Gene Transfer For Hemophilia Research Articles

Induction of factor VIII tolerance by hemophilia gene transfer to eradicate factor VIII inhibitors

Patients with hemophilia A can develop anti-factor antibodies to factor VIII. The incidence is ∼30% and such patients suffer worse morbidity and mortality. The only proven method to eradicate these inhibitors is via immune tolerance induction therapy which consists of infusing factor VIII concentrates at a regular intervals. This approach is effective ∼65% of the time leaving at least a third of patients who develop inhibitors with this lifelong problem. Although emicizumab has greatly improved the quality of life of inhibitor patients, eradicating the inhibitor remains an important treatment goal. Animal models have shown the potential for gene therapy to induce tolerance. A recent abstract describing a study in humans demonstrated the potential for successful tolerance induction. This article will describe the rationale for utilizing gene therapy to induce tolerance and provide this author's viewpoint on the importance and possible historic significance of attempting to eradicate inhibitors with this approach.

TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling

Upon viral infection of the liver, CD8+ Tcell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ Tcells. Surprisingly, we find local initiation of a CD8+ Tcell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1β contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ Tcells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral Tcell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

Gene transfer in hemophilia B: a big step forward

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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.

Long-Term Durable FVIII Expression with Improvements in Bleeding Rates Following AAV-Mediated FVIII Gene Transfer for Hemophilia A: Multiyear Follow-up on the Phase I/II Trial of SPK-8011

Background: Hemophilia A (HA) is an inherited blood disorder caused by deficiency or dysfunction of factor (F)VIII. The disease is characterized by recurrent bleeding, particularly into joints, which often results in arthropathy and functional disability. HA is currently managed with intravenous (IV) infusions of replacement FVIII or non-factor replacement therapy such as emicizumab. More recently, gene therapy, involving the use of a recombinant adeno-associated viral (AAV) vector to target hepatocytes, with the aim of safely imparting long-term stable FVIII expression to prevent bleeding, is being investigated for HA. SPK-8011 is an investigational AAV vector optimized to achieve clinically relevant and durable hepatocyte-derived FVIII expression at the lowest possible vector dose. Methods: This Phase I/II trial (NCT03003533/NCT03432520) of SPK-8011 is an open-label, multicenter, non-randomized trial, the full methodology of which has been reported previously (George et al. N Engl J Med 2021). Eligible participants were males ≥18 years of age, with FVIII levels ≤2%, >150 exposure days to FVIII concentrates, and no FVIII inhibitors. All participants were negative for neutralizing antibodies to SPK200. Participants were enrolled into four dose cohorts receiving a single IV dose of either 5×1011, 1×1012, 2×1012, or 1.5×1012 vector genomes (vg) per kilogram of body weight, with a maximum dose based on a BMI of 30 kg/m2. Several immunomodulation (IM) approaches were evaluated. Trial objectives included evaluation of the safety and efficacy of SPK-8011 and the extent and durability of FVIII expression. Results: At data cut-off (30 June 2022), 23 participants were included across dosing cohorts of 5×1011 vg/kg (n=2), 1×1012 vg/kg (n=3), 1.5×1012 vg/kg (n=9), or 2×1012 vg/kg (n=9). Median (range) observation time was 159.3 (6.9-259.9) weeks, based on both the primary study 8011-101 and the long-term follow-up study. A total of 49 treatment-related adverse events (AEs) were reported; 23 were considered vector-related, including one serious adverse event (Grade 2 alanine transaminase [ALT] elevation resulting in elective hospitalization for IV corticosteroid administration), and 26 were related to IM therapies. No FVIII inhibitors or thrombotic events were reported. Transient non-sustained asymptomatic ALT elevation occurred in 11 participants during the expected window of presumed capsid immune response and resolved with use of IM agents. In 16 participants with ≥1 year follow-up, a one-stage FVIII assay showed no apparent decrease in FVIII activity over time, with a majority expressing in the mild HA range (Table). Sustained expression of FVIII was maintained in 21/23 participants with several methods of IM; two participants lost FVIII expression following a presumed capsid immune response (George et al. N Engl J Med 2021). Across all 21 participants with sustained FVIII expression, a 91% (95% CI: 78-97%) reduction in annualized bleeding rate (ABR) for all bleeds was observed, with all-bleed ABR of 11.59 (7.39-18.17) before infusion vs. 0.99 (0.46-2.14) after infusion (Figure). The 16 participants on prophylactic treatment before gene therapy had an 82% (95% CI: 53-93%) reduction in ABR for all bleeds; 6.88 (4.16-11.36) before vs. 1.26 (0.57-2.77) after infusion. ABRs for spontaneous bleeds were 5.20 (2.99-9.04) and 0.52 (0.19-1.39) before and after infusion, respectively. The five participants using on-demand treatment before infusion had a 99% (95% CI: 98-100%) reduction in all-bleed ABR; 27.26 (18.80-39.53) vs. 0.20 (0.09-0.45) before and after infusion, respectively, and 20.98 (12.29-35.82) vs. 0.05 (0.01-0.29) for spontaneous bleeds. In total, 17/21 (81%) participants had an ABR of <1 for treated bleeds and 20/21 (95%) had an ABR of <1 for spontaneous treated bleeds following infusion of SPK-8011. Median (IQR) annualized FVIII infusion rates (AIRs) of 0.2 (0.0-1.2) were reported across all participants 28 days post vector infusion vs. 82.0 (36.0-106.0) before vector administration. Conclusions: With up to 5 years of follow-up, a single infusion of SPK-8011 resulted in durable year-to-year FVIII expression and clinically meaningful reductions in ABR and AIR. No major safety signals were reported, indicating SPK-8011 was well tolerated in this population of people with HA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gene transfer in hemophilia A: not cogent yet

The phase 3 clinical study of gene transfer in hemophilia A using the BioMarin vector AAV5-hFVIII-SQ (also identified as valoctocogene roxaparvovec) recently reported the results obtained in as many as 134 adult men with severe hemophilia A, so that it is at the moment the largest gene transfer study ever conducted in a rare monogenic disorder [...].

Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice

AbstractHemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Multiyear Factor VIII Expression after AAV Gene Transfer for Hemophilia A

BackgroundThe goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose.MethodsIn this phase 1–2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5×1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2×1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII.ResultsThe median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti–AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], −2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration).ConclusionsSustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.)

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.

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.

Stable Elevations in FIX Activity and Reductions in Annualized Bleeding Rate over up to 2 Years of Follow-up of Adults with Severe or Moderate-Severe Hemophilia B Treated with AMT-060 (AAV5-hFIX) Gene Therapy

▪Background: Gene transfer for hemophilia offers the potential to convert the disease from a severe to mild phenotype with a single treatment. AMT-060 consists of an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized wildtype human factor IX (FIX) gene under control of a liver-specific promoter.Aim: This phase 1/2 study investigates the safety and efficacy of AMT-060 at 2 dose levels in adults with moderate-severe or severe hemophilia B. Updated data with up to 2 years follow up will be presented.Methods: Multi-national, open-label, dose-escalating study in patients (pts) with FIX activity ≤2 IU/dL of normal, and a severe bleeding phenotype (prophylactic exogenous FIX; or on-demand exogenous FIX, plus ≥4 bleeds/year or hemophilic arthropathy). Pts received either 5x1012 gc/kg (Cohort 1, n=5) or 2×1013 gc/kg (Cohort 2, n=5) of AMT-060 iv. Efficacy assessments include FIX activity (measured ≥10 days after use of exogenous FIX); reduction of FIX use; and annualized spontaneous bleeding rates. Safety assessments include treatment related adverse events, immunological and inflammatory biomarkers.Results: Nine pts with severe (FIX <1%) and 1 with moderate-severe (FIX 1.5%) hemophilia B were enrolled and received AMT-060. At study entry, 9 pts were on FIX prophylaxis, and 1 pt with severe hemophilia B in Cohort 2 used FIX on demand. Following AMT-060, mean FIX activity in the lower dose cohort was 4.6 IU/dL (95% CI 1.6-7.6) during 1.5 year of follow-up, and 7.1% (95% CI 3.2-11.1) in the higher dose cohort during 1 year of follow-up with stable FIX protein expression across both cohorts. Disease severity improved in all pts: severe to mild (n=5), severe to moderate (n=4), and from moderate to mild (n=1). Eight of 9 pts on FIX prophylaxis at study entry discontinued use after AMT-060 infusion. Across all pts, the total annualized reduction of exogenous FIX use post-AMT-060 and discontinuation of prophylaxis was 79% (85% in Cohort 1 and 68% in Cohort 2). Mean annualized spontaneous bleeds were reduced from 9.8 in the year prior to AMT-060 to 4.2 in the 1.5 years after treatment for pts in Cohort 1 (58% reduction), compared with a 54% reduction at the previously-reported 1-year follow up, while total bleeds were reduced by 54%. In 4 pts in Cohort 2, annualized spontaneous bleeds were reduced from a mean of 3.0 to 0.5 (84% reduction), compared with a 70% reduction at the previously-reported 6-month follow up, with no spontaneous bleeds reported in the last 6 months of follow up. Total bleeds were reduced by 64%. One pt in Cohort 2 was not included in the calculation as historical bleed data was not available; he experienced 1 traumatic bleed after discontinuation of prophylaxis. No pts developed inhibitors to FIX and there were no detectable signs of sustained AAV5 capsid-specific T-cell activation. Six pts (3 in each cohort) experienced a total of 14 treatment-emergent adverse events classified as possibly/probably related to treatment, most of which were classified as mild (n=11) and some as moderate (n=3). Mild, temporary elevations in ALT not associated with changes in FIX activity or capsid-specific T-cell responses were observed in 3 pts with higher mean FIX activity (6.4-12.7 IU/dL; 1 in the lower- and 2 in the higher-dose cohort). Each received a tapering course of prednisolone. ALT elevations have not recurred. Additional efficacy and safety data will be presented up to 2- and 1.5-years of follow-up for Cohorts 1 and 2, respectively.Conclusions: Longer-term outcomes for up to 1.5 years following a single infusion of AMT-060 continue to indicate a positive safety profile coupled with stable and clinically meaningful elevations in FIX protein expression and activity without evidence of capsid-specific immune responses. Bleeding rates declined over time, with a near cessation of spontaneous bleeds in the high dose cohort. DisclosuresMeijer:BMS: Consultancy; Boehringer Ingelheim: Consultancy; Sanquin: Consultancy; Bayer: Consultancy; Uniqure BV: Consultancy; Aspen: Consultancy. Coppens:Bayer: Consultancy; CSL Behring: Consultancy; Uniqure BV: Consultancy. Kampmann:Uniqure BV: Research Funding. Klamroth:Shire: Consultancy; Biotest: Consultancy; SOBI: Consultancy; Pfizer: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Chiesi: Consultancy; Octapharma: Consultancy; Novo Nordisk: Consultancy. Schutgens:Uniqure BV: Research Funding; CSL Behring: Research Funding; Pfizer: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; Baxalta/Shire: Research Funding. Castaman:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Sawyer:Uniqure BV: Employment.

Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy.

Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector’s DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.

Hemophilia gene therapy-New country initiatives.

Gene therapy is an opportunity for haemophilia patients to receive a one-time treatment and have lasting factor levels for years or decades instead of dependence on repeated administration within short intervals and on sustained supply of drug. Great strides have been made in the development of gene therapy for haemophilia in the last decade. Adeno-associated virus (AAV) vector-mediated gene transfer in haemophilia A and B has entered the phase III trial stage. Gene transfer by lentiviral vector or gene editing technologies using factor VIII (FVIII) or IX (FIX) genes are now entering clinical evaluation. It is expected that the first FVIII and FIX gene therapy products will soon be approved and distributed in major markets. Global access to gene therapy is a critical goal. This review presents new and ongoing efforts towards this goal in countries other than North America and Europe. In Japan, researchers, regulators and funders have established a promising gene therapy development platform for multiple diseases including haemophilia. Decades of scientific and clinical research in haemophilia gene therapy in China have led to a recently registered clinical trial of AAV-mediated gene therapy for haemophilia B. Other countries are in earlier phases of building gene therapy programmes or participate in international trials. A phase 2 feasibility trial of AAV-mediated FIX gene therapy in low- and middle-income countries aims to demonstrate that gene therapy could become available in resource-constrained socio-economic settings. The different strategies for establishing gene therapy provide opportunities for closing the global gap in haemophilia care.

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

A Novel Lysine to Arginine Substitution at Position 301 Enhances Activity of Factor IX

Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex.Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration.Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants.Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. DisclosuresVerhoef:Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Phase 1/2 Trial of Investigational Spk-8011 in Hemophilia a Demonstrates Durable Expression and Prevention of Bleeds

A Phase 1/2 Trial of Investigational Spk-8011 in Hemophilia a Demonstrates Durable Expression and Prevention of Bleeds

Adeno-Associated Mediated Gene Transfer for Hemophilia B:8 Year Follow up and Impact of Removing “Empty Viral Particles” on Safety and Efficacy of Gene Transfer

▪Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients.Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX <1%) were enrolled into the first and mid-dose cohorts, with six patients treated at the high dose. In a follow-on study arm, two severe HB subjects received a dose of 2x1012vg/kg of the new scAAV2/8-LP1-hFIXco preparation (full: empty capsid ratio 1:3) whilst the next 2 patients were treated at a dose of 5x1012vg/kg. In both arms, vector was administered without prophylactic immunosuppression but corticosteroids (starting at 60mg/day) were commenced if liver enzymes increased to ≥2 fold over baseline levels after gene transfer.Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids.Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. DisclosuresNathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Hemophilia gene therapy comes of age

Concurrent with the development of recombinant factor replacement products, the characterization of the F9 and F8 genes over 3 decades ago allowed for the development of recombinant factor products and made the hemophilias a target disease for gene transfer. The progress of hemophilia gene therapy has been announced in 3 American Society of Hematology scientific plenary sessions, including the first "cure" in a large animal model of hemophilia B in 1998, first in human sustained vector-derived factor IX activity in 2011, and our clinical trial results reporting sustained vector-derived factor IX activity well into the mild or normal range in 2016. This progression to clinically meaningful success combined with numerous ongoing recombinant adeno-associated virus (rAAV)-mediated hemophilia gene transfer clinical trials suggest that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized. Although several novel therapeutics have recently emerged for hemophilia, gene therapy is unique in its potential for a one-time disease-altering, or even curative, treatment. This review will focus on the prior progress and current clinical trial investigation of rAAV-mediated gene transfer for hemophilia A and B.

Hemophilia gene therapy comes of age.

Concurrent with the development of recombinant factor replacement products, the characterization of the F9 and F8 genes over 3 decades ago allowed for the development of recombinant factor products and made the hemophilias a target disease for gene transfer. The progress of hemophilia gene therapy has been announced in 3 American Society of Hematology scientific plenary sessions, including the first "cure" in a large animal model of hemophilia B in 1998, first in human sustained vector-derived factor IX activity in 2011, and our clinical trial results reporting sustained vector-derived factor IX activity well into the mild or normal range in 2016. This progression to clinically meaningful success combined with numerous ongoing recombinant adeno-associated virus (rAAV)-mediated hemophilia gene transfer clinical trials suggest that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized. Although several novel therapeutics have recently emerged for hemophilia, gene therapy is unique in its potential for a one-time disease-altering, or even curative, treatment. This review will focus on the prior progress and current clinical trial investigation of rAAV-mediated gene transfer for hemophilia A and B.