Trials Of Gene Therapy For Hemophilia Research Articles

Adeno-associated virus vector-based gene therapies for pediatric diseases.

Gene therapy using adeno-associated virus (AAV) is a rapidly developing technology with widespread treatment potential. AAV2 vectors injected directly into the brain by stereotaxic brain surgery have shown good results in treating aromatic l-amino acid decarboxylase deficiency. Moreover, gene therapy using the AAV9 vector, which crosses the blood-brain barrier, has been performed in more than 2000 patients worldwide as a disease-modifying therapy for spinal muscular atrophy. AAV vectors have been applied to the development of gene therapies for various pediatric diseases. Gene therapy trials for hemophilia and ornithine transcarbamylase deficiency are underway. Clinical trials are planned for glucose transporter I deficiency, Niemann-Pick disease type C, and spinocerebellar ataxia type 1. The genome of AAV vectors is located in the episome and is rarely integrated into chromosomes, making the vectors safe. However, serious adverse events such as hepatic failure and thrombotic microangiopathy have been reported, and ongoing studies are focusing on developing more efficient vectors to reduce required dosages.

First-in-Human Dose-Finding Study of AAVhu37 Vector-Based Gene Therapy: BAY 2599023 Has Stable and Sustained Expression of FVIII over 2 Years

▪BackgroundGene therapy for hemophilia A has the potential to reduce the treatment burden for patients and their care providers by eliminating the need for regular factor VIII (FVIII) prophylaxis through long-term expression of endogenous FVIII at levels sufficient to provide bleed protection. Ongoing phase 3 gene therapy trials for hemophilia A show promise but can result in unpredictable FVIII expression of uncertain durability. Gene therapy must evolve to meet patient expectations of a durable, efficacious and safe treatment.BAY 2599023 (AAVhu37.hFVIIIco) is the first, clinical stage adeno-associated virus (AAV) gene therapy vector, based on the AAVhu37 serotype. BAY 2599023 is a non-replicating AAV vector and contains a single-stranded DNA genome encoding a B-domain-deleted FVIII, under the control of a liver-specific promoter/enhancer combination, optimized for transgenic expression. The AAVhu37 capsid is a member of the hepatotropic clade E family and was selected based on preclinical studies demonstrating efficient, liver-directed FVIII gene transfer, favorable biodistribution and durable FVIII expression. Here, we report safety and FVIII activity levels achieved to date in this first-in-human, dose-finding study of BAY 2599023.MethodsThe ongoing BAY 2599023 phase 1/2, open-label, dose-finding study (NCT03588299) included male patients aged ≥18 years with severe hemophilia A, no history of FVIII inhibitors, no detectable neutralizing immunity against AAVhu37 (neutralizing antibody titer ≤5), and ≥150 exposure days to FVIII products. Patients received a single intravenous infusion of BAY 2599023 and were enrolled sequentially into three dose cohorts (0.5 × 10 13 GC/kg, 1.0 × 10 13 GC/kg and 2.0 × 10 13 GC/kg), each comprising at least two patients. Patients to be enrolled in a fourth cohort will receive a single infusion of 4 × 10 13 GC/kg (Figure 1). Primary endpoints were adverse events (AEs), serious AEs (SAEs) and AEs/SAEs of special interest (S/AESIs). The secondary endpoint was FVIII activity over time. Informed patient consent and ethics committee approval were obtained.ResultsThree cohorts of ≥2 patients each (N = 9) were enrolled sequentially (Figure 1). At the data cutoff (May 2021), FVIII activity data were available for the first eight patients.BAY 2599023 delivered sustained FVIII expression levels for up to >23 months, with evidence of bleed protection. Patients in Cohorts 2 and 3 have all been off prophylaxis with FVIII products since approximately 6-12 weeks after gene transfer. To date, it has been observed that no spontaneous bleeds requiring treatment have been reported once FVIII levels >11 IU/dL were achieved. Of the 9 patients treated, 5 patients developed an AESI: mild/moderate alanine aminotransferase (ALT) elevations observed in Cohort 2 (n =1) and Cohort 3 (n = 3) were managed with corticosteroid treatment; another ALT elevation was reported as study-drug-related SAE in Cohort 3 (n = 1) but returned to normal a few weeks after interruption of the H2 blocker famotidine. The latest follow-up data for up to 28 months will be presented.ConclusionsBAY 2599023 was designed to enhance efficacy and durability of FVIII expression with a favorable safety profile. Sustained FVIII levels allowed suspension of FVIII prophylaxis in the majority of patients, with asymptomatic ALT elevations that respondedto corticosteroids, making BAY 2599023 a key candidate in the evolution of gene therapy in hemophilia A. [Display omitted] DisclosuresPipe: Genventiv: Consultancy; Regeneron/ Intellia: Consultancy; uniQure: Consultancy, Other; Spark Therapeutics: Consultancy; Takeda: Consultancy; Sanofi: Consultancy, Other; Sangamo Therapeutics: Consultancy; Roche/Genentech: Consultancy, Other; Pfizer: Consultancy; Novo Nordisk: Consultancy; Freeline: Consultancy, Other: Clinical trial investigator; HEMA Biologics: Consultancy; CSL Behring: Consultancy; Catalyst Biosciences: Consultancy; Biomarin: Consultancy, Other: Clinical trial investigator; Bayer: Consultancy; ASC Therapeutics: Consultancy; Apcintex: Consultancy; Grifols: Consultancy; Octapharma: Consultancy; Shire: Consultancy. Sheehan: BioMarin: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Bayer: Consultancy, Research Funding. Coppens: Portola/Alexion: Research Funding; CSL Behring: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Roche: Research Funding; Daiichi Sankyo: Research Funding; Sanquin Blood Supply: Research Funding; uniQure: Research Funding; Medcon International: Consultancy; MEDtalks: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Sobi: Consultancy. Eichler: Takeda: Consultancy, Honoraria; BioMarin: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Pfizer: Research Funding; Novo Nordisk: Consultancy, Research Funding; Biotest: Consultancy, Honoraria; Bayer: Consultancy, Research Funding. Linardi: Bayer: Current Employment. Wiegmann: Bayer: Current Employment. Hay: Pfizer: Consultancy, Research Funding; Inspiration: Consultancy, Honoraria; BioMarin: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Lissitchkov: Bayer: Other: Principal Investigator of Clinical Trials; Catalist: Other: Principal Investigator of Clinical Trials; Grifols: Other: Principal Investigator of Clinical Trials.

Regulation of Factor VIII By Activated Protein C: Assessment In Vitro and In Vivo

Factor VIII (FVIII) plays an essential role in the activation of factor X (FX), acting as a factor IXa (FIXa) cofactor in the intrinsic Xase complex. Activated protein C (APC) is a serine protease that inactivates FV/Va. Resistance of FV/FVa to APC due to the Leiden mutation is the most common inherited thrombophilia. By cleaving FVIII/FVIIIa at Arg336 and Arg 562, APC can also inactivate FVIII; however, the role of this reaction, especially in vivo, is not well defined. Further, thrombophilic mutations at either of these sites have yet to be identified, calling into question the importance of this reaction. Understanding the role of APC in FVIII regulation is important as there are several novel therapeutics in development for hemophilia A (HA) that either bypass FVIII regulatory pathways or target inhibition of APC. Additionally, while data using a high-specific activity factor IX variant has been successful in a hemophilia B gene transfer trial, there is no such described hyperactive FVIII protein. In principle, manipulation of APC cleavage sites may result in a more procoagulant FVIII protein with enhanced hemostastic function that may serve as a novel therapeutic for HA in the setting of a recombinant protein treatment or gene therapy.We sought to further understand the role of APC regulation of FVIII and to evaluate the in vivo hemostatic effect of an APC-resistant FVIII as a potential treatment strategy for HA. We conducted site-directed mutagenesis to introduce missense arginine to glutamine mutations at FVIII APC cleavage sites (R336Q and R562Q) on a B-domain deleted FVIII (FVIII-WT) backbone. The resulting recombinant, purified FVIII variant protein (FVIII-QQ) was then compared to FVIII-WT. Following incubation with APC, SDS-PAGE analysis of FVIII-QQ demonstrated no apparent cleavage by APC. In contrast, FVIII-WT demonstrated expected cleavage at R336 and R562, thereby confirming FVIII-QQ is resistant to APC cleavage. Surprisingly, one-stage clotting assay determination of FVIII-QQ specific activity was approximately 30% higher than FVIII-WT; this was unexpected given the absence of thrombomodulin or APC. We speculate this may be related to the ability of FXa to cleave FVIIIa at Arg336 and Arg562 inactivating FVIIIa that is likely disrupted in the FVIII-QQ variant (Eaton D et al. Biochemistry 1986 and Plantier JL et al. J Thromb Haemost 2010). To test the role of APC regulation of FVIII, we performed plasma-based in vitro thrombin generation assays in the presence of APC. Human FVIII deficient plasma was reconstituted to 1 nM (normal plasma FVIII concentration) with FVIII-QQ or FVIII-WT wherein increasing concentrations of APC were then added and thrombin generation was measured. Consistent with the known role of APC being an anticoagulant, the amount of thrombin generation decreased with increasing APC concentrations. However, the amount of APC necessary to inhibit 50% peak thrombin generation with FVIII-WT was 5.2 nM ± 0.3 nM versus FVIII-QQ was 11.3 nM ± 0.8 nM, which again demonstrated FVIII-QQ is resistant to APC inhibition. We suspect the observed decrease in thrombin generation with increasing APC concentrations is due to the role of APC in FV/FVa inhibition. Overall, our data support a role for APC inactivation of FVIIIa in this in vitro system. Interestingly, FVIII-QQ reliably demonstrated an approximate 2-fold increase in peak thrombin in the thrombin generation assay. This is being explored, but we again speculate this may be related to the effects of FXa on FVIII-WT at R336 and R562.We next evaluated the in vivo hemostatic efficacy of FVIII-WT vs. FVIII-QQ in HA mice. HA mice were infused with FVIII-WT or FVIII-QQ protein prior to tail clip assay. Comparison of both proteins at a dose of 3 µg/kg demonstrated greater blood loss in HA mice treated with FVIII-WT vs. FVIII-QQ (p=0.006). Further, blood loss in HA mice treated with FVIII-WT was greater than wild type mice (p=0.007); in contrast, there was no difference between wild type and HA mice treated with 3 µg/kg of FVIII-QQ (p=0.26).Our data demonstrate FVIII-QQ has a higher specific activity, requires 2-fold greater APC concentrations to inhibit thrombin generation in vitro and an approximate 2-fold greater in vivo hemostastic efficacy. Our results support that APC has physiologic significance in FVIII/FVIIIa inactivation and provide rationale for the study of APC in FVIII/FVIIIa regulation to develop potential novel HA therapeutics. DisclosuresDavidson:Spark Therapeutics: Consultancy. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy, Research Funding. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B.

Spk-8011: Preliminary Results from a Phase 1/2 Dose Escalation Trial of an Investigational AAV-Mediated Gene Therapy for Hemophilia a

▪Background: Hemophilia A therapy is currently based on intravenous administration of exogenous FVIII protein either on demand to treat bleeding or prophylactically to prevent bleeding. Prophylaxis has revolutionized health outcomes in hemophilia by significantly reducing the frequency of bleeding, preventing the development of arthropathy, improving health-related quality of life and enabling affected individuals to increase participation in physical activities enhancing psychosocial outcomes. The burden of repetitive infusions and the time required to perform them may be partially responsible for a strikingly high rate of non-compliance. A recent multi-center study revealed only 43% of individuals with hemophilia adhered to their prophylactic regimen (Schrijvers 2016) putting improved outcomes in jeopardy. Recombinant adeno-associated viral (rAAV) vectors have been in development for >25 years. No major safety concerns have emerged in the >100 previously conducted rAAV gene transfer clinical trials. A single administration of rAAV vector encoding human coagulation F8 or F9 gene may result in sustained expression of therapeutic factor activity levels sufficient to reduce or eliminate the need for exogenous factor infusions (Pasi 2017; George 2017).Objectives: 1) To safely obtain consistent, predictable and sustained FVIII activity (FVIII:C) >12% adequate to prevent spontaneous bleeding without the need for prophylactic FVIII infusions, manipulation of normal coagulant or anticoagulant pathways or increased thrombotic risk; 2) Minimize a dose-dependent capsid immune response by using the lowest possible vector dose that produces clinically relevant improvements in FVIII:C.Methods: SPK-8011 is a recombinant AAV vector composed of a bio-engineered capsid (AAV-Spark200) with liver specific enhanced tropism and a codon-optimized expression cassette that encodes the SQ-FVIII variant of a B-domain-deleted (BDD) human F8 gene (Lind 1995). This Phase 1/2 study is an open-label, non-randomized, dose-escalation study of SPK-8011 with a starting dose of 5x1011 vg/kg. The study is evaluating the safety, tolerability and efficacy of a single intravenous infusion of SPK-8011 in up to 18 adult men with hemophilia A (endogenous FVIII activity levels ≤2% of normal). Data on bleeding and factor infusions in the year prior to enrollment are retrospectively compiled. Laboratory values, bleeding frequency, and FVIII consumption are prospectively evaluated following vector infusion.Results: As of 8/1/17, we infused 3 subjects with SPK-8011, two at a dose of 5 x1011 vg/kg and one at a dose of 1x1012 vg/kg. Infused subjects were adult males ages 34-52 years with baseline FVIII:C <1% and Spark200 NAb titer of <1:1. The two participants infused with a vector dose of 5x1011 vg/kg have FVIII:C of 11% and 14% at 23 and 12 weeks, respectively, after infusion (Figure 1). Subject 3 is two-weeks post infusion with 1x1012 vg/kg and has not yet reached peak steady-state transgene expression. No subjects have experienced hepatic transaminase elevation, a decline in FVIII:C or required steroid intervention. ELISPOT reactivity to Spark200 capsid peptides revealed a transient low-level positivity to two different AAV peptides at week 3 and 4 in the second subject. By week 5 all ELISPOTs were negative. The positive ELISPOTs were not associated with a decrease in FVIII:C or increase in hepatic transaminases. No participants have developed a FVIII inhibitor. All subjects have discontinued FVIII prophylaxis. In the cumulative 258 days of follow up, there have been no vector or procedure-related adverse events.Conclusion: Our preliminary results from the ongoing Phase 1/2 study of SPK-8011 demonstrate FVIII:C levels in the range of 12%, sufficient in the trial to date to prevent spontaneous bleeding without the need for exogenous factor infusions, and with no evidence of a cellular immune response to transduced hepatocytes. Therapeutic transgene-derived FVIII:C was achieved at a 120-fold lower vector dose than that previously reported (Pasi 2017) in another ongoing AAV gene transfer trial for hemophilia A. Transduction in the participant dosed at 1x1012 vg/kg tracks at higher levels than observed at the same time point for participants in the low dose cohort, providing early evidence of a dose-response. No adverse events or safety concerns have been observed in the first 258 days of subject exposure to SPK-8011. [Display omitted] DisclosuresGeorge:Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B; Pfizer: Consultancy. Ragni:Alnylam, CSL Behring, BAYER, Biomarin, Biomarin, Bioverativ, Genetech/Roche, Pfizer, Shire, SPARK: Research Funding; A Anylam, Biomarin, Bioverativ, Shire: Honoraria. Cuker:Spark Therapeutics: Research Funding; T2 Biosystems: Research Funding. Cole:Spark Therapeutics: Employment. Wright:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chen:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment, Equity Ownership. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Reape:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Spk-9001: Adeno-Associated Virus Mediated Gene Transfer for Hemophilia B Achieves Sustained Factor IX with Minimal Immune Response

▪Background: Data from earlier hemophilia B (HB) AAV-mediated liver gene transfer trials demonstrated a dose-dependent, capsid-specific immune response that may result in clearance of transduced hepatocytes and loss of transgene expression (Manno et al. 2006, Mingozzi et al. 2007). This has not posed major safety concerns, but may limit efficacy. Prior work incorporated the use of steroids to abort this immune response and maintain factor IX (FIX) expression (Nathwani et al 2014). The percent of transgene expression lost increased with number of elapsed days from transaminase elevation to steroid initiation. Once vector responsiveness to steroids is established, the need for steroids is not a limitation in and of itself but highlights the requirement to reliably and expeditiously recognize an immune response and initiate steroids. Here we present our immunomonitoring data following infusion of SPK-9001, an AAV vector designed to achieve therapeutic FIX:C at a low vector dose to minimize capsid-specific T cell responses.Method: Ten HB subjects were infused with 5 x 1011 vg/kg SPK-9001, containing a transgene cassette encoding FIX-Padua that confers ~8-12 - fold higher specific activity than wild-type factor IX (Simioni et al. 2009, Crudele et al. 2016). T cell responses to the SPK-9001 AAV capsid and transgene product were monitored post-infusion using a validated interferon-γ enzyme-linked immunospot (ELISpot) assay, with a threshold for positivity of > 50 spot forming units (SFU) per 106 PBMC.Results: As of 7/26/17, 10 subjects are 26 -78 weeks post SPK-9001 infusion with mean sustained FIX:C of 33.8±18.1% (mean±SD). There were no vector or procedure-related adverse events. All subjects discontinued prophylaxis and had a significant reduction in the annualized bleeding rate (p<0.0001). No patients exhibited a significant T cell response to the transgene product, including the Padua mutation site. T cell responses against the viral capsid generally fell into 3 categories: 5 subjects had no detectable response to capsid; 3 subjects had a transient T cell response that peaked at week 2 that quickly resolved without intervention; 2 subjects demonstrated a T cell response that persisted for longer than 3 weeks. The persistent T cell responses observed in the 2 subjects correlated with an asymptomatic increase of liver transaminases. One subject was administered a course of prednisone starting on day 56, after which FIX:C levels stabilized and T cell reactivity quickly returned to baseline levels. An orthogonal peptide matrix approach identified 1 candidate AAV epitope, SEYQLPYVL, which is a MHC Class I predicted binder for the subject's HLA-B*18 haplotype according to bioinformatics prediction algorithms. This epitope is highly conserved across AAV serotypes. In response to grade I transaminase toxicity, the second subject was administered steroids starting on day 36. Concurrent ELISPOT values were elevated (1260 SFU/106 PBMC), but not associated with a decline in FIX:C and the subject maintained FIX:C in the normal range. Like the subject's LFTs, the ELISPOT response markedly decreased with steroids while his FIX:C remained stable at ~80%. This subject continues to demonstrate a low-positivity to AAV capsid peptides through 32 weeks and off steroids, but the persistent T cell response has not been associated with a decline in FIX:C or impact on overall efficacy. Further ELISPOT analysis on this subject identified 4 AAV T cell epitopes that are predicted binders for 2 of this subject's alleles, HLA-A*03 and HLA-B*07.Conclusion:We report consistent levels of sustained plasma FIX:C of 33.8±18.1% (mean±SD) in 10 subjects to date. Observed T cell responses to the AAV capsid occurred in 2/10 subjects that required steroid intervention. The early administration of immunosuppression and subsequent decrease in T cell response in the absence of a corresponding loss in FIX:C suggests that prompt recognition of a capsid immune response and early steroid intervention is critical. Furthermore, persistence of detectable T cell responses late in the time-course, with no association with a diminution in FIX:C, may suggest that circulating T cells may be present long after transduced hepatocytes cease presenting capsid peptides. Preliminary data suggest that our validated method for assessing T cell responses can detect responses that require management with steroids to ensure continuous FIX expression. DisclosuresHui:Spark Therapeutics: Employment, Equity Ownership. Liu:Spark Therapeutics: Employment. Patel:Spark Therapeutics: Employment. Chen:Spark Therapeutics: Employment. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B. Rasko:Genea: Equity Ownership; Novartis: Other: Clinical trials, Speakers Bureau; Spark: Equity Ownership, Other: clinical trials, Speakers Bureau; Rarecyte: Consultancy, Equity Ownership; President-Elect International Society for Cellular Therapy: Membership on an entity's Board of Directors or advisory committees; IMAGO Biosciences: Consultancy, Equity Ownership. Ducore:Octapharma: Research Funding; Bayer, Shire, HemaBiologics, Bioverativ, Octapharma, Spark Therapeutics: Other: Advisory board. Dasen:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Spk-9001: Adeno-Associated Virus Mediated Gene Transfer for Hemophilia B - 1 Year Follow up and Impact of Baseline Characteristics on Transgene-Derived Factor IX Activity and Persistence

▪Background: Prior work demonstrated long term expression of factor IX (mean FIX:C ~5.1%) after AAV8-mediated gene transfer in hemophilia B (Nathwani et al., 2014), but transgene-derived FIX:C fell short of trough values obtained by long-acting FIX prophylaxis (Santagostino et al . 2016) and natural history data supporting levels of ~12% sufficiently eliminate spontaneous hemarthroses (den Uijl et al. 2011). We developed an efficient capsid and expression cassette that could be administered at low doses to achieve FIX:C adequate to prevent bleeding without use of exogenous FIX. Here we report 1 year follow up data in 7 subjects following SPK-9001 infusion at a dose of 5 x 1011vg/kg. These data represent the largest cohort of hemophilia B subjects treated with gene transfer using the same vector and dose with available 1 year follow up data. We additionally report observations of subject baseline pre-vector characteristics relative to steady-state transgene-derived FIX:C and clinical outcomes.Objective: To obtain long term transgene-derived FIX:C > 12% able to prevent spontaneous bleeding without the need for prophylactic FIX infusions with consistent and predictable results among all subjects, irrespective of baseline characteristics.Methods: The study vector, SPK-9001, consists of a liver specific, bioengineered AAV capsid (Spark100) and a single-stranded codon optimized expression cassette encoding FIX-Padua. FIX-Padua is a naturally occurring variant that confers ~8-12-fold greater specific activity compared to wild-type FIX (Simioni et al. 2009, Crudele et al. 2015). Subject baseline characteristics were determined at study screening. FIX:C and clinical data were prospectively collected after vector infusion.Results: To date, 7 subjects completed 1 year follow up after SPK-9001 infusion at a dose of 5 x1011 vg/kg. No subjects developed a FIX inhibitor or experienced vector related adverse events (AEs). Subject mean FIX:C following vector infusion through 52 weeks is outlined in figure 1. Subjects were males ages 18-52 yrs with FIX:C ≤2%. One subject had a Spark 100 neutralizing antibody (NAb) of 1:1 and the remaining were <1:1. Five patients were CRM+ and 2 were CRM-. The FIX:C at steady state, defined as the average of all values measured starting at week 12, for the 5 CRM+ subjects was 32.3±11.0% (mean±SD) and 21.0±5.7% for the 2 CRM- subjects. However, the analysis is confounded by a 1:1 NAb in one CRM- subject and partial loss of FIX:C due to immune response in one of the CRM+ subjects. Steady state FIX:C for the 6 subjects with <1:1 NAb was 31.1±10.3% and 17% for the one subject with NAb 1:1. FIX:C in subjects with a history of HCV and stage 1-2 liver fibrosis (29.7±12.7%), was comparable to that of subjects with no history of HCV (28.2±10.2%). FIX:C in the one patient with HCV exposure on HIV anti-retroviral therapy did not differ from other participants (25% vs. 29.7%±11.7%). All subjects discontinued prophylaxis at vector infusion. One subject self-reported bleeding that was incongruent with the remaining cohort, including those with the similar vector-derived FIX:C and baseline hemophilic arthropathy, who did not experience bleeding events or require factor (Table 1). The 1 subject (subject 7) who experienced a suspected capsid immune response had no known baseline characteristics that differed from the cohort; the subject completed a course of steroids, had a stable, sustained FIX:C of 14% and did not experience bleeding post vector. All subjects reported significantly improved quality of life (QoL) at 1-year (p<0.023).Conclusion: As of 8/1/17, we report the highest, most consistent and sustained vector derived FIX:C following gene transfer. Despite heterogenous baseline characteristics, all subjects achieved consistent clinical results, including no vector related AEs, improved QoL, termination of prophylaxis, near complete elimination of bleeding and factor use. Low grade liver fibrosis and prior HCV exposure did not appear to affect steady-state FIX:C. Individual subject data may imply effects of CRM status and NAb titer on achieved FIX, but the current dataset is too small to define the relationship. In summary, these preliminary data suggest SPK-9001 safely, consistently, and independent of subject baseline characteristics, achieved FIX:C able to substantially ameliorate clinical manifestations of hemophilia. [Display omitted] DisclosuresGeorge:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B. Ducore:Octapharma: Research Funding; Bayer, Shire, HemaBiologics, Bioverativ, Octapharma, Spark Therapeutics: Other: Advisory board. Cuker:T2 Biosystems: Research Funding; Spark Therapeutics: Research Funding. Von Mackensen:LFB: Honoraria. McGuinn:Shire / Baxalta, Spark, Biogen, Roche/Genetehc: Research Funding; Shire/Baxalta: Consultancy. Wright:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dasen:Spark Therapeutics: Employment. Barber:Spark Therapeutics: Employment. Chen:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Patel:Spark Therapeutics: Employment. Liu:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Reape:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Genetic-Based Approaches to Inherited Metabolic Liver Diseases.

In vertebrates, the liver is the central metabolic organ of the body, which carries out an estimated 500 functions that range from general detoxification to protein synthesis, bile production, metabolism of fats, carbohydrates, proteins, bilirubin, vitamin and mineral storage and it even has an immune function. Hepatocytes are considered the professional liver cells, which carry out all of these functions. With such a variety of tasks to perform, it is not surprising that more than 400 rare monogenic disorders of hepatic origin have been described. For many of these, liver transplantation remains the only curative strategy, however, this is limited by organ availability and requires lifelong immune suppression. The fact that liver transplantation is curative led to the assumption that the restoration of the expression of the defective gene would result in the resolution of the disease. Indeed, liver-directed gene therapy trials for hemophilia A and B have demonstrated the potential of gene therapy to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Thus, liver-directed gene therapy and gene editing strategies have emerged as promising alternatives to transplantation in inherited monogenic liver disorders. Herein, we review the advances and limitations of gene therapy for such disorders, covering therapeutic strategies based on gene addition and gene editing and the exciting clinical results obtained with the use of ribonucleic acid as therapeutic molecules.

What´s new in Gene Therapy of Hemophilia.

Several methods have been investigated to effectively and safely transmit genes that stimulate cells to release therapeutic factor VIII (FVIII) and factor IX (FIX) into the circulation of people with hemophilia (PWH). To review the role of gene therapy (GT) in PWH. A Cochrane Library and PubMed (MEDLINE) search related to the role of GT in hemophilia was analyzed. The most promising vectors for hemophilia GT are adeno-associated virus (AAV) and lentivirus. Several gene methods are available to lessen risks related to random vector integration and insertional mutagenesis, based on designer nucleases or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated system). However, off-target issues need to be more meticulously and widely evaluated. Some clinical studies on hemophilia B based on AAV have obtained transitory or subtherapeutic levels of FIX expression. Another problem is possible transitory liver toxicity. Therefore, to reduce unintentional immune responses, transitory immunosuppression must be used, particularly when administering high-vector doses. Codon-optimized FVIII or FIX transgenes are able to promote clotting factor expression levels. The inclusion of a hyper-active gain-of-function R338L mutation in the FIX gene (FIX-R338L [FIX Padua]) makes the procedure more effective. Achieving a safe and efficient remedy for hemophilia A and B by means of GT vector engineering needs further improvement. No randomized or quasi-randomized clinical trials of GT for hemophilia have been found. Given it is in its incipient period, there is need for well-designed clinical trials to evaluate the long-term practicability, efficacy and risks of GT for PWH.

Perspectives on best practices for gene therapy programs.

With recent successes in gene therapy trials for hemophilia and retinal diseases, the promise and prospects for gene therapy are once again garnering significant attention. To build on this momentum, the National Institute of Neurological Disorders and Stroke and the Muscular Dystrophy Association jointly hosted a workshop in April 2014 on "Best Practices for Gene Therapy Programs," with a focus on neuromuscular disorders. Workshop participants included researchers from academia and industry as well as representatives from the regulatory, legal, and patient advocacy sectors to cover the gamut from preclinical optimization to intellectual property concerns and regulatory approval. The workshop focused on three key issues in the field: (1) establishing adequate scientific premise for clinical trials in gene therapy, (2) addressing regulatory process issues, and (3) intellectual property and commercialization issues as they relate to gene therapy. The outcomes from the discussions at this workshop are intended to provide guidance for researchers and funders in the gene therapy field.

Prevention of Inhibitor Development Using Apoptosis-Induced FVIII Producing ES Cells.

[Background] Hemophilia A is an X-linked bleeding disorder resulting from abnormality in the coagulation factor VIII (FVIII) gene. Development of inhibitors remains a significant problem for a treatment of hemophilia A. Approximately 25–30% of sever type hemophilia A patients develop inhibitor antibodies that reduce or completely negate the benefits of replacement therapy. Furthermore, a major issue that all gene therapy trials for hemophilia are facing is the risk of forming inhibitory antibodies to the transgene product. Although immune tolerance induction (ITI) is the effective method to decrease inhibitor concentration, it costs too expensively and there are some patients who do not show any response to ITI. Thus, it is desired to establish a novel immune tolerance induction method. Recent immunological studies have pointed out that “danger signal” is required to initiate immune reactions and immune tolerance is induced in the absence of danger signal. Apoptotic cell death is considered to be associated with immune tolerance because processing of intracellular antigen occurs without danger signal. Recently, Miyake et al. reported that intravenous injection of apoptotic cells expressing a fragment of myelin oligodendrocyte glycoprotein (MOG) on cell membrane reduced MOG-specific T cell response and prevented the development of experimental autoimmune encephalomyelitis (EAE) [J Clin Invest 2007; 117:2268–78.]. Based on these principles and findings, we investigated the novel immune tolerance approach with pretreatment of apoptotic embryonic stem (ES) cells secreting FVIII.[Methods] All animal experiments were performed in accordance with institutional and national regulations and approved by the Nara Medical University Animal Care Committee. We have already established mouse ES cells secreting human FVIII by integrating human FVIII gene [Kasuda et al, J Thromb Haemost 2008]. As a pretreatment, hypo-osmotic stress-induced apoptotic ES cells were injected intraperitoneally into the FVIII-KO mouse. After the various periods, administration of hFVIII concentrates (4 IU/body) was repeated every week. Then, titration of inhibitor was measured chronologically with Bethesda methods.[Results] In the non-pretreatment group (n=15), the inhibitor titer was 0.03 ± 0.00 BU/ml at 1 week after first administration of hFVIII concentrates. Then, the inhibitor titer rose to 0.18 ± 0.03 BU/ml and 3.03 ± 0.12 BU/ml with every following hFVIII administration. On the other hand, in the pretreatment group #1 (hFVIII administration was begun 1 week after pretreatment, n=9), inhibitor titer showed the significantly low levels (0.54 ± 0.27 BU/ml) even after thrice administration of hFVIII (p<0.05). Furthermore, in another pretreatment group #2 (hFVIII administration was begun 2 weeks after pretreatment, n=3), inhibitor titer showed much lower levels (0.09 ± 0.03 BU/ml) after thrice administration (p<0.01).[Discussion] We showed the possibility of reduction of inhibitor titer with pretreatment of apoptotic hFVIII secreting ES cells. The efficacy for the reduction of inhibitor titer depended on the period between pretreatment with apoptotic cells and first hFVIII administration, suggesting that a specified period is required to induce immune tolerance. Unlike the method using a peptide fragment of target protein (MOG) in the previous report by Miyake et al., our method features the utilization of a whole target protein (FVIII), by which tolerance to multiple epitopes of the protein would be induced. Identification of the precise peptide epitopes of a target protein would not be necessary since selection and presentation are executed by the host's own antigen presenting cells. We aspire for the complete suppression of inhibitor formation with an ingenious administration of apoptotic ES cells. We believe that this approach has potential for future clinical therapy.

FVIII Tolerance Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Encode a High-Expression Factor VIII Transgene in Hemophilia A Mice.

Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al., Blood 2007). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of >100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (>2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

Adeno-Associated Virus-Mediated Gene Transfer of Factor IX for Treatment of Hemophilia B by Gene Therapy

IntroductionPatients with severe hemophilia have circulating blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B) levels below 1% of normal due to a genetic defect in the respective X-linked gene. The resulting bleeding disorder is characterized by spontaneous joint bleeds or, in a more life-threatening situation, into critical closed spaces, such as the intracranial or retroperitoneal space. Current treatment for hemophilia is based on intravenous infusions of clotting factor concentrates. These can be episode-based in response to bleeds (which does not prevent ongoing tissue damage nor the risk of a life-threatening bleed) or prophylactic (an expensive and not always practical alternative). The goal of a gene-based therapy is to introduce a functional clotting factor gene into a patient in order to provide a continuous supply of factor levels above 1%.1,2 Clinical endpoints for the efficacy of potential gene therapy trials for hemophilia are, therefore, well-defined and unequivocal.The relatively small size of the factor IX coding sequence (1.4 kb) and the fact that a number of cell types other than hepatocytes (which normally synthesize factor IX) are capable of producing biologically-active factor IX have contributed to the development of hemophilia B into an important model for the treatment of genetic diseases by gene therapy. The factor IX gene can be incorporated into a variety of vector systems. Various target tissues can be chosen for gene transfer as long as the secreted factor IX reaches the circulation and tight regulation of transgene expression is not required.3 Possibly most important in research on gene therapy for coagulation factor deficiencies, and genetic disorders in general, is the availability of a large animal model with severe disease. In this case, it is the well-characterized hemophilia B dogs maintained at the University of North Carolina at Chapel Hill. The animals contain a point-mutation in the portion of the factor IX gene encoding the catalytic domain. This mutation results in an absence of circulating factor IX antigen and, consequently, severe hemophilia B that closely mimics the human disease.4 Gene therapy strategies for hemophilia B have typically established a method of gene transfer, resulting in expression of factor IX in mice, and subsequently, attempted scale-up to the dog model. These investigations have established experiments in the hemophilic dog model as a critical step for the assessment of the efficacy of gene therapy protocols showing initial promise in mice. For example, reimplantation of primary myoblasts that had been transduced ex vivo with a retrovirus was successful in mice, but not in the canine model.5 Adenoviral gene transfer, characterized by varying success in mice, depending on the strain and dose used, has persistently resulted in high, but transient expression following intravenous infusion into dogs.6,7 Cellular immune responses and hepatotoxicity have limited the expression of factor IX from adenoviral vectors to just a few weeks. Repeat administration of the vector was complicated by the induction of neutralizing antibodies to viral particles in injected animals following the first administration. Retroviral gene transfer to hepatocytes was successful in long-term expression of factor IX in hemophilia B dogs but required a partial hepatectomy prior to infusion of the vector through the portal vein. The resulting expression levels were no higher than 0.1% of normal human factor IX levels.8