scholarly journals Hemophilia A Dogs Tolerant to Human Factor VIII Provide a Unique Model to Determine Efficacy and Safety of AAV Delivery of Novel Factor VIII Variants

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3628-3628
Author(s):  
Giang N. Nguyen ◽  
Lauren E. Wimsey ◽  
Elizabeth P. Merricks ◽  
Katherine P. Ponder ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Immune Response ◽  
Animal Model ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Specific Activity ◽  
Large Animal Model ◽  
Large Animal ◽  
Efficacy And Safety ◽  
Therapy Studies

The hemophilia dog models are valuable for evaluating the efficacy of novel hemophilia therapeutics. The hemophilia B dog was predictive of the therapeutic dose of adeno-associated viral (AAV) vector delivery of human factor IX in clinical trials. In addition, it provides an opportunity to study the long-term efficacy and safety after AAV administration. However, there are several challenges in using the hemophilia A (HA) dog model for gene therapy studies. First, canine factor VIII (cFVIII) has higher specific activity and increased rate of secretion compared to human FVIII (hFVIII). This significant difference between cFVIII and hFVIII prevents the use of a species-specific transgene to predict the efficacy of AAV-hFVIII. Second, the HA dogs are immunocompetent and develop an immune response to the xenoprotein, hFVIII, that precludes the ability to measure transgene expression. Therefore, in order to employ this valuable model for gene therapy studies, we generated a unique cohort of HA dogs that are tolerant to B-domain deleted (BDD) hFVIII. We hypothesized that tolerizing dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response to a novel hFVIII variant. To tolerize the dogs to hFVIII, neonatal HA dogs were treated with a retrovirus (RV-hAAT-hFVIII-BDD-WPRE, 3x109 TU/kg) (n=5) on day 2 of life. The hFVIII expression was between 0.3%-6% at 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. To determine if the dogs were tolerant to hFVIII-BDD, the dogs were challenged with hFVIII-BDD protein at 5-6 months post-RV administration (Xyntha, 25IU/kg per wk x 6 wks, I.V.). Anti-hFVIII antibodies were monitored closely throughout the challenge and up to 8 weeks after the last challenge. In 4 out of 5 dogs, no anti-hFVIII immune response was observed based on IgG1, IgG2, total IgG or Bethesda titer. In contrast, naïve HA dogs (n=2) developed high level anti-hFVIII IgG2 (1.2-3.2 μg/mL), total IgG (3.4-5.0 μg/mL), and Bethesda titer (4.1-67.8 BU/mL) after the same challenge regimen. Interestingly, the hFVIII activity in one RV-treated dog (V06) was undetectable at 6 months post-RV administration. After the challenge, V06 had anti-hFVIII IgG2 (1.7 μg/mL), total IgG (2.6 μg/mL), and a Bethesda titer (9.5 BU/mL), suggesting that FVIII must be maintained to achieve tolerance. These dogs were used to evaluate the efficacy of AAV serotype 8 (AAV8) delivery of a hFVIII-BDD codon-optimized sequence driven by a hepatocyte promoter, modified transthyretin promoter (TTRm). S29 was delivered AAV8-TTRm-hFVIII-CO (2x1012 vg/kg). Prior to AAV delivery, the levels of hFVIII activity were 0.5-1% from the tolerization with the RV. After AAV administration the hFVIII activity was 3.8% at d168 and 4.7% at d387, resulting in a 4% increase in hFVIII expression. No anti-hFVIII antibodies were detected. The annual bleeding rate (ABR) for S29 post-RV delivery was 5 and after AAV delivery was 0, showing an improvement in the bleeding phenotype in contrast to untreated HA dogs (ABR=13, n=11). A hFVIII-tolerized littermate, S28, was recently treated with a hFVIII variant, AAV8-TTRm-hFVIII-CO-Δ3-SP/DE (2x1012 vg/kg). The hFVIII-Δ3-SP/DE variant has a deletion of the furin site (1645-47) and replaces residues SD at 1657-58 with PE. This variant showed higher specific activity (2-fold) in vitro and increased secretion (4-fold) compared to wild type hFVIII-BDD in the setting of AAV delivery in HA mice. Based on the results in S28, we will determine the dosing of V26 and V27. These studies demonstrate that sustained low level hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII, while allowing for the subsequent assessment of AAV efficacy. A clinically relevant dose of AAV8-TTRm-hFVIII-CO resulted in therapeutic levels of hFVIII expression while ongoing studies will allow investigation of the efficacy of the hFVIII-BDD variant, Δ3-SP/DE, in the setting of AAV administration in a large animal model. Overall, these studies demonstrate that RV-targeting of hFVIII-BDD expression to the liver in neonatal HA dogs leads to tolerance to this xenoprotein and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of novel FVIII variants. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

2001 ◽  
Vol 85 (01) ◽  
pp. 125-133 ◽  
Author(s):  
Huiyun Wu ◽  
Mark Reding ◽  
Jiahua Qian ◽  
David Okita ◽  
Ernie Parker ◽  
...  
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Th2 Cells ◽  
Tween 20 ◽  
Sorbitan Monolaurate ◽  
Human Factor Viii ◽  
Ifn Γ ◽  
Th1 And Th2

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

scholarly journals Generation of a Unique Cohort of Hemophilia A Dogs Tolerant to Human FVIII for Evaluating the Safety and Efficacy of AAV Delivery of Wild Type and Variant Human FVIII

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2453-2453
Author(s):  
Giang N. Nguyen ◽  
Lauren Wimsey ◽  
Elizabeth Merricks ◽  
Katherine P. Ponder ◽  
Timothy C. Nichols ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Hemophilia A ◽  
Factor Ix ◽  
Common Mutation ◽  
Large Animal ◽  
Efficacy And Safety ◽  
Aav Vector ◽  
Human Factor Ix

Abstract The hemophilia A (HA) dogs have been a valuable model for evaluating the efficacy of novel hemophilia therapeutics. These dogs have a mutation that is analogous to the most common mutation in humans with severe disease, the intron 22 inversion. The advantages of the hemophilia dog model include: (1) it was predictive of the therapeutic adeno-associated viral (AAV) vector dose in human factor IX (hFIX) clinical trials, (2) it is an outbred immunocompetent species, (3) it demonstrates a clinical bleeding phenotype consistent with patients, including hemarthrosis, and (4) it permits long-term investigation of both efficacy and safety. Our previous studies delivering AAV-canine factor VIII (cFVIII) in the HA dogs demonstrated long-term (up to 10 years of follow-up) dose-dependent cFVIII expression without evidence of an immune response to the cFVIII protein. In hemophilia B preclinical studies, the comparable biological characteristics between canine FIX and hFIX allows preclinical results using cFIX to be translated to clinical studies. In contrast, for hemophilia A, our studies of recombinant cFVIII and human FVIII (hFVIII) proteins demonstrate that cFVIII is a more stable protein that has higher biological activity and is secreted better than hFVIII (Sabatino et al. 2009). Thus, expression of cFVIII following AAV delivery does not accurately predict the therapeutic dose of AAV-hFVIII which is relevant for translation to clinical trials. The challenge of administering AAV-hFVIII to the HA dogs is that this expressed xenoprotein (hFVIII) results in inhibitor formation that precludes the ability to measure transgene (hFVIII) expression. We hypothesized that tolerizing HA dogs to hFVIII will (1) permit accurate evaluation of hFVIII expression and thus predict the therapeutic vector dose and (2) allow the evaluation of the potential immune response of AAV8-hFVIII versus a novel hFVIII variant that has increased activity and secretion (Nguyen et al. 2017). In this study we used a neonatal retroviral (RV) delivery approach to tolerize the HA dogs (n=5) to B-domain deleted hFVIII (hFVIII-BDD). HA neonatal male dogs (S28, S29, V06, V26, V27) were treated with the retrovirus (RV-hAAT-hFVIII-BDD-WPRE) (3x10e9 TU/kg) on day 2 of life. The levels of hFVIII expression after RV delivery were 0.3-6% 4 weeks after RV delivery and plateaued after 6 months to 0.8% (S28), 0.3% (S29), 0% (V06), 1.5% (V26) and 1.7% (V27) based on Coatest assay. At 5-6 months of life the dogs (S28, S29, V06) were challenged with hFVIII-BDD (Xyntha; 25IU/kg; I.V.) weekly for 6 consecutive weeks. Samples were collected before and 15 minutes after each protein challenge to demonstrate the successful infusion of the protein. At 4 weeks after the final challenge, no anti-hFVIII IgG1 or IgG2 antibodies were detected consistent with no evidence of an inhibitor. At 4.5 years of age, S28 and S29 had stable expression of 0.5-1% of hFVIII and were rechallenged with hFVIII-BDD (Xyntha; 25IU/kg per week x 6 wks). No anti-hFVIII IgG1 or IgG2 antibodies were detected 4 weeks after the final protein challenge. Thus, these data demonstrate that all of the RV-hAAT-hFVIII-BDD-WPRE treated dogs that have been challenged (n=3) have been tolerant to hFVIII-BDD. Since the goal of this study is to generate a cohort of HA dogs tolerant to hFVIII-BDD to address the efficacy and safety of AAV8-hFVIII-BDD versus a hFVIII-BDD variant, we treated the first dog (S29) 12 weeks after the second series of protein challenges with an optimized AAV vector cassette containing a codon-optimized hFVIII sequence with a modified transthyretin (TTRm) promoter, AAV8-TTRm-hFVIII-BDD (2x10e12 vg/kg). Prior to AAV administration the hFVIII activity was 0.2%. At 8 weeks post AAV administration, the hFVIII activity increased to 3.5%. No anti-hFVIII-BDD IgG1 or IgG2 was detected after AAV-hFVIII administration. These data demonstrate that low levels of sustained hFVIII expression of 0.2-2% up to 4 years post-retroviral delivery were able to induce and maintain tolerance to hFVIII. Overall, these studies demonstrate that the neonatal HA dogs treated with a retrovirus targeting hFVIII expression to the liver are tolerant to hFVIII and provide a unique large animal model to evaluate both efficacy as well as potential immunogenicity of our novel FVIII variants. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

Gene Transfer of Canine FVIIa Results in Partial Correction of Canine Hemophilia: A Model for FVIII/FIX Gene-Based Bypassing Agents.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 195-195 ◽  
Author(s):  
Paris Margaritis ◽  
Elise Roy ◽  
Harre D. Downey ◽  
Shangzen Zhou ◽  
Elizabeth Merricks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Animal Model ◽  
Gene Transfer ◽  
Hemophilia A ◽  
Active Form ◽  
Factor Ix ◽  
Factor Vii ◽  
Large Animal Model ◽  
Large Animal ◽  
Partial Correction

Abstract Despite its extensive use particularly in the management of hemophilic inhibitor patients, recombinant Factor VIIa (rhFVIIa) infusion has important limitations stemming from the nature of FVIIa itself, since its short half-life necessitates repeated injections and also carries high treatment costs. To overcome these, we have designed a gene transfer approach using a modified FVII transgene that is cleaved intracellularly and secreted in the active form, FVIIa. Using the human and murine analogue of this engineered transgene we have shown phenotypic correction of hemophilia B mice, following adeno-associated virus (AAV) - mediated, liver-directed gene delivery (Margaritis et al., 2004). In order to demonstrate efficacy in a large animal model of hemophilia, we cloned the canine Factor VII cDNA and generated the canine homologue of our modified transgene (cFVIIa). Recombinant cFVII zymogen and cFVIIa were purified and characterized in vitro in a clotting-based assay using canine reagents only (activated partial thromboplastin time [aPTT]). We found that cFVIIa had activity indistinguishable from rhFVIIa, while cFVII zymogen had negligible activity (5% rhFVIIa). In order to demonstrate in vivo efficacy, we produced 4 lots of an AAV8-based vector directing liver-specific expression of cFVIIa with similar vector titers (2–5 E13 vector genomes [vg]/ml). In hemophilia A (HA) or B (HB) mice, tail-vein delivery of 0.3 – 1.2 E12 vg/mouse (1.2 – 4.8 E13 vg/kg) resulted in long-term normalization of the hemophilic phenotype, demonstrating that cFVIIa can correct the defect in murine hemophilia. We proceeded to infuse 4 hemophilia dogs, with increasing vector doses: HB male (2.06 E13 vg/kg); HA male (6.25 E13 vg/kg); HA female (1.25 E14 vg/kg); HA male (1.25 E14 vg/kg). None of the dogs showed any adverse effects following vector delivery at any dose (the initial HB dog has been followed for almost 2 years [ongoing]). We followed the level of gene expression by clotting assays (prothrombin time [PT]/aPTT) and whole blood clotting time (WBCT). The initial dose of 2.06 E13 vg/kg resulted in a transient reduction in the PT/aPTT/WBCT. A considerable and sustained reduction in PT (18 sec, normal is ∼25 sec), aPTT (19 sec, normal is ∼30 sec, hemophilic is >40sec) and WBCT (25min, normal is ∼15min, hemophilic is >40min) was observed following administration of 6.25 E13 vg/kg in an HA male dog. Two more HA dogs were infused with 1.25 E14 vg/kg (male and female). The female HA dog exhibited only a modest decrease in aPTT (22sec), despite the vector dose increase, and a reduction in WBCT (30min), an observation that could be due to previously described gender-specific effects on gene expression. From preliminary and ongoing observations, the male HA dog infused also exhibited a decrease in WBCT. As an efficacy endpoint, the dogs exhibited a total of 3 bleeding episodes (none likely to be spontaneous, occurred in the lowest dose HB dog) in a cumulative time period of 38.5 months, compared to the expected 16 episodes (Brunetti-Pierri et al., 2005). In summary, our results demonstrate for the first time that gene transfer using a Factor VIII/Factor IX bypassing agent (canine FVIIa) can result in partial correction of the hemophilic phenotype in a large animal model of hemophilia.

scholarly journals Specific-Pathogen-Free Pigs as an Animal Model for Studying Chlamydia trachomatis Genital Infection

2005 ◽  
Vol 73 (12) ◽  
pp. 8317-8321 ◽  
Author(s):  
Daisy Vanrompay ◽  
Thi Q. T. Hoang ◽  
Liselotte De Vos ◽  
Kristel Verminnen ◽  
Taher Harkinezhad ◽  
...  
Keyword(s):  
Immune Response ◽  
Animal Model ◽  
Chlamydia Trachomatis ◽  
Immunoglobulin M ◽  
Large Animal Model ◽  
Genital Infection ◽  
Large Animal ◽  
Specific Pathogen Free ◽  
Humoral Immune ◽  
Specific Pathogen

ABSTRACT The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.

Mechanism and Kinetics of Factor VIII Inactivation: Study With an IgG4 Monoclonal Antibody Derived From a Hemophilia A Patient With Inhibitor

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 496-506 ◽  
Author(s):  
Marc G. Jacquemin ◽  
Benoı̂t G. Desqueper ◽  
Abdellah Benhida ◽  
Luc Vander Elst ◽  
Marc F. Hoylaerts ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immune Response ◽  
Factor Viii ◽  
Rate Constant ◽  
Hemophilia A ◽  
Specific Activity ◽  
Major Complication ◽  
Dissociation Rate ◽  
Igg Antibody ◽  
Cell Repertoire

Abstract The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4κ monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass ∼7.4 × 105M−1 s−1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss ≤1 × 10−5s−1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

scholarly journals Adeno-Associated Viral Vector Delivery of Optimized Human Factor VIII Achieves Therapeutic Factor VIII Levels in Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 199-199 ◽  
Author(s):  
Xavier M. Anguela ◽  
Liron Elkouby ◽  
Raffaella Toso ◽  
Marti DiPietro ◽  
Robert J. Davidson ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Ix ◽  
Large Animal ◽  
Wild Type ◽  
Employment Equity ◽  
Human Factor Viii ◽  
Equity Ownership

Abstract Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x1011 vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x1012 vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x1012 vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x1013 vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Elkouby:Spark Therapeutics, Inc.: Employment, Equity Ownership. Toso:Spark Therapeutics, Inc.: Employment, Equity Ownership. DiPietro:Spark Therapeutics, Inc.: Employment, Equity Ownership. Davidson:Spark Therapeutics: Consultancy. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Sabatino:Spark Therapeutics, Inc.: Research Funding.

scholarly journals The diversity of the immune response to the A2 domain of human factor VIII

Blood ◽  
2013 ◽  
Vol 121 (14) ◽  
pp. 2785-2795 ◽  
Author(s):  
Rebecca C. Markovitz ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Shannon L. Meeks ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Entire Surface ◽  
Human Factor Viii ◽  
Key Points ◽  
A2 Domain

Key Points The Abs to the human fVIII A2 domain in a murine hemophilia A model inhibit fVIIIa and activation of fVIII Epitopes targeted by hemophilia A mouse Abs cover nearly the entire surface of the human fVIII A2 domain

scholarly journals Recombinant canine B-domain–deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4562-4565 ◽  
Author(s):  
Denise E. Sabatino ◽  
Christian Furlan Freguia ◽  
Raffaella Toso ◽  
Andrey Santos ◽  
Elizabeth P. Merricks ◽  
...  
Keyword(s):  
Factor Viii ◽  
Immune Tolerance ◽  
Hemophilia A ◽  
Specific Activity ◽  
High Specific Activity ◽  
Pharmacokinetic Profile ◽  
Preclinical Studies ◽  
Novel Therapies ◽  
Efficacy And Safety ◽  
Disease Phenotype

Abstract Production of recombinant B-domain–deleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia A dogs resulted in correction of the disease phenotype with a pharmacokinetic profile similar to clinical experience with hFVIII-BDD. Notably, immune tolerance challenges with cFVIII-BDD in young and adult hemophilia A dogs did not induce the formation of neutralizing or nonneutralizing antibodies to cFVIII. These data establish the framework to quantitatively investigate the efficacy and safety in preclinical studies of novel therapies for hemophilia A.

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