/kg; n = 7) and 5 (4 × 1013 vg/kg; n = 6) years. Safety, including investigation of potential associations between a malignancy and gene therapy, and efficacy are reported. RESULTS: No new treatment-related safety signals emerged. During year 6, a participant in the 6 × 1013  vg/kg cohort was diagnosed with grade 2 parotid gland acinar cell carcinoma; definitive treatment was uncomplicated parotidectomy with lymph node dissection. Target enrichment sequencing of tumour and adjacent healthy tissue revealed low vector integration (8.25 × 10-5 per diploid cell). Integrations were not elevated in tumour samples, no insertions appeared to drive tumorigenesis, and no clonal expansion of integration-containing cells occurred. During all follow-ups, >90% decreases from baseline in annualised treated bleeds and FVIII infusion rates were maintained. At the end of years 6 and 5, mean FVIII activity (chromogenic assay) was 9.8 IU/dL (median, 5.6 IU/dL) and 7.6 IU/dL (median, 7.1 IU/dL) for the 6 × 1013 and 4 × 1013 vg/kg cohorts, respectively, representing proportionally smaller year-over-year declines than earlier timepoints. CONCLUSIONS: Valoctocogene roxaparvovec safety and efficacy profiles remain largely unchanged; genomic investigations showed no association with a parotid tumour.

Hemophilia Healing with AAV: Navigating the Frontier of Gene Therapy.

Gene therapy for hemophilia has advanced tremendously after thirty years of continual study and development. Advancements in medical science have facilitated attaining normal levels of Factor VIII (FVIII) or Factor IX (FIX) in individuals with haemophilia, thereby offering the potential for their complete recovery. Despite the notable advancements in various countries, there is significant scope for further enhancement in haemophilia gene therapy. Adeno-associated virus (AAV) currently serves as the primary vehicle for gene therapy in clinical trials targeting haemophilia. Subsequent investigations will prioritize enhancing viral capsid structures, transgene compositions, and promoters to achieve heightened transduction efficacy, diminished immunogenicity, and more predictable therapeutic results. The present study indicates that whereas animal models have transduction efficiency that is over 100% high, human hepatocytes are unable to express clotting factors and transduction efficiency to comparable levels. According to the current study, achieving high transduction efficiency and high levels of clotting factor expression in human hepatocytes is still insufficient. It is also crucial to reduce the risk of cellular stress caused by protein overload. Despite encountering various hurdles, the field of haemophilia gene therapy holds promise for the future. As technology continues to advance and mature, it is anticipated that a personalized therapeutic approach will be developed to cure haemophilia effectively.

An intronic RNA element modulates Factor VIII exon-16 splicing.

Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure-function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3'-end of intron-15 (TWJ-3-15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3-15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3-15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing.

Giroctocogene fitelparvovec gene therapy for severe hemophilia A: 104-week analysis of the phase 1/2 Alta study.

ABSTRACT: Patients with hemophilia A require exogenous factor VIII (FVIII) or nonfactor hemostatic agents to prevent spontaneous bleeding events. Adeno-associated virus (AAV) vector-based gene therapy is under clinical investigation to enable endogenous FVIII production. Giroctocogene fitelparvovec is a recombinant AAV serotype 6 vector containing the coding sequence for the B-domain-deleted human F8 gene. In the ongoing phase 1/2, dose-ranging Alta study, 4 sequential cohorts of male participants with severe hemophilia A received a single IV dose of giroctocogene fitelparvovec. The primary end points are safety and changes in circulating FVIII activity. Interim results up to 214 weeks after treatment for all participants are presented. Eleven participants were dosed. Increases in alanine and aspartate aminotransferases were the most common treatment-related adverse events (AEs), which resolved with corticosteroid administration. Two treatment-related serious AEs (hypotension and pyrexia) were reported in 1 participant within 6 hours of infusion and resolved within 24 hours after infusion. At the highest dose level (3 × 1013 vg/kg; n = 5), the mean circulating FVIII activity level at week 52 was 42.6% (range, 7.8%-122.3%), and at week 104 it was 25.4% (range, 0.9%-71.6%) based on a chromogenic assay. No liver masses, thrombotic events, or confirmed inhibitors were detected in any participant. These interim 104-week data suggest that giroctocogene fitelparvovec is generally well tolerated with appropriate clinical management and has the potential to provide clinically meaningful FVIII activity levels, as indicated by the low rate of bleeding events in the highest dose cohort. This trial was registered at www.clinicaltrials.gov as #NCT03061201.

Deciphering conundrums of adeno-associated virus liver-directed gene therapy: focus on hemophilia.

Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies have been approved by regulatory agencies. Most have not progressed beyond phase 1/2 due to toxicity, lack of efficacy, or both. The liver is a natural target for adeno-associated virus since most serotypes have a high degree of tropism for hepatocytes due to cell surface receptors for the virus and the unique liver sinusoidal geometry facilitating high volumes of blood contact with hepatocyte cell surfaces. Recessive monogenic diseases such as hemophilia represent promising targets since the defective proteins are often synthesized in the liver and secreted into the circulation, making them easy to measure, and many do not require precise regulation. Yet, despite initiation of many disease-specific clinical trials, therapeutic windows are often nonexistent, resulting in excess toxicity and insufficient efficacy. Iterative progress built on these attempts is best illustrated by hemophilia, with the first regulatory approvals for factor IX and factor VIII gene therapies eventually achieved 25 years after the first gene therapy studies in humans. Although successful gene transfer may result in the production of sufficient transgenic protein to modify the disease, many emerging questions on durability, predictability, reliability, and variability of response have not been answered. The underlying biology accounting for these heterogeneous responses and the interplay between host and virus is the subject of intense investigation and the subject of this review.

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

Targeted delivery of miR125a-5p and human Factor VIII attenuates molecular mediators of hemophilic arthropathy.

Hemophilic arthropathy (HA) due to repeated bleeding into the joint cavity is a major cause of morbidity in patients with hemophilia. The molecular mechanisms contributing to this condition are not well characterized. MicroRNAs (miRs) are known to modulate the phenotype of multiple joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Since miR125a is known to modulate disease progression in OA and RA, we performed a targeted screen of miR125a-5p and its target genes in a murine model of chronic HA. A digital PCR analysis demonstrated significant downregulation of miR125a-5p (2-fold vs control joint). Further molecular evaluation revealed elevated expression of the immunological markers STAT1 (7.6-fold vs control joint) and TRAF6 (10.6 fold vs control joint), which are direct targets of miR125a-5p. We then studied the impact of targeted overexpression of miR125a-5p using an Adeno-associated virus (AAV) vector in modulating the molecular mediators of HA. AAV5-miR125a vectors were administered intra-articularly either alone or in combination with a low dose of AAV8-based human factor 8 (F8) gene in a murine model of HA. We observed significantly increased expression of miR125a-5p in AAV5-miR125a administered mice (~12 fold vs injured joint) or in combination with AAV8-F8 vectors (~44 fold vs injured joint). The activity assay revealed ~17 %-20 % FVIII levels in mice that received low dose liver-directed F8 gene therapy. Further immunohistochemical analysis, demonstrated a decrease in inflammatory markers (STAT1 and TRAF6) and cartilage-degrading matrix metalloproteinases (MMPs) 3, 9, 13 in the joints of treated animals. These data highlight the crucial role of miR125a-5p in the development of HA.

Potential role for oral tolerance in gene therapy.

Oral immunotherapies are being developed for various autoimmune diseases and allergies to suppress immune responses in an antigen-specific manner. Previous studies have shown that anti-drug antibody (inhibitor) formation in protein replacement therapy for the inherited bleeding disorder hemophilia can be prevented by repeated oral delivery of coagulation factor antigens bioencapsulated in transplastomic lettuce cells. Here, we find that this approach substantially reduces antibody development against factor VIII in hemophilia A mice treated with adeno-associated viral gene transfer. We propose that the concept of oral tolerance can be applied to prevent immune responses against therapeutic transgene products expressed in gene therapy.

Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

BACKGROUND: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa. AIM: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798. METHODS AND RESULTS: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC50; 19.2 and 42.9 µM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction. CONCLUSION: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.

Maladaptive lymphangiogenesis is associated with synovial iron accumulation and delayed clearance in factor VIII-deficient mice after induced hemarthrosis.

BACKGROUND: Mechanisms of iron clearance from hemophilic joints are unknown. OBJECTIVES: To better understand mechanisms of iron clearance following joint bleeding in a mouse model of hemophilia. METHODS: Hemarthrosis was induced by subpatellar puncture in factor VIII (FVIII)-deficient (FVII-/-) mice, +/- periprocedural recombinant human FVIII, and hypocoagulable (HypoBALB/c) mice. HypoBALB/c mice experienced transient FVIII deficiency (anti-FVIII antibody) at the time of injury combined with warfarin-induced hypocoagulability. Synovial tissue was harvested weekly up to 6 weeks after injury for histological analysis, ferric iron and macrophage accumulation (CD68), blood and lymphatic vessel remodeling (αSMA; LYVE1). Synovial RNA sequencing was performed for FVIII-/- mice at days 0, 3, and 14 after injury to quantify expression changes of iron regulators and lymphatic markers. RESULTS: Bleed volumes were similar in FVIII-/- and HypoBALB/c mice. However, pronounced and prolonged synovial iron accumulation colocalizing with macrophages and impaired lymphangiogenesis were detected only in FVIII-/- mice and were prevented by periprocedural FVIII. Gene expression changes involved in iron handling (some genes with dual roles in inflammation) and lymphatic markers supported proinflammatory milieu with iron retention and disturbed lymphangiogenesis. CONCLUSION: Accumulation and delayed clearance of iron-laden macrophages were associated with defective lymphangiogenesis after hemarthrosis in FVIII-/- mice. The absence of such findings in HypoBALB/c mice suggests that intact lymphatics are required for removal of iron-laden macrophages and that these processes depend on FVIII availability. Studies to elucidate the biological mechanisms of disturbed lymphangiogenesis in hemophilia appear critical to develop new therapeutic targets.

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Objective: Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). Materials and Methods: A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. Results: The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). Conclusion: The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII.

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

Lack of germline transmission in male mice following a single intravenous administration of AAV5-hFVIII-SQ gene therapy.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.

Progress, and prospects in the therapeutic armamentarium of persons with congenital hemophilia. Defining the place for liver-directed gene therapy.

In persons with congenital severe hemophilia A (HA) living in high-income countries, twice weekly intravenous infusions of extended half-life (EHL) factor VIII (FVIII) products, or weekly/biweekly/monthly subcutaneous injections of emicizumab are the gold standard home treatments to grant days without hurdles and limitations. Once weekly/twice monthly infusions of EHL Factor IX (FIX) products achieve the same target in severe hemophilia B (HB). Gene therapy, which is likely to be licensed for clinical use within 1-2 years, embodies a shift beyond these standards. At an individual patient level, a single functional gene transfer leads to a > 10-yr almost full correction of the hemostatic defect in HB and to a sustained (3-6-yrs) expression of FVIII sufficient to discontinue exogenous clotting factor administrations. At the doses employed, the limited liver toxicity of systemically infused recombinant adeno-associated virus (rAAV) vectors is documented by long-term (12-15 yrs) follow-ups, and pre-existing high-titer neutralizing antibodies to the AAV5 vector are no longer an exclusion criterion for effective transgene expression with this vector. A safe durable treatment that converts a challenging illness to a phenotypically curable disease, allows persons to feel virtually free from the fears and the obligations of hemophilia for years/decades. Along with patient organizations and health care professionals, communicating to government authorities and reimbursement agencies the liberating potential of this substantial innovation, and disseminating across the Centers updated information on benefits and risks of this strategy, will align expectations of different stakeholders and establish the notion of a potentially lifelong cure of hemophilia.

Gene therapy for hemophilia.

The cloning of the factor VIII (FVIII) and factor IX (FIX) genes in the 1980s has led to a succession of clinical advances starting with the advent of molecular diagnostic for hemophilia, followed by the development of recombinant clotting factor replacement therapy. Now gene therapy beckons on the back of decades of research that has brought us to the final stages of the approval of 2 products in Europe and United States, thus heralding a new era in the treatment of the hemophilias. Valoctocogene roxaparvovec, the first gene therapy for treatment of hemophilia A, has been granted conditional marketing authorization in Europe. Another approach (etranacogene dezaparvovec, AMT-061) for hemophilia B is also under review by regulators. There are several other gene therapy approaches in earlier stages of development. These approaches entail a one-off infusion of a genetically modified adeno-associated virus (AAV) engineered to deliver either the FVIII or FIX gene to the liver, leading to the continuous endogenous synthesis and secretion of the missing coagulation factor into the circulation by the hepatocytes, thus preventing or reducing bleeding episodes. Ongoing observations show sustained clinical benefit of gene therapy for >5 years following a single administration of an AAV vector without long-lasting or late toxicities. An asymptomatic, self-limiting, immune-mediated rise in alanine aminotransferase is commonly observed within the first 12 months after gene transfer that has the potential to eliminate the transduced hepatocytes in the absence of treatment with immunosuppressive agents such as corticosteroids. The current state of this exciting and rapidly evolving field, as well as the challenges that need to be overcome for the widespread adaptation of this new treatment paradigm, is the subject of this review.

Autologous bone marrow-derived MSCs engineered to express oFVIII-FLAG engraft in adult sheep and produce an effective increase in plasma FVIII levels.

Introduction: Hemophilia A (HA) is the most common X-linked bleeding disorder, occurring in 1 in 5,000 live male births and affecting >1 million individuals worldwide. Although advances in protein-based HA therapeutics have improved health outcomes, current standard-of-care requires infusion 2-3 times per week for life, and 30% of patients develop inhibitors, significantly increasing morbidity and mortality. There are thus unmet medical needs requiring novel approaches to treat HA. Methods: We tested, in a highly translational large animal (sheep) model, whether the unique immunological and biological properties of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) could enable them to serve as cellular delivery vehicles to provide long-term expression of FVIII, avoiding the need for frequent infusions. Results: We show that autologous BM-MSCs can be isolated, transduced with a lentivector to produce high levels of ovine (o)FVIII, extensively expanded, and transplanted into adult animals safely. The transplanted cells engraft in multiple organs, and they stably produce and secrete sufficient quantities of FVIII to yield elevated plasma FVIII levels for at least 15 weeks. Discussion: These studies thus highlight the promise of cellular-based gene delivery approaches for treating HA.