Mouse Model Of Hemophilia Research Articles

Preclinical Immunogenicity Assessment of Baxter‘s Longer-Acting FVIII Candidate BAX 855 Using Novel Preclinical Models,

Abstract 3323Baxter is developing a PEGylated recombinant human factor VIII conjugate (BAX 855) based on the modification of full-length FVIII with polyethylene glycol (PEG). The FVIII molecule is derived from a CHO cell line using a plasma-protein-free method.Any chemical modification of FVIII might alter its immunogenic potential. Therefore, careful preclinical immunogenicity studies of PEGylated FVIII in comparison to unmodified FVIII are important. We evaluated the immunogenicity of BAX 855 in comparison to ADVATE, Baxter‘s unmodified recombinant full-length FVIII concentrate. For this purpose, we used different in vitro and in vivo models to s assess the potential impact of BAX 855 and ADVATE on both the innate and the adaptive immune system.The assessment of the potential impact of BAX 855 and ADVATE on the human innate immune system was based on their potential to induce the release of pro- inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) in an in vitro human whole blood assay and on its potential to activate the human complement system in human plasma in vitro.The assessment of the potential impact of BAX 855 and ADVATE on the adaptive immune system was based on their potential to induce antibodies in 3 different hemophilic mouse models and in cynomolgous monkeys. The hemophilic moue models included a hemophilic mouse model with a knockout of the murine FVIII that express a human F8 cDNA as a transgene, a hemophilic mouse model that expresses the human MHC-class II protein HLA-DRB1*1501 on the background of a knockout of the murine MHC-class II complex and a conventional hemophilic mouse model with a knockout of the murine FVIII gene. Hemophilic mice that express a human F8 cDNA as a transgene are immunologically tolerant to human FVIII and develop antibodies against human FVIII only if the immune tolerance breaks down. Using this model, we asked if BAX 855 is able to maintain immune tolerance to human FVIII. The hemophilic mouse models that expresses the human MHC-class II protein HLA-DRB1*1501 mimics the situation in an important fraction of hemophilia A patients with FVIII inhibitors. The human MHC-class II haplotype HLA-DRB1*1501 was previously shown to be associated with an increased risk for patients to develop FVIII inhibitors. Using this model, we asked if BAX 855 expresses an immunogenicity profile similar to ADVATE.BAX 855 and ADAVTE induced similar low levels of cytokine release (IL-1β, IL-6, IL-8 and TNF-α) that were similar to the cytokine release observed in the buffer control group after incubation with human whole blood in vitro. In addition, BAX 855 and ADVATE induced similar low levels of complement activation that were not different from the buffer control group after incubation with human plasma in vitro.Importantly, BAX 855 and ADVATE induced similar levels and incidences of antibodies against FVIII and against PEG-FVIII in all mouse models. In addition, immune tolerance to FVIII was maintained in hemophilic mice expressing the human F8 cDNA. Finally, no major differences in antibody titers were seen after treatment of cynomolgus monkeys with BAX 855 and ADVATE.We conclude that results obtained in comparative immunogenicity studies in vitro and in vivo demonstrate that BAX 855 and ADVATE have a similar immunogenicity profile. Therefore, we expect that BAX 855 will express a similar safety profile as ADVATE in patients. Disclosures:Horling:Baxter Innovations GmbH: Employment. Schwele:Baxter Innovations GmbH: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter Innovations GmbH: Employment. Weiller:Baxter BioScience: Employment. Spatzenegger:Baxter Innovation GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment.

Efficacy of Baxter's Longer Acting rFVIII (BAX 855) in Mouse Models of Hemophilia A

Abstract 4350Factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. FVIII concentrates are used in patients with hemophilia A to provide a hemostatic FVIII level sufficient to treat and prevent bleeding episodes. Multiple prophylactic administrations per week are necessary to maintain a FVIII level of at least 1% of normal to effectively prevent or reduce spontaneous bleeding episodes. A longer acting FVIII concentrate would reduce the frequency of infusions. Baxter and Nektar have developed a longer-acting PEGylated variant (BAX 855) of Baxter's recombinant FVIII (ADVATE process). The aim of the presented studies was to evaluate the efficacy of BAX 855 in two primary pharmacodynamics in hemophilia A mice: a tail-tip bleeding model (TTBM) and a carotid occlusion model (COM).BAX 855 was tested at a dose of 200 IU/kg rFVIII. ADVATE™ served as the active reference item, and formulation buffer as the negative control item. Mice received intravenous treatment with either BAX 855 or ADVATE™ 12–40h prior to the experiment. Buffer was administered 5–15min prior to the experiment.Animals were anesthetized using ketamine and xylazine. In the TTBM (n=16) the tip of the tail was cut off and total blood loss [mg] was assessed over 60 minutes. In the COM study (n=10) the left carotid artery was exposed and the endothelium was denuded by topical application of FeCl3. Time to occlusion [min] was assessed.Buffer-treated animals had a median blood loss of 951 mg in the TTBM and did not show vessel occlusion within 30 min in the COM. In the TTBM, a prophylactic effect of ADVATE™ could be observed for up to 18 h, leading to a reduction in blood loss to 121 mg after this treatment interval. Prophylactic efficacy after treatment with BAX 855 could be observed for up to 40 h with a median blood loss of 73 mg after 30 h and 436 mg after 40 h. In the COM, treatment with ADVATE™ 12 h before the experiment shortened the time to occlusion to 3.8 min. With BAX 855, a similar efficacy could be observed for up to 24 h after administration (5.2 min).In summary, the results of our studies show that BAX 855 is efficacious in these two mouse models of hemophilia A and that the efficacy of BAX 855 is prolonged compared with an unmodified rFVIII. Furthermore, the intravenous injection of BAX 855 was well tolerated in all animals across all treatment groups without any signs of acute toxicity. Disclosures:Schiviz:Baxter Innovations GmbH: Employment. Barbara:Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment.

Modification of An Interactive Surface in the C1 Domain Reduces Immune Responses to FVIII in a Mouse Model of Hemophilia A

Abstract 199▪FN2▪This icon denotes a clinically relevant abstractThe X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. The formation of FVIII-neutralizing antibodies in hemophilia A patients is initiated by the endocytosis of FVIII by professional antigen-presenting cells. Endocytosis of FVIII by human monocyte-derived dendritic cells can be significantly blocked by monoclonal antibody KM33, directed towards the C1 domain of FVIII. We have previously shown that C1 domain residues 2092 and 2093 are required for KM33 binding to FVIII (Meems et al., 2009). However, replacement of both these residues by Ala did not completely abolish KM33 binding. We created an additional C1 domain variant which included an arginine at position 2090. A FVIII variant in which three residues, 2090, 2092 and 2093 were substituted by alanine, showed only minimal binding to KM33. Functional analysis revealed that this variant designated FVIII-2090/2092/2093 retains pro-coagulant activity as measured by a chromogenic assay. FVIII-2090/2092/2093 displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine bone marrow-derived dendritic cells. These results emphasize a role for C1 domain residues 2090, 2092 and 2093 in FVIII endocytosis by antigen-presenting cells. We subsequently investigated the in vivo ability of this variant to induce inhibitors in FVIII−/− mice (E17KO). We show that E17KO mice treated with the FVIII variant have significantly lower anti-FVIII antibody titers when compared to mice treated with wild type FVIII. In accordance with these findings reduced numbers of FVIII-specific antibody-secreting cells were detected in the spleen of mice treated with FVIII-2090/2092/2093. Also, FVIII-specific CD4+ T cell responses of splenocytes derived from FVIII-2090/2092/2093 infused mice were greatly reduced when compared to that of splenocytes derived from wild type FVIII infused mice. These findings show that alanine substitutions at positions 2090, 2092 and 2093 result in a FVIII molecule that is significantly less immunogenic when compared to wild type FVIII. Collectively, our data suggest that FVIII variants displaying a reduced uptake by antigen-presenting cells show a reduced immunogenicity in vivo. Based on our findings we hypothesize that FVIII variants displaying a reduced uptake by antigen-presenting cells provide a novel therapeutic approach to reduce inhibitor development in patients with hemophilia A. Disclosures:No relevant conflicts of interest to declare.

Robust Factor IX Expression Following ZFN-Mediated Genome Editing in An Adult Mouse Model of Hemophilia B

Abstract 668As a therapeutic strategy, site-specific modification of the genome has the potential to avoid some of the disadvantages of traditional gene replacement approaches such as insertional mutagenesis and lack of endogenous regulatory control of expression. We have recently reported that zinc finger nuclease (ZFN) driven gene correction can be achieved in vivo in a neonatal mouse model of hemophilia by combining AAV-mediated delivery of both the ZFNs and a Factor IX donor template with homology to the targeted F.IX gene (Li et al., Nature, 2011). The mouse model carries a mutant human F.IX mini-gene (hF9mut) knocked into the ROSA26 locus and ZFN-mediated cleavage followed by donor-dependent repair results in restoration of functional F.IX expression. AAV-ZFN and AAV-Donor vectors were administered to neonatal mice, where the rapid proliferation of hepatocytes in the growing animal may promote genome editing through homology directed repair (HDR). Here we sought to investigate whether ZFN-mediated genome editing is feasible in adult animals with predominantly quiescent hepatocytes. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type factor IX insert flanked by arms of homology to the target site, into adult (8 week old) mice (n=17) resulted in stable (>10wk) circulating F.IX levels of 730–1900 ng/mL (15-38% of normal), whereas mice receiving ZFN alone (n=9) exhibited F.IX levels below detection (<15 ng/mL). Co-delivery of AAV-Mock (luciferase expressing) & AAV-Donor (n=9), yielded <65 ng/mL F.IX. Importantly, mice lacking the hF9mut gene averaged less than 100 ng/mL after receiving AAV-ZFN and AAV-Donor (n=8), suggesting that F.IX expression was derived from on-target genome editing. To eliminate the potential for hF.IX expression resulting from episomal (non-integrated) AAV genomes we performed a two-thirds partial hepatectomy two days after AAV administration. Liver regeneration following hepatectomy is known to substantially reduce expression from non-integrated AAV genomes yet no significant differences in transgene expression were observed compared to non-hepatectomized mice: circulating F.IX levels in the AAV-ZFN + AAV-Donor group (n=13) ranged between 678–1240 ng/mL, whereas mice receiving ZFN alone (n=8) or Mock + AAV-Donor (n=8) had no detectable F.IX expression, or <100 ng/mL F.IX, respectively. Taken together, these data suggest that the F.IX expression in ZFN + Donor treated mice was derived from stable correction of the genome at the intended target site.In summary, we have shown that synchronized cell proliferation of hepatocytes, either in neonatal mice or following partial hepatectomy, is not necessary to achieve highly efficient genome editing and resultant high levels of transgene expression in vivo. These findings substantially expand the potential of ZFN-mediated genome editing as a therapeutic modality. Disclosures:Doyon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Abstract 2280Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics.We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells.The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains.Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures:Steinitz:Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We describe a surprisingly useful approach to improve hemostasis in vivo using a variant of coagulation factor Xa (FXaI16L). This conformationally pliant derivative is partially inactive due to a defect in transitioning from zymogen to protease 1,2. Using mouse models of hemophilia, we show that FXaI16L has a prolonged half-life, relative to wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa which is used to treat bleeding in hemophilia inhibitor patients3. Because of its underlying mechanism of action, FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model

AbstractReplacement of the missing factor VIII (FVIII) is the current standard of care for patients with hemophilia A. However, the short half-life of FVIII makes frequent treatment necessary. Current efforts focus on the development of longer-acting FVIII concentrates by introducing chemical and genetic modifications to the protein. Any modification of the FVIII protein, however, risks increasing its immunogenic potential to induce neutralizing antibodies (FVIII inhibitors), and this is one of the major complications in current therapy. It would be highly desirable to identify candidates with a high risk for increased immunogenicity before entering clinical development to minimize the risk of exposing patients to such altered FVIII proteins. In the present study, we describe a transgenic mouse line that expresses a human F8 cDNA. This mouse is immunologically tolerant to therapeutic doses of native human FVIII but is able to mount an antibody response when challenged with a modified FVIII protein that possesses altered immunogenic properties. In this situation, immunologic tolerance breaks down and antibodies develop that recognize both the modified and the native human FVIII. The applicability of this new model for preclinical immunogenicity assessment of new FVIII molecules and its potential use for basic research are discussed.

Stable expression of native Coagulation factor VIII using the 2A self-processing sequence and furin cleavage site

Hemophilia A is an X-linked recessive bleeding disorder, widely prevalent throughout the world, for which, replacement therapy is the current treatment done by infusion of either recombinant FVIII or human plasma derived FVIII. The expression of FVIII is limited by many aspects, bioengineered FVIII with increased the activity and/or the stability can overcome some of these limitations. We demonstrate that a furin cleavage site (RKRR) and a 2A self-processing peptide derived from FMDV can efficiently and apparently facilitate the equimolar expression of full-length FVIII heavy and light chains from a single ORF, and the FVIII heavy and light chain can self-assemble to form a functional molecule in vivo and in vitro. In addition, our results shown that retaining the 6 N-linked oligosaccharides within a short B-domain spacer associated with 2A and furin cleavage site is expressed more efficiently both in vitro in traditional heterologous expression systems as well as in vivo in a mouse model of hemophilia A.

Transgenic mouse model of hemophilia as a screen for immunogenicity of Factor VIII–based hemophilia therapies

Transgenic mouse model of hemophilia as a screen for immunogenicity of Factor VIII–based hemophilia therapies

In vivo genome editing restores haemostasis in a mouse model of haemophilia

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.

Catalytic domain modification and viral gene delivery of activated factor VII confers hemostasis at reduced expression levels and vector doses in vivo

AbstractCatalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.

Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ΦC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with ΦC31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding ΦC31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when ΦC31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in ΦC31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

AbstractWe herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti–mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.

B-Cell Depletion Using IgG1 Anti-CD20 Spares MZ B Cells and Promotes Tolerance to Human FVIII In a Murine Model of Hemophilia A

AbstractAbstract 2206Preventing and reversing inhibitor formation remains one of the major challenges for hemophilia A therapy. Anti-CD20 mAb (Rituximab) has been reported to be beneficial for hemophilia A patients who failed immune tolerance induction (ITI). However, the evaluation of anti-CD20 therapy often is complicated in the clinical setting by concomitant use of other immune modulating drugs, such as hydrocortisone and IVIG. In this study, we tested the effect of B-cell depletion per se on tolerance induction to FVIII in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential effects were used. We previously showed that IgG1 anti-CD20 selectively depleted follicular (FO) B cells and spared marginal zone (MZ) B cells, while IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice (inhibitor titer = 30.7 ± 4.8 BU/ml), a single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice given daily, high dose FVIII i.v. injection as a model for ITI. Surprisingly, only a marginal effect was achieved when we repeated the same protocol using IgG2a anti-CD20 for B-cell depletion, which efficiently depletes both FO and MZ B cells. To examine tolerance to FVIII, we re-challenged the treated mice with 2 μg FVIII intraperitoneally three months after the initiation of B cell depletion using IgG1 anti-CD20 when the number of peripheral B cells had recovered 60 % or more. The inhibitor titers remained significantly lower in the IgG1 anti-CD20 group after this FVIII boost injection (60.9 ± 33.2 versus 190.3 ± 33.5 BU/ml in control IgG1 group; p = 0.02). Importantly, after the mice were subcutaneously challenged with an unrelated antigen, OVA in CFA, there was no significant difference in anti-OVA IgG titers between the two groups. Taken together, these results suggested that selectively depletion of FO B cells by IgG1 subtype anti-CD20 mAb treatment may facilitate the tolerance induction to FVIII. (Supported by NIH R01 HL061883 and a fellowship from the American Heart Association) Disclosures:No relevant conflicts of interest to declare.

Phenotypic Correction of a Mouse Model of Hemophilia B by In Vivo Genetic Correction of the F9 Gene

AbstractAbstract LBA-5Inherited hematologic disorders have the potential to be effectively treated by gene therapy, with recent successes reported for several genetic disorders using viral vector-mediated gene transfer (ADA-SCID, NEJM 2009; β-thalassemia, Nature 2010). However, these trials and others illustrate some of the disadvantages and risks of using viral vector-based gene addition strategies, including loss of endogenous gene regulation and random insertion leading to potential for insertional mutagenesis. An alternative approach is gene correction, where in situ correction of a gene mutation allows endogenous gene regulation and decreases risks related to random integration. Gene correction is based on gene targeting, the therapeutic utility of which has historically been limited to mouse embryonic stem cells due to low homologous recombination rates in other cell types. However, a recently developed class of fusion proteins, zinc finger nucleases (ZFNs), have been shown to increase targeting efficiency 2–3 logs by inducing site-specific DNA double strand breaks at the intended targeting site. ZFNs have permitted high efficiency therapeutic gene targeting in a variety of cultured cells previously thought intractable to these processes, but ZFN-mediated gene correction has yet to be successfully achieved in vivo in an animal model of disease. Here we show ZFN-mediated therapeutic gene targeting of a mutated F9 gene in vivo, resulting in phenotypic correction of a mouse model of hemophilia B (HB). We first generated ZFNs targeting intron 1 of the human F9 gene (F9 ZFNs). We hypothesized the F9 ZFNs would mediate insertion of a wild-type F9 exons 2–8 minigene into intron 1 via gene targeting, thus bypassing the 95% of F9 mutations that occur in exons 2–8. We next generated a humanized HB mouse model with a deletion of the mouse F9 gene and knock-in (at the ROSA 26 locus) of a catalytic domain-deleted human F9 mini-gene (hF9mut) transgene. Adeno-associated viral (AAV) vector delivery of the F9 ZFNs to hF9mut mouse liver resulted in cleavage of the intron 1 target site in 45% of hepatocytes. We then generated an AAV donor vector containing a w.t. exons 2–8 insert flanked by arms of homology. Co-delivery of the AAV-ZFN and AAV-donor vectors to neonatal hF9mut mice (n=16) resulted in circulating F.IX levels of 120–350 ng/mL (2-7% of normal), whereas mice receiving AAV-ZFN alone (n=17) or AAV-mock & AAV-donor (n=15) had no detectable F.IX expression (detection limit 15 ng/mL), or <25 ng/mL F.IX, respectively. PCR analysis of liver DNA from ZFN+donor mice demonstrated genomic evidence of gene targeting at a rate of 2–7% of alleles. F.IX expression in ZFN+donor mice was shown to be stable after 5 months, with follow-up ongoing. In addition, there was no loss of expression following partial hepatectomy, which causes loss of expression from non-integrated episomes upon subsequent hepatocyte proliferation. F.IX expression was also shown to be specific, as opposed to resulting from random integration, as mice lacking the hF9mut gene averaged less than 30 ng/mL after receiving AAV-ZFN and AAV-donor. hF.IX RT-PCR on 10 different tissues confirmed liver-specific expression. To assess phenotypic correction, we performed aPTTs on mice that received ZFN+donor or mock+donor, as well as wild-type (WT) mice and HB mice. WT mice averaged 36 seconds, ZFN+donor mice averaged 44 seconds, mock+donor mice averaged 60 seconds, and HB mice averaged 67 seconds. There was no significant difference in aPTT between WT and ZFN+donor, or mock+donor and HB (p = 0.086 and 0.11, respectively). However, the aPTT for ZFN+donor mice was significantly shortened compared to mock+donor mice (p=0.0014), demonstrating phenotypic correction of the defect in clot formation in HB mice. To our knowledge this is the first demonstration of ZFN-driven gene correction in vivo, and the first demonstration of the in vivo use of ZFNs to correct an animal model of human disease. These results establish a novel paradigm for in vivo gene correction as a method for treating inherited hematologic diseases. Disclosures:Doyon:Sangamo Biosciences: Employment. Li:Sangamo Biosciences: Employment. Wong:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Rebar:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo: Employment. High:Sangamo Biosciences: Consultancy; Children's Hospital of Philadelphia: Patents & Royalties.

Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

AbstractThe natural cell type(s) that synthesize and release factor VIII (FVIII) into the circulation are still not known with certainty. In vitro studies indicate that artificial expression of FVIII in endothelial cells produces an intracellular pool of FVIII that can be mobilized together with its carrier protein, von Willebrand factor (VWF), by agonists. Here, we show that expression of human B-domain deleted FVIII (hFVIII) in the vascular endothelium of otherwise FVIII-deficient mice results in costorage of FVIII and VWF in endothelial Weibel-Palade bodies and restores normal levels and activity of FVIII in plasma. Stored FVIII was mobilized into the circulation by subcutaneous administration of epinephrine. Human FVIII activity in plasma was strictly dependent on the presence of VWF. Endothelial-specific expression of hFVIII rescued the bleeding diathesis of hemophilic mice lacking endogenous FVIII. This hemostatic function of endothelial cell–derived hFVIII was suppressed in the presence of anti-FVIII inhibitory antibodies. These results suggest that targeting FVIII expression to endothelial cells may establish a releasable pool of FVIII and normalize plasma FVIII level and activity in hemophilia A, but does not prevent the inhibitory effect of anti-FVIII antibodies on the hemostatic function of transgene-derived hFVIII as is seen with platelet-derived FVIII expression.

Engineering Liver Tissues under the Kidney Capsule Site Provides Therapeutic Effects to Hemophilia B Mice

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5-2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Wound healing in haemophilia – breaking the vicious cycle

Our group has been studying how haemostasis interacts with repair processes and also how to optimize treatment of bleeding disorders in a mouse model of haemophilia B. We have found that cutaneous wounds heal more slowly in haemophilic mice than in wild-type mice, and also exhibit histological abnormalities, even after closure of the skin defect. The haemophilic wounds showed reduced influx of inflammatory cells and increased angiogenesis. Even after surface closure, the haemophilic animals experienced repeated episodes of re-bleeding and progressive accumulation of iron in the wound bed and deeper tissues. A dose of replacement or bypassing therapy sufficient to establish initial haemostasis did not normalize wound healing. In fact, daily dosing for 7 days was required to normalize wound closure. Thus, normal healing requires adequate haemostatic function for an extended period of time. We have hypothesized that this is because angiogenesis during healing predisposes to bleeding, especially in the setting where haemostasis is impaired. Thus, normalizing haemostasis, until the process of angiogenesis has resolved, may be required to prevent re-bleeding and additional tissue damage.

Effects of a novel mucosal tolerogen on critical T cell cytokine secretion and development of anti-FVIII inhibitor Ab in a mouse model of Hemophilia A (87.27)

Abstract The development of anti-FVIII (F8) antibodies (Abs) in Hemophilia A patients who receive therapeutic supplemental F8 results in significant morbidity. T cell help is required for anti-F8 inhibitor formation, however, the role of secreted cytokines is not yet fully understood. To ascertain the T cell cytokines important in the development of inhibitor Abs, F8-deficient mice were immunized 4x with 2μg recombinant human F8 at weekly intervals. To abate the production of inhibitor Ab, mice were intranasally immunized with 100μg of E. coli colony-forming antigen I (CFA/I), a novel tolerogen which reduces inflammatory immune responses. Plasma was assayed for the presence of secreted cytokines via multiplex and for functional anti-F8 Ab via Bethesda assay. Inhibitor Ab was detectable by day 14 post-primary immunization, and Th1 (IFNγ and IL-12), Th2 (IL-4), and Th17 (IL-17) cytokines were present in control and CFA/I treated plasma as early as 7 days post-primary immunization. There was no apparent correlation between Th1 vs Th2 responses and increased inhibitor titer. However, mice that were resistant to the effects of CFA/I treatment displayed increased anti-F8 Ab as well as IL-6 and IL-17 by day 21 post-primary immunization. In contrast, IL-13 secretion was associated with significantly decreased inhibitor antibody in CFA/I treated mice. Our results suggest that modification of Th cytokine response may amerliorate inhibitor Ab formation.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.