Factor VIII Expression Research Articles

Rescue of the endogenous FVIII expression in hemophilia A mice using CRISPR Cas9 mRNA LNPs

Gene editing provides a promising alternative approach that may achieve long-term FVIII expression for hemophilia A (HemA) treatment. In this study, we investigated in vivo correction of a mutant factor VIII (FVIII) gene in HemA mice. We first developed MC3-based LNPs for efficient mRNA delivery into liver sinusoidal endothelial cells (LSECs), the major site of FVIII biosynthesis. To target a five base pair deletion in FVIII exon 1 in a specific HemA mouse strain, we injected LNPs encapsulating Cas9 mRNA and specifically designed sgRNAs intravenously for in vivo gene editing of the mutant FVIII. Indel variants generated at the mutant site contained mostly a single base pair deletion , resulting in frame-shift correction of FVIII gene. Sustained endogenous FVIII activity up to 6% was achieved over 26 weeks in treated HemA mice. Sequencing data indicated an average gene editing rate of 15.3% in LSECs. Our study suggests that optimized MC3 LNP formulations, combined with CRISPR/Cas9 technology, can effectively correct the mutant FVIII gene in LSECs and restore FVIII activity for therapeutic treatment of HemA.

Efficacy, safety, and quality of life 4 years after valoctocogene roxaparvovec gene transfer for severe hemophilia A in the phase 3 GENEr8-1 trial

BackgroundValoctocogene roxaparvovec, an adeno-associated virus-mediated gene therapy for severe hemophilia A, enables endogenous factor VIII (FVIII) expression and provides bleed protection. ObjectivesDetermine valoctocogene roxaparvovec durability, efficacy, and safety 4 years post-treatment. MethodsIn the phase 3 GENEr8-1 trial, 134 adult males with severe hemophilia A without inhibitors and previously using FVIII prophylaxis received a 6x1013 vg/kg infusion of valoctocogene roxaparvovec. Efficacy endpoints included annualized bleed rate (ABR), annualized FVIII infusion rate, FVIII activity, and the Haemophilia-Specific Quality of Life Questionnaire for Adults (Haemo-QOL-A). Adverse events (AEs) and immunosuppressant use were assessed. Change from baseline was assessed after participants discontinued prophylaxis (scheduled for week 4). ResultsMedian follow-up was 214.3 weeks; 2 participants discontinued since the previous data cutoff. Declines from baseline in mean treated ABR (−82.6%; P<0.0001) and annualized FVIII infusion rate (−95.5%; P<0.0001) were maintained from previous years in the primary analysis population of 112 participants who enrolled from a noninterventional study. During year 4, 81/110 rollover participants experienced 0 treated bleeds. Week 208 mean and median chromogenic FVIII activity were 16.1 IU/dL and 6.7 IU/dL, respectively, in 130 modified intention-to-treat (mITT) participants. Seven participants resumed prophylaxis since the previous data cutoff. Mean change from baseline to week 208 in Haemo-QOL-A Total Score (P<0.0001) remained clinically meaningful for mITT participants. Alanine aminotransferase elevation was the most common AE during year 4 (56/131 participants); none required immunosuppressants. ConclusionsValoctocogene roxaparvovec provides persistent FVIII expression, hemostatic control, and health-related quality of life improvements with no new safety signals.

Gene Therapy in Hemophilia A: Achievements, Challenges, and Perspectives.

Strides in advancements of care of persons with hemophilia include development of long-acting factor replacement therapies, novel substitution and hemostatic rebalancing agents, and most recently approved gene therapy. Several decades of preclinical and clinical trials have led to development of adeno-associated viral (AAV) vector-mediated gene transfer for endogenous production of factor VIII (FVIII) in hemophilia A (HA). Only one gene therapy product for HA (valoctocogene roxaparvovec) has been approved by regulatory authorities. Results of valoctocogene roxaparvovec trial show significant improvement in bleeding rates and use of factor replacement therapy; however, sustainability and duration of response show variability with overall decline in FVIII expression over time. Further challenges include untoward adverse effects involving liver toxicity requiring immunosuppression and development of neutralizing antibodies to AAV vector rendering future doses ineffective. Real-life applicability of gene therapy for HA will require appropriate patient screening, infrastructure setup, long-term monitoring including data collection of patient-reported outcomes and innovative payment schemes. This review article highlights the success and development of HA gene therapy trials, challenges including adverse outcomes and variability of response, and perspectives on approach to gene therapy including shared decision-making and need for future strategies to overcome the several unmet needs.

Increased Expression Level of Human Blood Clotting Factor VIII Using NS0 Cell Line as a Host Cells.

Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields. The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest. We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL-1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA. A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030). The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry.

GO-8: Stable Expression of Factor VIII over 5 Years Following Adeno-Associated Gene Transfer in Subjects with Hemophilia a Using a Novel Human Factor VIII Variant

Background: GO-8 (ClinicalTrials.gov: NCT03001830) is a study of liver-directed adeno-associated virus (AAV) gene therapy for haemophilia A (HA) that uses a factor VIII (FVIII) variant containing a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain (AAV-HLP-hFVIII-V3). In preclinical studies, AAV-HLP-hFVIII-V3 mediated a 3-fold higher FVIII expression when compared to an identical AAV construct encoding the hFVIII-SQ variant used in most HA gene therapy trials. Methods: In a multi-centre, open-label, non-randomised, phase I/II clinical trial, we assessed the safety and efficacy of escalating doses of AAV-HLP-hFVIII-V3 pseudotyped with an AAV8 capsid in adults with severe haemophilia A (FVIII activity ≤1%). All participants received prophylactic glucocorticoids, with or without tacrolimus, with the aim of reducing the risk of vector-related transaminase elevation. The primary endpoints were safety and efficacy. Efficacy was assessed by measuring FVIII activity (FVIII: C) using both chromogenic and one-stage clotting assays and factor consumption pre and post-gene therapy. Results: As of May 31 2023, 12 participants were enrolled sequentially into one of four vector doses: 6×1011 vector genomes (vg)/kg body weight (n=1), 2×1012 vg/kg (n=3), 4×1012 vg/kg (n=3), or 6×1012 vg/kg (n=5). All participants were on FVIII prophylaxis prior to gene therapy. The most common vector-related adverse event was an elevation in liver aminotransferase levels, which occurred in 10 of 12 participants. In 7 of the 8 participants treated at doses ≥4×1012 vg/kg, recurrent elevation in aminotransferase levels was observed during the first 12 months, often associated with tapering of immunosuppression. This resulted in a reduction in transgene expression from peak levels in all participants, with a complete loss of transgenic protein in one participant. Vector-related elevation in aminotransferase was not observed after the 12-month time point in long-term follow-up. Mean chromogenic FVIII: C levels at 12 months after gene therapy were 3 IU/dL in the 6×1011 vg/kg cohort, 13±9IU/dL (range: 2-19 IU/dl) in the 2×1012 vg/kg cohort, 8±1IU/dl in the 4×1012 vg/kg cohort (range: 7-9 IU/dl) and 22±34 IU/dl in the 6×1012 vg/kg cohort (range 1-82 IU/dl). Transgene expression was then stably maintained over a median follow-up of 3 years (range: 0.2-5 years) from the level achieved 1-year post-infusion, best illustrated by the data from the 2×1012 and 4×1012 vg/kg cohorts shown in Figure 1. FVIII: C was, on average 2-fold higher when measured using a one-stage clotting assay compared to the chromogenic method. Nine of the 12 participants remained off prophylaxis after gene therapy for the duration of the follow-up period. Baseline mean and median annualised factor VIII use was 4097 and 4657 IU/kg per year before gene therapy. Following gene therapy, the mean and median annualised factor VIII concentrate use reduced across all participants to 1186 and 61 IU/kg (One sample t-test p=0.0009), respectively. No FVIII inhibitors or thrombotic events were reported for the duration of the study. Conclusion: A single infusion of AAV-HLP-hFVIII-V3 resulted in stable FVIII expression over a follow-up period of up to 5 years in participants with severe haemophilia A. A high rate of liver aminotransferase elevation following gene transfer impacted transgene expression. However, 9 of the 12 participants were able to discontinue FVIII prophylaxis over the duration of the study, resulting in a significant reduction in FVIII concentrate usage.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Childhood Treatment with Adeno-Associated Viral Gene Therapy Results in Stable FVIII Expression and Improved Bleeding Phenotype in Adult Severe Hemophilia A Dogs

Introduction: In patients with severe hemophilia A, adeno-associated virus (AAV)-mediated factor VIII (FVIII) gene therapy is associated with reduced bleeding compared with FVIII prophylaxis. AAV5-FVIII gene therapy is approved in the U.S. and conditionally approved in Europe for the treatment of adults with severe hemophilia A. Gene therapy during childhood could potentially prevent the development of arthropathy and improve quality of life. However, there are unknowns regarding long-term safety and whether the predominant episomal nature of AAV persistence would lead to loss of transgene expression with hepatocyte divisions and liver expansion during childhood. The inflammatory response to AAV gene therapy in children is also largely uncharacterized. We have previously reported the treatment of five neonatal or infant dogs with a single vector infusion of a codon-optimized AAV5-B-domain deleted canine FVIII (cFVIII) construct with a hybrid liver promoter (AAV5-cFVIII). Dogs treated at 2 weeks of age demonstrated improved whole blood clot time (WBCT) over a 6-month period despite minimal cFVIII expression (&amp;lt;3%). Dogs treated at 2 months of age demonstrated stable cFVIII expression measured over a 6-month period and decreased WBCT. Aims: To describe the early inflammatory response to AAV5-cFVIII in infant and neonatal dogs, and to provide an update on the safety and efficacy of AAV5-cFVIII in these animals 12-16 months post-treatment. Methods: Hemophilia A dogs were treated with a single vector infusion (dose=2.0e14 vg/kg) of AAV5-cFVIII at 2 weeks (n=2) or 2 months (n=3) of age. Epigenomic data suggests that 2 months of age in dogs is equivalent to 9 months in humans. Liver volume was measured by magnetic resonance imaging at baseline, 3, 6, and 12 months post-AAV5-cFVIII treatment. Plasma cytokine levels were measured using a cytokine array pre-treatment (day 0) and post-treatment (days 2-28). Percutaneous liver biopsy samples were obtained at 2 weeks, 6 months, and 12 months post-treatment. Results: Cytokine array analyses of samples demonstrated that one dog who received AAV5-cFVIII at 2 weeks experienced transient post-treatment plasma elevation of IL-6, KC-like, MCP-1, TGFβ-1, and TGFβ-2 that returned to baseline levels by day 18. Two dogs treated at 2 months experienced a transient increase in GMC-SF, IL-2, IL-6, IL-7, IL-15, and IL-18 that returned to baseline levels by day 21. No changes in levels of IFNγ, IL-8, IL-10, IP-10, TNFα, or TGFβ-3 were observed. Changes in plasma cytokine levels were not associated with an elevation in plasma alanine transaminase levels. Hemophilia A dogs treated with AAV5-cFVIII at 2 weeks of age maintained an improved WBCT 12 to 16 months post-treatment, although cFVIII expression as measured by one-stage (OSA) and chromogenic substrate assays (CSA) remained minimal (&amp;lt;3%). Body weight and liver volume increased by 10.6-fold (measured at 16 months) and 12-fold (measured at 12 months) respectively. Dogs treated with AAV5-cFVIII at 2 months of age demonstrated sustained cFVIII expression by OSA and CSA after 12 to 16 months (8.9-13.39% measured at 16 months by OSA), despite a 3.2-fold increase in body weight (measured at 16 months), and a 3.9-fold increase in liver volume (measured at 12 months). This was associated with improved WBCT observed over the 12 to 16-month period for all three dogs. Overall, a reduction in bleeding events (BEs) was observed in all dogs. No BEs were recorded prior to AAV5-cFVIII treatment for dogs treated at 2 weeks of age. During the 16 months post-treatment, one dog experienced two spontaneous BEs, while no BEs were observed for the second dog. The dogs treated at 2 months experienced 4 (3 spontaneous and 1 traumatic) BEs prior to AAV5-cFVIII treatment, and 1 traumatic BE 48 hours post-treatment prior to the detection of FVIII expression. For the remaining 16 months post-treatment, no BEs were observed in this group. Conclusions: Treatment using AAV5-cFVIII at 2 months of age in a hemophilia A dog model resulted in stable FVIII expression into adult life despite significant liver expansion. For some dogs, AAV-cFVIII treatment resulted in an early transient increase in plasma proinflammatory cytokine levels with no evidence of transaminitis that resolved within three weeks.Further studies on liver biopsy samples are ongoing to evaluate cellular implications, vector genome distribution, and mechanisms of AAV persistence.

AAV-Mediated Expression of a Single-Chain FVIII Mimetic Antibody Efficiently Corrects Bleeding in Haemophilia a Mice

Background: The recent approval of gene therapy for haemophilia A (HA) using adeno-associated viruses (AAVs) marks an important and long-awaited landmark as it is associated with markedly reduced bleeding events whilst overcoming the demands of regular life-long administration of factor concentrates or antibodies with FVIII mimetic activity. However, the benefits of AAV gene therapy in severely affected HA patients are likely to be short-lived as clinical data from most of the advanced trials show a gradual decline in transgenic Factor VIII (FVIII) levels over time. The reasons for this decline in expression remain unexplained but may be due in part to the packaging of an oversized FVIII expression cassette into AAV vectors. This hypothesis is supported by the fact that such decline in transgene expression has not been observed in other gene therapy approaches in clinic where the transgene expression cassette is within the packaging limits of AAV vectors. Therefore, we explored an alternative therapy approach for HA that utilises an expression cassette encoding a novel FVIII mimetic bispecific antibody (Bi8) that remains within the packaging constrains of AAV vectors, thus potentially improving the prospects of long-term control of the bleeding diathesis in severely affected HA subjects. . Methods: Bi8, is a small 54kDa single-chain bispecific antibody designed as a tandem of single-chain fragment variables (scFvs) targeting Factors IXa (FIXa) and X (FX). FVIII mimetic activity of recombinant Bi8 protein was evaluated in vitro using (1) the catalytic conversion of purified FX into FXa in a chromogenic assay, and (2) factor XIa (FXIa)-triggered thrombin generation assay (TGA) and activated partial thromboplastin time (aPTT) using spiked human HA plasma. Bi8 sequences were cloned in an AAV expression cassette downstream of a strong liver-specific promoter, pseudotyped with AAV serotype 8 capsid and evaluated in vitro as well as in vivo, including in FVIIIKO mice to assess potency in a tail vein transection assay, kinetics of Bi8 expression and the occurrence of anti-drug antibodies (ADAs). Results: In preliminary experiments, we demonstrated that the single-chain format used for Bi8 retained haemostatic functions. When tested in a chromogenic assay, recombinant Bi8 protein showed kinetics of FXa formation identical to that of emicizumab. Furthermore, Bi8 spiked in human HA plasma at 350nM corrected the clotting time (17.2 sec) in an aPTT assay to levels observed with emicizumab (22.7 sec). TGA with recombinant Bi8 and emicizumab showed comparable efficacy with peaks of thrombin generation at 354.2 and 358.4nM respectively. Next, human hepatocyte HuH7 cells were transduced with AAV8 encoding Bi8 (AAV8_Bi8) vectors in vitro, leading to the expression of a fully functional bispecific antibody with an activity/antigen ratio of 0.99 when compared to the recombinant Bi8 protein. Three doses of AAV8_Bi8 vector were then evaluated in 6-8 weeks old, male FVIIIKO mice, with a single intravenous infusion of 4e 11; 4e 12 and 1.2e 13 vector genome (vg) per kilo. Expression of the bispecific antibody was detectable in plasma for all 3 doses and peaked 2 weeks post-infusion with respective concentrations of 4.5; 47.4 and 178.8nM (Panel A). Transgenic expression was then stably maintained for 12 weeks and ADAs were not detected in any of the treated animals. When challenged in a bleeding assay, haemostasis was secured in all cohorts of mice infused with AAV-Bi8 vectors, including at the lowest dose of 4e 11 vg/kg where the average total blood loss was reduced to 401.2μL (median 406.2μL) compared to 760.4μL (median 773.1μL) in non-treated FVIIIKO mice (Panel B). Conclusion: This study establishes the feasibility of using AAV vectors to deliver an expression cassette encoding Bi8, a novel FVIII mimetic antibody formatted as a single-chain tandem-scFv, leading to circulating concentrations of bispecific antibody at levels that can secure haemostasis and reduce blood loss in a murine model of HA. This non FVIII-based expression cassette meets the packaging capacity of AAVs and therefore provides an alternative approach for AAV-mediated gene therapy for HA that may be better suited for stable long-term expression than FVIII transgenes.

Hemophilia Gene Therapy: The End of the Beginning?

Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

BackgroundHemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.MethodsTo investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.ResultsTo examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice.ConclusionsThe LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

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

Hemophilia A is an X-linked recessive bleeding disorder caused by deficiency of plasma coagulation factor VIII (FVIII), and accounts for about 80-85% of patients with hemophilia. Plasma-derived therapies or recombinant factor VIII concentrates and also FVIII mimicking antibodies are used to prevent and treat the bleeding symptoms. Recently, EMA granted a conditional marketing approval for the first gene therapy of Hemophilia A. Aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII secreting transgenic mesenchymal stem cells (MSC). A lentiviral vector encoding B-domain-deleted Factor VIII cDNA sequence with CD45 R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII expressing primary cell line by transducing MSC. Efficacy and functionality of secreted FVIII from MSCs was assessed with anti- FVIII ELISA, CD45R0t flow cytometry, FVIII Western Blot and Mixing Test analysis in vitro. Results of this study showed that the transgenic MSCs maintain a persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. Functionality of FVIII protein secreted in MSC supernatant was demonstrated by using Mixing Test in coagulation analysis. In the Mixing Test analysis, FVIIIdeficient human plasma products were mixed either with saline control or FVIII-secreted MSC supernatant. Mean FVIII levels of saline control group were 0.41±0.03 IU/dL whereas mean FVIII levels were 25.41±33.38 IU/dL (p<0.01) in FVIII-secreting MSC supernatant mixed group. Mean activated partial thromboplastin time (aPTT) of saline control group was 92.69±11.38 seconds whereas in FVIII-secreting MSC supernatant mixed group, mean aPTT levels were decreased to 38.60±13.38 seconds (p<0.001). Findings of this in vitro study shows a promising new method applicable to treat Hemophilia A. Accordingly, a FVIII secreting transgenic mesenchymal stem cell study will be initiated in FVIII knock-out animal model.

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

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). 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. 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). 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.

Characteristics of BAY 2599023 in the Current Treatment Landscape of Hemophilia A Gene Therapy.

Hemophilia A, a single gene disorder leading to deficient Factor VIII (FVIII), is a suitable candidate for gene therapy. The aspiration is for single administration of a genetic therapy that would allow the production of endogenous FVIII sufficient to restore hemostasis and other biological processes. This would potentially result in reliable protection from bleeding and its associated physical and emotional impacts. Gene therapy offers the possibility of a clinically relevant improvement in disease phenotype and transformational improvement in quality of life, including an opportunity to engage in physical activities more confidently. Gene therapy products for hemophilia A in advanced clinical development use adeno-associated viral (AAV) vectors and a codon-optimized B-domain deleted FVIII transgene. However, the different AAV-based gene therapies have distinct design features, such as choice of vector capsid, enhancer and promoter regions, FVIII transgene sequence and manufacturing processes. These, in turn, impact patient eligibility, safety and efficacy. Ideally, gene therapy technology for hemophilia A should offer bleed protection, durable FVIII expression, broad eligibility and limited response variability between patients, and long-term safety. However, several limitations and challenges must be overcome. Here, we introduce the characteristics of the BAY 2599023 (AAVhu37.hFVIIIco, DTX 201) gene therapy product, including the low prevalence in the general population of anti-AAV-hu37 antibodies, as well as other gene therapy AAV products and approaches. We will examine how these can potentially meet the challenges of gene therapy, with the ultimate aim of improving the lives of patients with hemophilia A.

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing.

Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region. Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs). Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs. Discussion: Thus, we firstly rescued the 140kb reversion mutation by gene addition of a 975bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.

Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells

BackgroundWe previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIIInull mice transplanted with 2bF8-transduced Sca-1+ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost. ObjectiveIn this study, we aimed to describe the mechanism of this platelet-FVIII loss. MethodsWe monitored platelet-FVIII expression in FVIIInull mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1+ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell–deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity. ResultsPlatelet-FVIII expression was sustained in 2bF8Tg bone marrow transplantation but not in 2bF8Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8Tg-Sca-1+ cells and secreted interferon gamma, but were not stimulated by 2bF8Tg platelets/megakaryocytes, suggesting that 2bF8Tg-Sca-1+ cells are targets for FVIII-primed CD8 T cells. When 2bF8Tg-Sca-1+ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice. ConclusionPre-existng immunity can alter the engraftment of 2bF8Tg-Sca-1+ cells through the cytotoxic CD8 T-cell–mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.

The Effects of Immunomodulation with Corticosteroids to Manage an AAV Capsid Immune response in the Phase I/II Study of SPK-8011

Background: Hemophilia A (HA) is an inherited blood disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII). SPK-8011 is an adeno-associated viral (AAV) vector gene therapy targeting hepatocyte expression of FVIII, aiming to provide durable FVIII expression and long-term bleed prevention. A Phase I/II trial of SPK-8011 (NCT03003533/NCT03432520) has demonstrated multiyear sustained FVIII expression and notably reduced bleeding in people with HA, with no major safety concerns. There are challenges around preventing and managing cellular immune responses to transduced hepatocytes. Here, we present the outcomes of participants who received corticosteroids following vector administration to prevent and/or treat a presumed AAV capsid immune response. Methods: In this open-label, multicenter, non-randomized, dose-escalation trial, people with HA were infused with SPK-8011 at one of four doses ranging from 5×1011 to 2×1012 vg/kg (full methods published in George et al. New Engl J Med 2021). Participants had baseline FVIII activity of ≤2% of normal levels and were negative for neutralizing antibodies to SPK200. Participants were monitored for 52 weeks after vector administration and enrolled in a 4-year long-term follow-up trial. Corticosteroids were used reactively at the investigators’ discretion upon a presumed capsid immune response, i.e., decrease in FVIII expression, increase in alanine aminotransferase (ALT) level, and/or positive peripheral blood mononuclear cells using interferon-γ enzyme-linked immunospot (ELISpot) to SPK200 peptides. Following loss of FVIII expression in two participants, a prophylactic regimen was explored. Assessments included duration of corticosteroid use and associated adverse events (AEs). Results: Twenty-three males with HA were dosed with SPK-8011 and 17 received corticosteroids (12 reactively, 5 prophylactically); five did not have evidence of a capsid immune response, and one remains in the window of a capsid immune response in the follow-up period (data cut-off: 30 Jun 2022). The median (range) time to initiation of reactive corticosteroids was 9.3 (4.3-12.1) weeks post vector infusion in the 12 participants, with a median (range) duration of treatment of 8.5 (2-18) weeks. Two of these 12 participants completely lost FVIII expression following a presumed capsid immune response (one initiated emicizumab; one resumed FVIII prophylaxis); FVIII expression was maintained in the remaining 10 participants. Corticosteroids were administered prophylactically (2-4 weeks post vector) in five participants, with transgene expression maintained in all five. In four of these five participants, length of exposure was prolonged (31-49 weeks), with attempted corticosteroid tapering resulting in decreased FVIII expression, increased ALT and/or positive ELISpot assays outside the typical window of capsid immune responses. Four participants had AEs related to corticosteroid therapy (Table), with weight gain (8.7%; n=2) and insomnia (8.7%; n=2) being the most common. The occurrence of AEs attributed to corticosteroids increased with duration of use, particularly in those with exposure of >30 weeks. Prophylactic corticosteroids did not consistently prevent a presumed capsid immune response in these five participants and four experienced prolonged courses of corticosteroids necessitating steroid-sparing agents (Figure). All five participants who received prophylactic corticosteroids and four participants who received reactive corticosteroids experienced a recurrence of one or more laboratory trigger(s) potentially linked to a presumed capsid immune response during their corticosteroid taper. Subsequent increased corticosteroid use did not consistently resolve these laboratory changes. Conclusions: Overcoming the challenge of capsid immune responses to achieve long-term stable and predictable FVIII expression remains a goal of AAV gene transfer for HA. Reactive oral corticosteroids were well tolerated but did not prevent loss of FVIII expression in all participants. Early prophylactic corticosteroids did not consistently prevent presumed capsid immune responses and resulted in prolonged immunomodulatory courses with associated AEs. Data so far have permitted refinement of laboratory triggers and investigation is ongoing to identify an alternative, safe and optimal immunomodulatory regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibiting Activated Protein C Cleavage of Factor VIII for Hemophilia a Gene Therapy

Background Despite progress in adeno-associated virus (AAV) mediated factor VIII (FVIII) gene transfer for hemophilia A (HA), durable and sustained FVIII expression sufficient to ameliorate bleeding in all participants is an unrealized goal. Following the success of using the gain of function FIX-Padua variant in hemophilia B gene therapy, we hypothesized that use of an enhanced hemostatic function FVIII would similarly improve HA gene therapy by: 1) permitting use of lower vector doses, thereby overcoming dose-dependent safety and efficacy limitations and 2) achieving efficacy with lower FVIII antigen. We previously published a FVIII variant resistant to activated protein C (APC) cleavage (FVIII-R336Q/R562Q or FVIII-QQ) with normal specific activity and 4-5 fold improved hemostatic function in recombinant protein experiments (Wilhelm et al. Blood 2021). Aims We evaluated the possible clinical translation of FVIII-QQ for HA AAV-mediated gene therapy by determining the hemostatic benefit and potential prothrombotic and immunological risks in mice. Additionally, FVIII-QQ steady-state expression was 2.5 to 5-fold higher than FVIII-WT. We hypothesized this was due to disrupted cleavage by APC or other serine proteases at R336 and/or R562. Results Survival studies in HA/CD4KO and WT/CD4KO mice treated with AAV vectors to express (h)uman FVIII-WT and hFVIII-QQ in the range of mild HA, normal, and supratherapeutic FVIII levels thus far demonstrate no significant differences in survival at 7-11 month follow up or D-dimer levels after 8 months. Additionally, FVIII-QQ mice were generated using CRISPR/Cas9 to introduce R562Q (WT mice are Q336) for endogenous (m)ouse FVIII-QQ expression. Endogenous mFVIII-QQ mice were viable, fertile, normal weight, and D-dimers did not significantly differ from WT mice, while FVIII activity was ~2-fold higher than WT mice. HA immune competent mice treated with either 6 weekly doses of hFVIII-WT or hFVIII-QQ protein (0.2 μg) or AAV vector to express hFVIII-QQ or hFVIII-WT had no significant differences in inhibitor titers. To evaluate hemostasis, HA/CD4KO mice were treated with AAV vector only differing by 2 amino acids (R336, R362) at 3 different doses (n=6-12 mice/cohort) to express hFVIII-QQ or hFVIII-WT and underwent tail clip challenge. Estimated EC50 and EC80 of vector doses required to normalize blood loss to WT mice were 5 and 10-fold lower, respectively, for hFVIII-QQ versus hFVIII-WT supporting a hemostatic benefit to using the hFVIII-QQ transgene. Interestingly, across 3 different vector constructs differing only by F8 codon optimization, we observed hFVIII-QQ antigen values 2.5 to 5-fold higher than in mice expressing hFVIII-WT. Analysis of liver tissue from HA/CD4KO mice treated with the same vector construct and dose to express hFVIII-WT or hFVIII-QQ demonstrated approximately the same mRNA values, but plasma hFVIII-QQ antigen was 4-fold higher than hFVIII-WT and intracellular hFVIII-QQ antigen was the same or lower than hFVIII-WT antigen. Consistent with our in vivo AAV experiments, preliminary studies of intracellular versus secreted hFVIII antigen in BHK stable cell lines expressing hFVIII-WT or hFVIII-QQ demonstrated similar mRNA values with higher relative secreted hFVIII-QQ protein. To further probe the mechanism of higher hFVIII-QQ steady-state expression, we evaluated a panel of serine proteases (APC, FIXa, FXa, plasmin, PACE/furin and thrombin) for cleavage of FVIII-QQ relative to FVIII-WT protein. As expected, hFVIII-QQ was resistant to APC cleavage. Surprisingly, all other serine proteases tested demonstrated cleavage of hFVIII-WT, but not hFVIII-QQ, at R336 and/or R562 suggesting protease attack at these cleavage sites may occur more than previously appreciated, though the physiologic significance remains to be determined. Conclusion Our studies outline the use of hFVIII-QQ transgene expression thus far does not demonstrate safety concerns and confers hemostatic benefit over hFVIII-WT via: 1) improved hemostatic function and 2) higher steady-state expression. Enhanced steady state hFVIII-QQ expression relative to hFVIII-WT was observed across multiple model systems. Preliminary data demonstrate multiple serine proteases can cleave at the described FVIII APC cleavage sites (R336, R562) suggesting hFVIII-QQ may generally be more proteolytically stable thereby permitting improved steady-state expression.

Heterogeneity and reciprocity of FVIII and VWF expression, and the response to shear stress in cultured human endothelial cells

BackgroundSubstantial phenotypic heterogeneity exists in endothelial cells and while much of this heterogeneity results from local microenvironments, epigenetic modifications also contribute. MethodsCultured human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, human hepatic sinusoidal endothelial cells, human lymphatic endothelial cells (hLECs), and two different isolations of endothelial colony forming cells (ECFCs) were assessed for levels of factor VIII (FVIII) and von Willebrand factor (VWF) RNA and protein. The intracellular location and co‐localization of both proteins was evaluated with immunofluorescence microscopy and stimulated release toof FVIII and VWF from Weibel–Palade bodies (WPBs) was evaluated. Changes in expression of FVIII and VWF RNA after hLECs and ECFCs were exposed to 2 or 15 dynes/cm2 of laminar shear stress were also assessed. ResultsWe observed considerable heterogeneity in FVIII and VWF expression among the endothelial cells. With the exception of hLECs, FVIII RNA and protein were barely detectable in any of the endothelial cells and a reciprocal relationship between levels of FVIII and VWF appears to exist. When FVIII and VWF are co‐expressed, they do not consistently co‐localize in the cytoplasm. However, in hLECs where significantly higher levels of FVIII are expressed, FVIII and VWF co‐localize in WPBs and are released together when stimulated. Expression of both FVIII and VWF is markedly reduced when hLECs are exposed to higher or lower levels of laminar shear stress, while in ECFCs there is a minimal response for both proteins. ConclusionsVariable levels of FVIII and VWF RNA and protein exist in a subset of cultured human endothelial cells. Higher levels of FVIII present in hLECs co‐localize with VWF and are released together when exposed to a secretagogue.

Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.

The Arrival of Gene Therapy for Patients with Hemophilia A.

Historically, the standard of care for hemophilia A has been intravenous administration of exogenous factor VIII (FVIII), either as prophylaxis or episodically. The development of emicizumab, a humanized bispecific monoclonal antibody mimicking activated FVIII, was a subsequent advance in treatment. However, both exogenous FVIII and emicizumab require repeated and lifelong administration, negatively impacting patient quality of life. A recent breakthrough has been the development of gene therapy. This allows a single intravenous treatment that could result in long-term expression of FVIII, maintenance of steady-state plasma concentrations, and minimization (or possibly elimination) of bleeding episodes for the recipient’s lifetime. Several gene therapies have been assessed in clinical trials, with positive outcomes. Valoctocogene roxaparvovec (an adeno-associated viral 5-based therapy encoding human B domain-deleted FVIII) is expected to be the first approved gene therapy in European countries, including Italy, in 2022. Some novel challenges exist including refining patient selection criteria, managing patient expectations, further elucidation of the durability and variability of transgene expression and long-term safety, and the development of standardized ‘hub and spoke’ centers to optimize and monitor this innovative treatment. Gene therapy represents a paradigm shift, and may become a new reference standard for treating patients with hemophilia A.