Gene Therapy in Hemophilia: An Assessment of Hematologists' Knowledge Gaps and Attitudes

Abstract BACKGROUND: Currently available treatments for hemophilia A and B require frequent intravenous infusions. Gene therapy, by contrast, offers the hope of a functional cure through endogenous expression of the factor VIII or factor IX genes. To identify and address practice gaps among healthcare providers who manage patients with hemophilia, a collaboration between the National Hemophilia Foundation (NHF), European Haemophilia Consortium (EHC), World Federation of Hemophilia (WFH), and Medscape Education was established. The objective of the current study is to assess clinicians' needs with regard to understanding the science and mechanism for gene therapy, the different technologies being proposed, identifying candidates for gene therapy, disease areas for which gene therapy is being explored, and the latest data on the potential role of gene therapy in hemophilia A and B. METHODS: A continuing medical education (CME)-certified clinical practice assessment comprising 25 multiple choice questions that measured knowledge, attitudes, and perspectives about gene therapy was developed. The survey instrument was made available online to physicians without monetary compensation or charge. Respondent confidentiality was maintained and responses were de-identified and aggregated prior to analyses. The activity launched on March 03, 2018; data through July 27, 2018 are presented, but data collection is ongoing. RESULTS: At the time of this analysis, 194 physician participants indicated that they actively manage patients with hemophilia. Demographics are presented in Table 1; confidence and attitudes about gene therapy in Table 2. Among hematologists/oncologists (n=54; hem/onc), identified knowledge gaps include, 46% could not identify the liver as the tissue/cell type that is the primary target for gene therapy in hemophilia46% thought that current approaches to gene therapy in hemophilia involved some form of host gene editing39% did not realize that gene therapy strategies only affect somatic cells and not the germline22% thought current gene therapy strategies would prevent or reduce the severity of hemophilia in subsequent generations of patients33% were unable to correctly identify adeno-associated virus (AAV) as the viral vector that is the current basis for the majority of gene therapy trials in hemophilia81% had misconceptions regarding the key characteristics of the AAV vector56% did not recognize that the AAV vector does not routinely integrate into the host genome19% were unfamiliar with the maximum gene cassette capacity of AAV57% were unfamiliar with the results from the pivotal University College London (UCL)/St Jude trial of scAAV2/8-LP1-hFIXcovector in patients with hemophilia B63% were unfamiliar with the clinical trial data for SPK-8011 in hemophilia A67% were unfamiliar with the results at the 12 month time-point for valoctocogene roxaparvovec in hemophilia A trials57% had misconceptions with regard to the causes for and treatment of ALT elevations seen in hemophilia AAV gene therapy trials CONCLUSIONS: Gene therapy has the potential to fundamentally alter the management of hemophilia. This educational activity identified clear deficits about - and attitudes toward - gene therapy among healthcare providers who currently care for patients with hemophilia. Furthermore, the great majority of healthcare providers lacked confidence in their understanding of gene therapy for hemophilia A. These findings will be used to inform the development of educational programs and to prepare providers for anticipated changes to the hemophilia treatment landscape. Disclosures Pasi: Catalyst Bio: Honoraria; Octapharma: Honoraria; NovoNordisk: Speakers Bureau; Bioverativ: Honoraria, Research Funding; Apcintex: Honoraria; Shire: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; Sobi: Honoraria; Biomarin: Honoraria, Research Funding; Alnylam: Honoraria, Research Funding.

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Gene therapy for hemophilia

Hematology ◽

10.1182/hematology.2019000007 ◽

2019 ◽

Vol 2019 (1) ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Amit C. Nathwani

Keyword(s):

Gene Therapy ◽

Hemophilia A ◽

Phase 1 ◽

Substantial Reduction ◽

Factor Ix ◽

Clotting Factor ◽

High Dose ◽

Single Administration ◽

Spontaneous Bleeding ◽

Endogenous Expression

Abstract Gene therapy offers the potential for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX (FIX) following transfer of a functional gene to replace the hemophilic patient’s own defective gene. The hemophilias are ideally suited for gene therapy because a small increment in blood factor levels (≥5% of normal) is associated with significant amelioration of bleeding phenotype in severely affected patients. In 2011, the St. Jude/UCL phase 1/2 trial was the first to provide clear evidence of a stable dose-dependent increase in FIX levels in patients with severe hemophilia B following a single administration of adeno-associated viral (AAV) vectors. Transgenic FIX expression has remained stable at ∼5% of normal in the high-dose cohort over a 7-year follow-up period, resulting in a substantial reduction in spontaneous bleeding and FIX protein usage without toxicity. This study has been followed by unparalleled advances in gene therapy for hemophilia A and B, leading to clotting factor activity approaching normal or near-normal levels associated with a “zero bleed rates” in previously severely affected patients following a single administration of AAV vectors. Thus, AAV gene therapies are likely to alter the treatment paradigm for hemophilia A and B. This review explores recent progress and the remaining limitations that need to be overcome for wider availability of this novel treatment of inherited bleeding disorders.

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Human-Cl Rhfviii Effectively and Safely Prevents Bleeding Episodes in Previously Treated Adult Patients with Severe Haemophilia A

Blood ◽

10.1182/blood.v120.21.1132.1132 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1132-1132

Author(s):

Sigurd Knaub ◽

Toshko Lissitchkov ◽

Kingsley Hampton ◽

Mario Von Depka ◽

Savita Rangarajan ◽

...

Keyword(s):

Gene Therapy ◽

Research Funding ◽

Prophylactic Treatment ◽

Hemophilia A ◽

Haemophilia A ◽

Severe Haemophilia ◽

Open Label ◽

Bleeding Rate ◽

Previously Treated

Abstract Abstract 1132 The main purpose of this prospective, multi-center, open-label phase 3 study was to assess the efficacy of prophylactic treatment with Human-cl rhFVIII, the first human cell-line derived recombinant FVIII, in previously treated patients (PTPs) with severe haemophilia A. Patients were to receive 30–40 international units (IU) FVIII of Human-cl rhFVIII per kg every other day for 6 months. Efficacy of preventing and treating bleeds were judged using objective criteria taking the monthly bleeding rate and the number of infusions needed to manage a break-through bleed into account. In-vivo recovery (IVR) was determined at the beginning of the study and after 3 and 6 months. FVIII:C was measured by validated chromogenic (CHR) and one-stage (OS) assays in a central laboratory, which also assigned drug potencies. Inhibitor activity was determined using the Nijmegen modification of the Bethesda assay before the first administration and at defined intervals thereafter. Thirty-two patients between 18 and 75 years of age were enrolled from 11 centres in Europe and treated prophylactically for 6.0±0.9 months (mean ± SD) with a mean prophylactic dose of 32.8 IU/kg. Sixteen patients never bled, 11 patients bled once and 5 more than once. The mean total and spontaneous monthly bleeding rate was 0.188±0.307 and 0.095±0.211, respectively. Efficacy of the prophylactic treatment was “excellent” in all patients for spontaneous BEs and “excellent” or “good” in all patients but one for all types of bleeds. All treatments of bleeds were rated as “excellent” (71.4%) or “good” (28.6%). The IVR at baseline was 2.6±0.5 % per IU/kg for the CHR and 2.2±0.5 % per IU/kg for the OS assay and remained stable during the study. A total of 2921 infusions were given in the study. Human-cl rhFVIII was well tolerated and no patient experienced a related serious adverse event. No FVIII inhibitors were detected. Conclusion: The data indicate that Human-cl rh FVIII is safe and efficacious in preventing and treating bleeds in PTPs with severe haemophlia A. Disclosures: Knaub: Octapharma AG: Employment. Lissitchkov:Octapharma AG: PI Other. Tuddenham:College London: Consultancy, Employment, Gene therapy for hemophilia A, Gene therapy for hemophilia A Patents & Royalties, Research Funding. Collins:Octapharma AG: Consultancy. Oldenburg:d and e: Baxter, Bayer, Biotest, CSL-Behring, Grifols, Inspiration, NovoNordisk, Octapharma, Pfizer e: Biogen IDec, Swedish Orphan Biovitrum: Honoraria, Research Funding. Bichler:Octapharma AG: Employment.

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Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy

Blood ◽

10.1182/blood-2010-06-288001 ◽

2010 ◽

Vol 116 (26) ◽

pp. 5842-5848 ◽

Cited By ~ 100

Author(s):

Jonathan D. Finn ◽

Margareth C. Ozelo ◽

Denise E. Sabatino ◽

Helen W. G. Franck ◽

Elizabeth P. Merricks ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Neutralizing Antibodies ◽

Viral Vectors ◽

Hemophilia A ◽

Major Complication ◽

Complete Disappearance ◽

Anamnestic Response ◽

Endogenous Expression ◽

Liver Gene

Abstract Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

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Emizicumab Promotes Factor Xa Generation on Activated Endothelium in a Blood Cell-Independent Manner

Blood ◽

10.1182/blood-2021-148556 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3182-3182

Author(s):

Patrick Ellsworth ◽

Dougald Monroe ◽

Maureane Hoffman ◽

Nigel S Key

Keyword(s):

Clinical Trial ◽

Endothelial Cells ◽

Growth Medium ◽

Research Funding ◽

Hemophilia A ◽

Coagulation Factor ◽

Standard Of Care ◽

Factor Ix ◽

Adhesive Film ◽

Trial Investigator

Abstract Introduction Hemophilia A (HA) is an inherited bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII) resulting in severe hemorrhage if untreated. Recombinant and plasma derived FVIII products have long been the standard of care in hemophilia. However, approximately 25-30% of patients with severe HA develop inhibitors, neutralizing alloantibodies to FVIII, a significant complication in the treatment of patients with HA that leads to bleeding despite factor therapy. First approved for bleed prophylaxis in HA with inhibitors in the US by the FDA in 2018, emicizumab (Genentech, USA) has initiated a new era of HA treatment. This drug is a bispecific, monoclonal antibody that binds to activated Factor IX (FIXa) and Factor X (FX), mimicking activated FVIII (FVIIIa) by bringing FIXa and FX into proximity to enable FX activation, even in the presence of inhibitors. Emicizumab prophylaxis drastically reduces bleed episodes. However, thromboses and thrombotic microangiopathy (TMA) were observed in trials, all associated with concomitant use of activated prothrombin complex concentrates (aPCC) (Callaghan et al., 2021). The mechanism of this devastating condition is uncertain, as emicizumab is not known to bind to phospholipid or vascular surfaces. We report that FX is more readily activated by FIXa and emicizumab on endothelium that has been activated by tumor necrosis factor alpha (TNF). This finding may partially explain the development of TMA in these patients. Methods We utilized novel, microfluidic devices that are inexpensive to manufacture and were modified from a technique previously described (Alapan et al. 2016). These devices are fabricated using a laser cut double-sided adhesive film sandwiched between a clear, gas-permeable polymer (Ibidi, Germany) and an acrylic top that is laser cut (Universal Laser Systems Inc., USA) (Figure 1). Human umbilical endothelial cells (HUVEC, Lonza, Switzerland) were harvested at passage 3 to 4 and seeded into the devices. These were then cultured under flow conditions using a non-peristaltic, air-driven pump (Ibidi GmbH, Germany) to achieve a confluent and quiescent endothelial surface. HUVEC are then activated by incubating with 5 nM TNF in serum-free growth medium for 4 hours. This treatment induced markers of endothelial activation without gross apoptosis. Non-activated HUVEC were incubated with endothelial cell growth medium (2% serum) until time of experiments. Factors IXa, X (Haemtech, USA), and/or emicizumab (discarded clinical material) were mixed in HEPES-buffered saline with 5 mM calcium chloride for all experimental conditions. Concentrations used of FIXa (30 nM), FX (170 nM), and emicizumab (55 ug/mL) were constant for all conditions. Combinations of factors and emicizumab were then incubated in the endothelialized device for 30 minutes at 37° C. The entire volume of the mixture was then aspirated (20 uL) and stored at -80° C. FXa activity was assayed on the effluent for 60 minutes using a chromogenic FXa substrate (Pefachrome, Pentapharm, Switzerland). Results No significant generation of Xa was noted in the presence of healthy or activated endothelium with emicuzumab alone, emicizumab and FIXa, emicizumab and FX, or factors IXa and X. Median Xa generation observed with the combination of emicizumab, FIXa, and FX on healthy endothelium was 2 nM. Median Xa generation with the same combination on activated endothelium was 8.1 nM, a four-fold increase (P = 0.028, Mann-Whitney test) (Figure 2). Discussion Emicizumab represents an evolving standard of care for hemophilia A. Considering data showing diminishing FVIII expression in the months to years after AAV gene therapy, (Pasi et al., 2020) it may well be the dominant treatment paradigm in HA for some time. However, much remains to be answered in the use of emicizumab, and the mechanism of thrombosis and TMA with concomitant aPCC use has resulted in the avoidance of aPCC use for breakthrough bleeding in patients on emicizumab therapy, even up to 6 months after cessation. Our data demonstrate that activated endothelial cells promote FX activation more readily than quiescent endothelial cells in the presence of FIXa and emicizumab. These findings demonstrate the potential of thrombotic angiopathy in an in vitro system. Further investigation of the interaction of endothelium with FIXa, FX, and FVIIIa-mimetic antibodies is warranted. Figure 1 Figure 1. Disclosures Ellsworth: Takeda: Other: Salary supported as part of NHF-Takeda Clinical Fellowship Award. Monroe: Medexus Pharmaceuticals: Consultancy; Takeda: Consultancy; Otello Medical: Current equity holder in publicly-traded company. Hoffman: Takeda: Research Funding; CSL Behring: Consultancy; Sanofi: Consultancy; BPL (Bio Products Laboratory): Consultancy. Key: BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator; Sanofi: Consultancy.

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Phase 1-2 Clinical Trial of a Recombinant AAV5 Vector Containing the Human FIX Gene in Patients with Severe or Moderately Severe Haemophilia B

Blood ◽

10.1182/blood.v124.21.5948.5948 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5948-5948 ◽

Cited By ~ 3

Author(s):

Wolfgang A. Miesbach ◽

Christian Meyer ◽

Bart Nijmeijer ◽

Florence Salmon ◽

Nadina Grosios ◽

...

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Research Funding ◽

Factor Ix ◽

Activity Level ◽

Severe Haemophilia ◽

Haemophilia B ◽

Commercial Scale ◽

Number Of Patients ◽

History Of

Abstract Gene therapy has been successfully used in a research setting in a limited number of patients with severe haemophilia B, primarily using vectors based on adeno-associated virus (AAV) serotype 2 and 8. Several research groups and companies are exploring new developments to improve current treatment strategies to eventually make gene therapy available for a larger number of patients. Pre-existing immunity to AAV capsid proteins may limit the availability of such therapies to small sub-groups of patients. A low prevalence of natural neutralizing antibodies against AAV5 compared with other serotypes has been demonstrated (Calcedo et al., Clin Vaccine Immunol 2009, 18:1586-1588). We use a recombinant AAV5 containing the codon optimized human factor IX gene (AAV5-hFIX), using a baculovirus production process that allows commercial scale manufacturing of the AAV5-hFIX drug product. Human FIX expression levels in macaques treated i.v. with AAV5-hFIX at a dose of 5 × 10^12 gc/kg were high enough to expect therapeutic effect on the haemophiliac phenotype in haemophilia B patients. Such levels were achieved in humans during AAV8-hFIXco clinical study (Nathwani et al., NEJM 2011, 365:2357-2365). hFIX levels in dose-escalating GLP toxicity studies in cynomolgus monkeys and mice showed linear dose responses using doses up to 1 × 10˄14 and 2.3 × 10^14 cg/kg, respectively, and no specific safety concerns. In human studies with AAV5 containing the human porbilinogen deaminase (PBGD) gene, administered in doses up to 2 x 10^13 gc/kg to patients with acute intermittent porphyria, no safety concerns were raised (D’Avola et al., ASGCT, Washington DC, 2014). Importantly, in this study no liver enzyme perturbations were observed following administration of AAV5. The primary objective of the proposed study is to investigate the safety of systemic administration of AAV5-hFIX to adult patients with severe or moderately severe haemophilia B. The multicentre trial has an open-label, uncontrolled, single-dose, dose-ascending design and consists of two cohorts. The study population consists of male patients, aged ≥18 years, with severe or moderately severe haemophilia B and a severe bleeding phenotype [i.e. known FIX deficiency with plasma FIX activity level <1% (severe), or plasma FIX activity level ≥1% and ≤2% (moderately severe) and currently on prophylactic FIX replacement therapy for a history of bleeding, or currently on on-demand therapy with a current or past history of frequent bleeding defined as four or more bleeding episodes in the last 12 months, or chronic haemophilic arthropathy. Patients should have had more than 150 previous exposure days of treatment with FIX protein. Subjects are allocated to one of two cohorts with the following planned dose levels: Cohort 1 (5 subjects) with AAV5-hFIX 5.0 × 10^12 gc/kg and Cohort 2 (5 subjects) with AAV5-hFIX 2.0 × 10^13 gc/kg. Key efficacy assessments include factor IX plasma levels, the need for FIX replacement therapy, incidence of spontaneous bleedings and health-related quality of life measurements. In conclusion, AAV5-hFIX produced in commercial scale represents a novel approach to gene therapy of haemophilia B. Disclosures Miesbach: uniQure: Consultancy; Bayer: Research Funding; Baxter: Research Funding; CSL Behring: Research Funding; Biotest: Research Funding; Octapharma: Research Funding. Meyer:uniQure B. V.: Employment. Nijmeijer:uniQure B. V.: Employment. Salmon:uniQure B. V.: Employment. Grosios:uniQure B. V.: Employment. Petry:uniQure B. V,: Employment. Leebeek:uniQure B. V.: Consultancy; CSL Behring: Research Funding; Baxter: Research Funding.

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Employing Factor IX Variants to Avoid Limitations Imposed by Immune Recognition of AAV Vector in Hemophilia B Gene Therapy

Blood ◽

10.1182/blood.v118.21.3124.3124 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3124-3124 ◽

Cited By ~ 1

Author(s):

Paul E. Monahan ◽

Junjiang Sun ◽

Tong Gui ◽

David G Wichlan ◽

Scott W McPhee ◽

...

Keyword(s):

Clinical Trial ◽

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Ongoing Clinical Trial ◽

Advisory Committees

Abstract Abstract 3124 Persistent factor IX expression and phenotypic improvement have been achieved in a human clinical trial for hemophilia B using liver-directed adeno-associated virus (AAV) gene therapy vectors. An ongoing clinical trial uses a vector incorporating self-complementing AAV (scAAV) genome form, factor IX codon optimization (FIXopt) and AAV serotype 8 capsid. As was seen in a previous single-strand AAV serotype 2 trial, dose escalation has been associated with apparent immune-mediated transient inflammation of vector-transduced liver, although in contrast to the previous trial persistent FIX expression has been maintained for the first time. Taken together, these important trials define a consistent threshold load of AAV capsid that has stimulated capsid-specific cytotoxic lymphocyte recognition and potential transaminitis. To advance the successes achieved in these trials while providing a clear margin of safety so that this immunogenic threshold need not be approached, we have pursued steps to limit further the AAV capsid load. Single amino acid substitutions at arginine 338 in the FIX catalytic domain generate FIX variants with increased specific activity. We separately substituted either R338A, R338Q, or R338L (FIX Padua) into a codon optimized human factor IX cDNA and evaluated F.IX expression in tissue culture following plasmid DNA transfection of HEK 293t cells. Each R338 substitution improved FIX specific activity, up to 10 times increased over wild type using the R338LFIXopt cDNA. We next generated scAAV8 vectors incorporating a liver-specific transthyretin (TTR) promoter to express optimized codon F.IX cDNA with or without the R338L substitution. FIX−/− mice receiving portal vein injection of 1 × 1010 vg/animal (4 ×1011 vg/kg) expressed 86.5% of normal FIX activity at 2 months post-transduction from the WTopt vector and 330% normal from the R338LFIXopt. Incorporation of R338Lopt variant resulted in at least 6 to 10 fold increase in FIX specific activity over a follow-up of > 40 weeks. At ten months following FIX gene delivery, mice underwent a tail transection bleeding challenge. FIX vector mice demonstrated therapeutic protection from this major bleeding challenge and furthermore all survived with no late rebleeding (a hallmark of hemophilic phenotype). Greater than 100% normal human FIX activity was maintained for >40 weeks following treatment with the R338LFIX vector (v. 26.3% at euthanasia in WTopt vector group). The prolonged follow-up permitted extended safety evaluation. Factor IX inhibitor antibodies were not detected in any mice throughout the follow-up; FIX-binding IgG1 and IgG2 were negative also. Thrombin/antithrombin III complexes (TAT) examined at 12 weeks and at >30 weeks of age in R338LFIXopt vector mice did not differ from levels in WTFIXopt vector-treated or age-matched C57Bl/6 hemostatically normal mice. Necropsy at 40–44 weeks after vector (1 year of age) showed only age-related changes with no microvascular or macrovascular thrombosis on H&E staining or specific immunostaining for fibrin/fibrinogen deposition; specific staining for fibrosis within myocardium or other sites was negative. We next synthesized a R338LFIXopt expression cassette containing the LP1 promoter/enhancer/intron sequence being used in the ongoing clinical trial and demonstrated equivalent FIX activity from either promoter construct. We then established that the R338LFIXopt vector gives a predictable dose-response across a range of doses as low as 1x 1010 vg/kg I.V. and as high as 4 × 1012 vg/kg I.V. Hemarthrosis is the most common bleeding complication in hemophilia and leads to chronic joint destruction. Bleeding was induced in the joint of FIX−/− mice that had been transduced 4 weeks earlier with the R338LFIX vector. Joints were collected at 2 weeks after induced bleed and the bleeding-induced joint damage was graded using an established histologic score. I.V. R338LFIXopt vector pretreatment resulted in protection against joint degeneration in a dose-dependent fashion in this most relevant clinical scenario. These preclinical studies demonstrate a safety :efficacy profile to advance hemophilia gene therapy using the scAAV8.R338LFIXopt vector. Disclosures: Monahan: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asklepios BioPharmaceutical: Patents & Royalties, Research Funding; CSL Behring: Honoraria; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaIN: Research Funding; Prolor-Biotech: Research Funding. McPhee:Asklepios Biopharmaceutical: Employment. Samulski:Asklepios Biopharmaceutical: Employment, Patents & Royalties.

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Bioengineering of coagulation factor VIII for improved secretion

Blood ◽

10.1182/blood-2003-10-3591 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3412-3419 ◽

Cited By ~ 135

Author(s):

Hongzhi Z. Miao ◽

Nongnuch Sirachainan ◽

Lisa Palmer ◽

Phillip Kucab ◽

Michael A. Cunningham ◽

...

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Hemophilia A ◽

Coagulation Factor ◽

Mrna Levels ◽

Golgi Transport ◽

Recombinant Fviii ◽

Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

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Updated Follow-up of the Alta Study, a Phase 1/2 Study of Giroctocogene Fitelparvovec (SB-525) Gene Therapy in Adults with Severe Hemophilia a

Blood ◽

10.1182/blood-2020-137648 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12

Author(s):

Andrew D. Leavitt ◽

Barbara A. Konkle ◽

Kimo Stine ◽

Nathan Visweshwar ◽

Thomas J. Harrington ◽

...

Keyword(s):

Gene Therapy ◽

Stock Options ◽

Research Funding ◽

Hemophilia A ◽

Phase 1 ◽

Severe Hemophilia ◽

Bleeding Events ◽

Dose Cohort ◽

Fviii Activity

Introduction: Hemophilia A is a rare bleeding disorder caused by pathogenic variants in the F8 gene, resulting in insufficient factor VIII (FVIII) activity. Adeno-associated virus (AAV)-mediated gene transfer enables the delivery of a modified functional F8 gene to hepatocytes that subsequently synthesize FVIII at levels that may prevent bleeding events in the absence of exogenous FVIII. Updated results and follow-up from the Alta study, an ongoing gene therapy study in patients with severe hemophilia A, are presented. Methods: The Alta study is a phase 1/2 dose-ranging, single-dose study of giroctocogene fitelparvovec (also known as SB-525 and PF-07055480), a recombinant AAV serotype 6 (rAAV6) vector encoding a modified F8 gene. Adults aged ≥18 years with severe hemophilia A were eligible for inclusion. Giroctocogene fitelparvovec was infused into patients in 4 cohorts of 2 patients each across 4 ascending doses (9e11, 2e12, 1e13, and 3e13 vg/kg). The 3e13 vg/kg dose cohort was expanded with 3 additional patients. Key end points included safety, circulating FVIII activity, use of FVIII replacement therapy, and frequency of bleeding events. Presented data are from the ongoing Alta study (NCT#03061201; data cutoff date, 26 May 2020; database not locked; data reflect those at time of data cutoff, have not undergone standard quality checks, and may be subject to change). Results: Eleven male patients participated in the study (mean [SD] age, 30.3 [7.8] years; white, 81.8%). As of the cutoff date, patients have been followed for 35 to 144 weeks; one patient in the 1e13 vg/kg cohort discontinued from the study. Overall, the most commonly reported adverse events (AEs; n) included increased alanine aminotransferase (ALT; 8 [72.7%]), increased aspartate aminotransferase (AST; 5 [45.5%]), upper respiratory tract infection (4 [36.4%]), and pyrexia (4 [36.4%]). Treatment-related serious AEs were reported in 1 patient (in the 3e13 vg/kg cohort) who experienced hypotension and fever ≈6 hours after giroctocogene fitelparvovec infusion; the events fully resolved with treatment and did not delay post-infusion discharge. In the 3 lower-dose cohorts, no ALT elevation requiring more than 7 days of corticosteroid treatment was observed. Of the 5 patients in the 3e13 vg/kg cohort, 4 had elevations in ALT that were managed with a tapering course of corticosteroids (ranging from 10-134 days) without loss of clinically relevant FVIII activity through 40 weeks, as evidenced by a lack of bleeding events before and after treatment with corticosteroids. Increases in FVIII activity from baseline were generally dose-dependent. Patients in the 3e13 vg/kg cohort achieved a mean normal-range of FVIII activity within 5 weeks post-infusion, with mean FVIII activity maintained through week 40, which is the last time point with data for all 5 patients in this cohort (Table). Following the initial prophylactic period of up to ≈3 weeks after giroctocogene fitelparvovec administration, no bleeding events occurred in any patient treated in the 3e13 vg/kg cohort. Use of FVIII replacement therapy ≥3 weeks after giroctocogene fitelparvovec administration was reported in 5/6 patients in the lower-dose cohorts (range: 9-115 infusions); none of the patients in the 3e13 vg/kg cohort required FVIII replacement beyond initial use of prophylactic factor for up to ≈3 weeks (prophylactic coverage stopped 3 weeks and 2 days after giroctocogene fitelparvovec administration in 1 patient in the 3e13 vg/kg cohort). Conclusions: To date, a single infusion of giroctocogene fitelparvovec gene therapy in patients with severe hemophilia A resulted in dose-dependent and sustained increases in FVIII levels without administration of exogenous FVIII, bleeding episodes or sustained adverse events in the highest-dose cohort (3e13 vg/kg). Additionally, patients treated in the highest-dose cohort achieved a mean FVIII activity in the normal range within 5 weeks, which was maintained through week 40. Data on all patients with more than 1 year of follow-up will also be presented. The study is ongoing, and these interim results support further development of giroctocogene fitelparvovec for the treatment of patients with severe hemophilia A. Disclosures Leavitt: BioMarin: Membership on an entity's Board of Directors or advisory committees. Konkle:Sanofi: Consultancy, Research Funding; Takeda: Research Funding; Uniquire: Research Funding; CSL Behring: Consultancy; BioMarin: Consultancy; Baxalta: Research Funding; Spark: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Sigilon: Consultancy, Research Funding; Roche: Consultancy. Stine:Biomarin: Consultancy; Applied Stem Cell Therapeutics: Consultancy. Visweshwar:Biogen Idec: Membership on an entity's Board of Directors or advisory committees. Giermasz:uniQure: Consultancy, Research Funding; Sangamo Therapeutics: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding, Speakers Bureau; BioMarin: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, Research Funding, Speakers Bureau. Arkin:Pfizer: Current Employment, Current equity holder in publicly-traded company, Other: own stock/options in the company. Fang:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Plonski:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Smith:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Tseng:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Di Russo:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Cockroft:Sangamo Therapeutics: Current Employment, Other: Shareholder of Sangamo Therapeutics. Rupon:Pfizer Inc.: Current Employment, Other: own stock/options in the company. Rouy:Sangamo Therapeutics: Current Employment, Other: Shareholder of Sangamo Therapeutics.

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Etranacogene Dezaparvovec (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B: Two Year Data from a Phase 2b Trial

Blood ◽

10.1182/blood-2020-139295 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 13-13

Author(s):

Annette von Drygalski ◽

Adam Giermasz ◽

Giancarlo Castaman ◽

Nigel S. Key ◽

Susan U. Lattimore ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Factor Ix ◽

Hemophilia B ◽

Advisory Committees ◽

Adeno Associated Virus ◽

Human Factor Ix ◽

Virus Serotype ◽

Serotype 5

Background: Gene therapy for hemophilia offers the possibility to ameliorate disease severity to a mild or functionally curative state through a single administration. Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter. Aims: We have previously shown a single dose of etranacogene dezaparvovec to provide sustained FIX activity into the mild-to normal range for up to 52 weeks post-dose in participants with severe or moderate-severe hemophilia B. This time, 2 years of follow-up data will be presented for the first time. Methods: A Phase 2b, open-label, single-dose, single-arm, multi-center trial (NCT03489291) in adult hemophilia B subjects. Interestingly, participants were not excluded based on neutralizing antibodies to AAV5. All subjects received a single intravenous dose of etranacogene dezaparvovec (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly to a mean of 31% at Week 6. At Week 52, mean FIX activity increased further to 41% with FIX activity levels of 50%, 31% and 41% in participants 1-3 respectively. There was no relationship between the presence of anti-AAV5 NAbs and response to etranacogene dezaparvovec. As of 52 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to etranacogene dezaparvovec and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 2 years of follow-up will be presented with the main focus on FIX activity, FIX replacement therapy use and reported bleeds. Conclusions: Patients with AAV5 NAbs were included in the Phase 2b etranacogene dezaparvovec trial and have shown sustained FIX activity into the mild-to normal range. All participants were able to discontinue routine prophylaxis, and there have been no bleeds post-treatment with etranacogene dezaparvovec. Disclosures Giermasz: BioMarin: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, Research Funding, Speakers Bureau; uniQure: Consultancy, Research Funding; Sangamo Therapeutics: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding, Speakers Bureau. Castaman:Alexion: Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Baxalta/Shire: Honoraria; Bayer: Honoraria; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kedrion: Speakers Bureau; Werfen: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau. Key:Novo Nordisk: Other: Chair of Grants Committee; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy. Miesbach:Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Recht:Spark: Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; uniQure: Consultancy, Other: personal fees, Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Pfizer: Consultancy, Other: personal fees; Genentech: Consultancy, Other: personal fees, Research Funding; CSL Behring: Consultancy, Other: personal fees. Gomez:Global Blood Therapeutics: Speakers Bureau. Gut:uniQure: Current Employment. Pipe:Siemens: Research Funding; Medical and Scientific Advisory Council to the National Hemophilia Foundation; Medical Advisory Board to World Federation of Hemophilia: Membership on an entity's Board of Directors or advisory committees; Apcintex, Bayer, BioMarin, Catalyst Biosciences, CSL Behring, HEMA Biologics, Freeline, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, Inc., Sangamo Therapeutics, Sanofi, Takeda, Spark Therapeutics, uniQure: Consultancy. OffLabel Disclosure: Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter.

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Gene Therapy for Hemophilia A and B: Patient Selection and Follow-up, Requirements for a Cure

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615882 ◽

1999 ◽

Vol 82 (08) ◽

pp. 572-575 ◽

Cited By ~ 5

Author(s):

Jeanne Lusher

Keyword(s):

Gene Therapy ◽

Factor Viii ◽

Hepatitis A ◽

Hemophilia A ◽

The United States ◽

Factor Ix ◽

Clotting Factor ◽

R Factor ◽

New Variant ◽

Clotting Factor Concentrates

IntroductionThe treatment of hemophilia A and B has improved considerably in recent years. The availability of hepatitis A and B vaccines, safer clotting factor concentrates (particularly recombinant factor VIII and recombinant factor IX concentrates), and synthetic agents, such as desmopressin,1 has resulted in earlier, more aggressive treatment and prophylactic regimens aimed at preventing chronic, debilitating joint disease.2-8There have been no new cases of human immunodeficiency virus (HIV) disease attributable to clotting factor in North America since 1987, and documented instances of hepatitis transmission by clotting factor concentrates have been rare in the 1990s. Concerns remain that certain nonenveloped viruses, such as human parvovirus B19 and hepatitis A virus, can still be transmitted by some plasma-derived clotting factor concentrates,9and questions linger as to whether the agents causing Creutzfeld-Jacob disease (CJD) and new variant CJD might also be transmitted. Overall, however, the products available to treat hemophilia today are safer than ever before.An increasing number of persons with hemophilia are receiving exclusively recombinant (r) products, and manufacturers are now producing new, second-generation r-factor VIII products that are stabilized with sugars, rather than albumin, or are smaller, truncated molecules.10 Scientists are now designing specific changes into the factor VIII genes in an attempt to derive unique and improved forms of r-factor VIII.11 The next logical areas of focus are to bring to fruition the promise of an “unlimited supply” of r-factor VIII and r-factor IX products, to meet the needs of persons with hemophilia, not only in developed countries, but throughout the world, and to be able to cure hemophilia through gene therapy.As gene therapy trials begin in humans with hemophilia, the scientists involved, the United States Food and Drug Administration (FDA), and perhaps most importantly, members of the hemophilia community must decide which categories of affected individuals should be entered in these trials, particularly the earliest, Phase I trials. Who is most likely to benefit if gene therapy proves to be both effective and safe? Who should be the first patients to be enrolled in each new trial? Who is at greatest risk if something unexpected happens? What would be considered a good outcome? Clearly, some of these questions are more difficult to answer than others.

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