Canine Factor VIII Research Articles

Childhood Treatment with Adeno-Associated Viral Gene Therapy Results in Stable FVIII Expression and Improved Bleeding Phenotype in Adult Severe Hemophilia A Dogs

Introduction: In patients with severe hemophilia A, adeno-associated virus (AAV)-mediated factor VIII (FVIII) gene therapy is associated with reduced bleeding compared with FVIII prophylaxis. AAV5-FVIII gene therapy is approved in the U.S. and conditionally approved in Europe for the treatment of adults with severe hemophilia A. Gene therapy during childhood could potentially prevent the development of arthropathy and improve quality of life. However, there are unknowns regarding long-term safety and whether the predominant episomal nature of AAV persistence would lead to loss of transgene expression with hepatocyte divisions and liver expansion during childhood. The inflammatory response to AAV gene therapy in children is also largely uncharacterized. We have previously reported the treatment of five neonatal or infant dogs with a single vector infusion of a codon-optimized AAV5-B-domain deleted canine FVIII (cFVIII) construct with a hybrid liver promoter (AAV5-cFVIII). Dogs treated at 2 weeks of age demonstrated improved whole blood clot time (WBCT) over a 6-month period despite minimal cFVIII expression (<3%). Dogs treated at 2 months of age demonstrated stable cFVIII expression measured over a 6-month period and decreased WBCT. Aims: To describe the early inflammatory response to AAV5-cFVIII in infant and neonatal dogs, and to provide an update on the safety and efficacy of AAV5-cFVIII in these animals 12-16 months post-treatment. Methods: Hemophilia A dogs were treated with a single vector infusion (dose=2.0e14 vg/kg) of AAV5-cFVIII at 2 weeks (n=2) or 2 months (n=3) of age. Epigenomic data suggests that 2 months of age in dogs is equivalent to 9 months in humans. Liver volume was measured by magnetic resonance imaging at baseline, 3, 6, and 12 months post-AAV5-cFVIII treatment. Plasma cytokine levels were measured using a cytokine array pre-treatment (day 0) and post-treatment (days 2-28). Percutaneous liver biopsy samples were obtained at 2 weeks, 6 months, and 12 months post-treatment. Results: Cytokine array analyses of samples demonstrated that one dog who received AAV5-cFVIII at 2 weeks experienced transient post-treatment plasma elevation of IL-6, KC-like, MCP-1, TGFβ-1, and TGFβ-2 that returned to baseline levels by day 18. Two dogs treated at 2 months experienced a transient increase in GMC-SF, IL-2, IL-6, IL-7, IL-15, and IL-18 that returned to baseline levels by day 21. No changes in levels of IFNγ, IL-8, IL-10, IP-10, TNFα, or TGFβ-3 were observed. Changes in plasma cytokine levels were not associated with an elevation in plasma alanine transaminase levels. Hemophilia A dogs treated with AAV5-cFVIII at 2 weeks of age maintained an improved WBCT 12 to 16 months post-treatment, although cFVIII expression as measured by one-stage (OSA) and chromogenic substrate assays (CSA) remained minimal (<3%). Body weight and liver volume increased by 10.6-fold (measured at 16 months) and 12-fold (measured at 12 months) respectively. Dogs treated with AAV5-cFVIII at 2 months of age demonstrated sustained cFVIII expression by OSA and CSA after 12 to 16 months (8.9-13.39% measured at 16 months by OSA), despite a 3.2-fold increase in body weight (measured at 16 months), and a 3.9-fold increase in liver volume (measured at 12 months). This was associated with improved WBCT observed over the 12 to 16-month period for all three dogs. Overall, a reduction in bleeding events (BEs) was observed in all dogs. No BEs were recorded prior to AAV5-cFVIII treatment for dogs treated at 2 weeks of age. During the 16 months post-treatment, one dog experienced two spontaneous BEs, while no BEs were observed for the second dog. The dogs treated at 2 months experienced 4 (3 spontaneous and 1 traumatic) BEs prior to AAV5-cFVIII treatment, and 1 traumatic BE 48 hours post-treatment prior to the detection of FVIII expression. For the remaining 16 months post-treatment, no BEs were observed in this group. Conclusions: Treatment using AAV5-cFVIII at 2 months of age in a hemophilia A dog model resulted in stable FVIII expression into adult life despite significant liver expansion. For some dogs, AAV-cFVIII treatment resulted in an early transient increase in plasma proinflammatory cytokine levels with no evidence of transaminitis that resolved within three weeks.Further studies on liver biopsy samples are ongoing to evaluate cellular implications, vector genome distribution, and mechanisms of AAV persistence.

A Novel Variant of Factor VIII Yields Cofactor Activity without Proteolysis between the A1 and A2 Domains

Factor VIII (FVIII) has a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2), and intracellular processing within the B domain results in its secretion as a heterodimeric procofactor consisting of a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2). Proteolytic cleavage by thrombin at R372, R740, and R1689 removes the B-domain, releases it from vWF by cleaving the a3-acidic region and activates FVIII to the heterotrimeric cofactor, FVIIIa (A1-a1/A2-a2/A3-C1-C2). The requirement for cleavage following a1 to generate active cofactor sets FVIII/FVIIIa apart from factor V, its structural and functional homolog. FVIII binds its cognate protease, factor IXa (FIXa) with high affinity in a membrane-dependent way. However, this complex does not efficiently activate factor X (FX) until R372 is cleaved, indicating that this cleavage reaction is important in facilitating FX recognition and its enhanced activation by intrinsic tenase complex. We speculated that separation of A1-a1 and A2-a2 domains through cleavage at R372 might be replicated by inserting a linker between A1-a1 and A2-a2 domains even when the 372 site was rendered uncleavable. We chose canine FVIII (cFVIII) to test our ideas due to its higher expression levels in cell culture compared to human FVIII. We made cDNA constructs encoding cFVIII variants with flexible (GGGGS, [GGGGS]3, GGGGGG) or rigid (EAAAK, [EAAAK]3, PAPAP) linkers between A1-a1 and A2-a2 domains on the R366Q backbone (corresponding to R372 in human FVIII). All constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Clotting times of cFVIII variants with flexible linkers were very similar to cFVIII R366Q without linker (~64 sec). In contrast, clotting times of cFVIII variants with rigid linkers were consistently lower (16 - 26 sec). cFVIII variants (R366Q, R366Q-EAAAK, R366Q-PAPAP) were purified from 15L of expression media each. In one stage aPTT assays purified cFVIII variants (10 nM) showed clotting times (R366Q-EAAAK, 33.3 s, R366Q-PAPAP, 26 s) that were considerably shorter than the R366Q variant (52.2 s) and more in line with wild type cFVIII (cFVIII-WT,19.3 s). To assess possible cleavage at the 366 site, purified cFVIII variants were analyzed by SDS-PAGE following treatment with a high concentration of thrombin. As expected, cFVIII-WT was quantitatively cleaved at R366, R734 and R1689 to produce bands corresponding to A1-a1, A2-a2 and A3-C1-C2. In contrast, both R366Q and R366Q-PAPAP variants were resistant to cleavage at 366, yielding bands corresponding to A1-a1-A2-a2 and A3-C1-C2. The cFVIII-R366Q-EAAAK variant was susceptible to some cleavage, likely at the lysine present within linker. We also performed steady state kinetic studies of FX activation using limiting concentrations of IXa, saturating concentrations of cFVIII variants pretreated with thrombin, membranes and increasing concentrations of FX. Vmax/[E] for FXa formation was 140 ± 7 min-1 with cFVIIIa-WT, 4 ± 0.3 min-1 with cFVIIIa-R366Q, and 42 ± 4 min-1 with cFVIIIa-R366Q-PAPAP. The Km for FX remained unaffected for all variants. The results indicate that the impaired cofactor activity of the FVIII variant that is not cleaved at the 366 site can be substantially rescued by insertion of the PAPAP linker after 366Q. Further optimization of the linker might completely correct defective FX activation associated with the lack of proteolytic cleavage between A1-a1 and A2-a2 domains. Our study indicates that cleavage at the 366 site is not an absolute determinant of FVIII activation and provides new insights into the function of FVIIIa as a cofactor within the intrinsic tenase complex. DisclosuresGoolyam Basavaraj:Bayer: Research Funding; Grifols: Research Funding. Krishnaswamy:Portola: Research Funding; Baxalta: Consultancy; Janssen Research & Development: Research Funding.

Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Successful Long-Term Phenotypic Correction Of Hemophilia A Mice By Engineering Functional Factor VIII Delivery System In Vivo Using Autologous Endothelial Cell Sheets

Recently, preclinical and clinical studies using adeno-associated viral (AAV) vectors for hemophilia B showed that the safety profile is vector dose-dependent and that immune responses to AAV-capsid proteins with subsequent hepatocyte toxicity required transient immunosuppression for sustained transgene expression. However, there still remain concerns over the safety of systemic vector injection. Potential side effects include adverse immunological reactions, vector-mediated cytotoxicity, germ-line transmission, and insertional oncogenesis. Moreover, especially in hemophilia A, an alternative transgene delivery approach may be necessary due to the large size of the factor VIII (FVIII) cDNA.Blood outgrowth endothelial cells (BOECs) are considered to be an attractive candidate to treat hemophilia A, because BOECs express von Willebrand factor, which is known to act as a carrier protein for FVIII and prevents its proteolytic degradation. We previously reported that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 6 months, in which lentivirally-engineered BOECs mixed with Matrigel were implanted subcutaneously. However, the gradual loss of plasma FVIII was observed probably due to the breakdown of the scaffold material or cell death. To overcome this issue, we employed a novel tissue engineering approach, in which BOECs were formed into a contiguous monolayer sheet that can readily used for transplantation into the subcutaneous space for the FVIII production. To evaluate alternative scaffold material that can enhance the viability of the implanted cells, FVIII-transduced BOECs were cultured on temperature-responsive poly (N-isopropylacrylamide) (PIPAAm)-grafted dish to engineer BOECs sheet. This dish allows for simple detachment of the cultured cells without the use of proteolytic enzymes such as trypsin and enables us to harvest the cell sheet as a contiguous monolayer that retains its native intercellular communications and intracellular microstructure, which are indispensable for normal cellular function. The lentivirally-modified BOECs that expressed canine FVIII at the level of 2,000-3,000 mU/mL /1x10 6 cells/24hrs in vitro were seeded on the temperature-responsive culture dishes. When the cultured BOECs reached confluency, the cultured cells were detached from the PIPPAm dish as a uniformly connected tissue sheet by lowering the culture temperature to 20°C for 30 minutes. Despite requiring a single plasma infusion at the time of the small surgical procedure and transient immunosuppression with cyclophosphamide, 2-11% FVIII levels of normal plasma were detected 3 weeks after cell transplantation and sustained up to 300 days without the development of anti-canine FVIII antibodies in immunocompetent hemophilia A mice receiving the transplanted BOECs sheet. In addition, by histological examination, we also confirmed that transplanted the BOECs differentiate into mature endothelial cells and contribute to new blood vessel formation. There is a clear difference in cell viability between our previous Matrigel transplantation approach and this new cell sheet strategy. Although BOECs are capable of engrafting and expressing FVIII following transplantation into the same subcutaneous space, BOECs implanted through the cell sheet approach exhibit prolonged cell viability that results in both long-term and 3-5 times higher expression of FVIII.In conclusion, we have succeeded in the long-term correction of hemophilia A in a mouse model by ex vivo engineering of genetically-modified autologous endothelial cells in an ectopic subcutaneous space. Our cell sheet engineering approach could contribute to clinical translational studies for hemophilia A in a variety of therapeutic settings. Disclosures:No relevant conflicts of interest to declare.

Bioengineering Factor VIII B-Domain Sequences Improves Function and Efficacy in Hemophilia A Models.

Abstract 2208We previously reported that recombinant canine B-domain deleted factor VIII (FVIII) was expressed predominantly (>75%) as a single-chain protein and, upon activation with thrombin, yielded an active cofactor species with increased stability due to delayed A2 domain dissociation. We hypothesized that this could be in part due to a unique recognition sequence (HHQR) for intracellular PACE/Furin protease present in the canine FVIII, but absent in other mammals including human, porcine and murine FVIII where the sequence is RHQR. Here, we changed position 1645 from R to H in the B-domain deleted human FVIII (hFVIII-RH) and found that much more of the protein was expressed in a single chain form (3-fold) compared to hFVIII-wild type (hFVIII-WT). Importantly, the variant hFVIII-RH was biologically active exhibiting a 2-fold higher activity measured in a 2-stage activated partial thromboplastin time (p<0.05) likely due to slower dissociation of the A2-domain upon thrombin activation compared to hFVIII-WT (t½ 3.48 vs. 1.48 minutes, respectively).We next sought to determine the potential in vivo efficacy and safety of circulating hFVIII-RH in murine models of hemophilia A (HA) by hepatocyte-restricted transgene expression using adeno-associated viral (AAV serotype 8) vector and by exogenous administration of the recombinant protein. HA mice received varying doses (8×1012-4×1013 vg/kg) of AAV-hFVIII-WT or AAV-hFVIII-RH and exhibited a corresponding dose-dependent response of FVIII with plateau antigen levels 2–3 fold higher in hFVIII-RH than hFVIII-WT expressing mice (n = 5/group, p < 0.05 at all doses). At the low dose cohort, circulating FVIII antigen levels were 79±6.6 and 50±9 ng/ml for the FVIII-RH and FVIII-WT, respectively. In the mid dose cohort, FVIII levels were 165±54 and 84±9 ng/ml and in the high dose 274±39 and 120±35 ng/ml. We monitored the blood loss following tail-clip assay in groups of mice stratified into groups determined by expression to be low (28–60 ng/mL), mid (60–100 ng/mL), or high (above 100 ng/mL) for analysis (n= 3–9/group). Expression of either hFVIII variant was capable of decreasing the blood loss of mice in the low group compared to untreated HA mice, but did not reach that of hemostatically normal mice. Notably, at the mid expression group only hFVIII-RH expressing mice had corrected blood loss to normal hemostasis (hFVIII-RH vs hFVIII-WT p < 0.02). In the high dose all mice (FVIII-RH and FVIII-WT) exhibited blood loss similar to that of hemostatically normal mice. The increased effect of hFVIII-RH is a result of more stable clot as seen in the FeCl3- carotid artery injury model. At comparable FVIII levels, 15/16 mice expressing hFVIII-RH form stable occlusive thrombi whereas only 14/21 in hFVIII-WT group.Laser-induced injury at microcirculation resulted in increased fibrin deposition by 2–3 fold in HA mice expressing FVIII-RH (n=4 mice, 20 injuries) compared to those expressing FVIII-WT (n=3, 10 injuries). Similar findings were obtained upon injection of recombinant protein to achieve ∼40% of normal for both FVIII-RH (n=3, 13 injuries) and FVIII-WT (n=3, 18 injuries). Moreover, we assessed the immunogenicity of hFVIII-RH by administering AAV vectors to transgenic HA mice tolerant to B-domain deleted hFVIII-WT. At 4 weeks post AAV administration all mice exhibited stable hFVIII-WT/hFVIIl-RH levels (n=5/group) with no evidence of inhibitor by Bethesda assay or by mouse specific-hFVIII-IgG titers. In summary, FVIII-RH is superior to FVIII-WT in terms of expression levels and total hemostastic function without evidence of increased immunogenicity. Therefore FVIII-RH is an attractive molecule for protein replacement and gene/cell-based strategies for HA. Disclosures:No relevant conflicts of interest to declare.

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.

Efficacy and Safety of Long-term Prophylaxis in Severe Hemophilia A Dogs Following Liver Gene Therapy Using AAV Vectors

Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

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

AbstractInhibitory 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.

Structural Elements of FVIII Required for Accelerating Proteolytic Cleavage of Von Willebrand Factor by ADAMTS13.

Abstract 849ADAMTS13 (ADisintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 sessile bond. An inability to cleave ultra large vWF by ADAMTS13 on endothelial cell surface and in blood may result in disseminated microvascular thromboses seen in patients with thrombotic thrombocytopenic purpura. Recent study has demonstrated that human coagulation factor VIII (FVIII) can accelerate proteolytic cleavage of human vWF by ADAMTS13 under fluid shear stress (Cao et al, PNAS, 2008, 105:7416-21). However, the structural elements of FVIII required for such rate enhancing effect are not known. Using recombinant technology, biochemical assays and an animal model, we were able to show that an isolated light chain of FVIII (FVIII-LC) was sufficient to accelerate proteolytic cleavage of vWF by ADAMTS13 under fluid shear stress in a concentration-dependent manner. At concentration of 5 nM, FVIII-LC increased the proteolytic cleavage product of VWF (350 kDa) by approximately 5-7 fold, similar to wild type FVIII and B-domain deleted FVIII-SQ. The concentration of FVIII-LC achieving the half maximal rate enhancing effect (IC50) was estimated to be ∼2.0 nM. In contrast, an isolated heavy chain of FVIII (FVIII-HC) or a FVIII-LC with an acidic region being deleted (FVIII-LCda3) did not exhibit significant rate enhancing effect under the same conditions. A microtiter binding assay demonstrated that the FVIII-LC bound vWF with a dissociation constant (KD) of ∼17 nM, whereas FVIII-HC and FVIII-LCda3 did not bind the immobilized vWF detectably. These results suggest that binding of FVIII-LC to vWF may be necessary and sufficient to increase the susceptibility of vWF to proteolytic cleavage by ADAMTS13 under fluid shear stress. To corroborate our biochemical findings, we performed animal studies by investigating plasma vWF antigen levels and multimer distribution using mini-agarose gel electrophoresis and Western blot. We showed that even at baseline plasma vWF antigen and high molecular weight (HMW) vWF multimers in fVIII knockout mice (fVIII-/-) (n=18) were modestly increased compared with those of wild-type mice (n=14). Upon challenge by a hydrodynamic injection of phosphate-buffered saline (2 ml in 5 seconds), plasma vWF antigen and HMW-vWF multimer were dramatically increased in the fVIII−/− mice (n=10) at 48-hour post-injection as a result of endothelial cell activatin and injury. Supprisingly, this dramatic increase in plasma vWF antigen and HMW-vWF multimers were abrogated in the fVIII−/− mice with the similar hydrodynamic injection of saline containing a plasmid encoding canine FVIII-SQ. At 48-h post injection, plasma canine FVIII-SQ ranged from 150% to 700% of normal canine FVIII activity based on a Coatest assay (n=20). A microtiter binding assay further showed that canine FVIII-SQ bound immobilized murine vWF with a dissociation constant (KD) of ∼1.7 nM. These findings provide further evidence that FVIII may be a cofactor that regulates proteolytic processing of vWF by ADAMTS13 under both physiological and pathological conditions. The cofactor activity of FVIII depends on high affinity interactions between the FVIII light chain and vWF under fluid shear stress. Disclosures:No relevant conflicts of interest to declare.

Liver‐restricted expression of the canine factor VIII gene facilitates prevention of inhibitor formation in factor VIII‐deficient mice

Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII. AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous beta-actin promoter, the liver-specific human alpha1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human alpha1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii(-/-)) mice. Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVIII transgene expression was achieved, resulting in increased blood cFVIII activities to more than 100% of the normal canine FVIII levels in fviii(-/-) mice at a 1 : 10 lower dose of the AAV8 vector carrying the cFVIII gene driven by the HAAT promoter. Under short-term immunosuppression, neutralizing antibodies against cFVIII developed in only one out of six mice when the HAAT promoter was used for cFVIII expression, whereas all the mice developed neutralizing antibodies against cFVIII when the beta-actin promoter was used for cFVIII expression. No neutralizing antibodies against cFVIII developed in fviii(-/-) mice that received the AAV8 vector carrying the cFVIII gene driven by the HCRHAAT enhancer/promoter complex without immunosuppression. These data suggest that AAV8 vector-mediated liver-restricted cFVIII gene expression is sufficient for immune hypo-responsiveness to transgene-derived cFVIII in fviii(-/-) mice.

A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells

AbstractUnder certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice. We used lentiviral vectors to deliver the canine FVIII transgene to neonatal hemophilic mice and demonstrated that induction of long-term FVIII tolerance could be achieved. Hemophilia A mice are capable of mounting a robust immune response to FVIII after neonatal gene transfer, and tolerance induction is dependent on the route of delivery and type of promoter used. High-level expression of FVIII was not required for tolerance induction and, indeed, tolerance developed in some animals without evidence of detectable plasma FVIII. Tolerance to FVIII could be adoptively transferred to naive hemophilia recipient mice, and FVIII-stimulated splenocytes isolated from tolerized mice expressed increased levels of interleukin-10 and decreased levels of interleukin-6 and interferon-γ. Finally, induction of FVIII tolerance mediated by this protocol is associated with a FVIII-expandable population of CD4+CD25+Foxp3+ regulatory T cells.

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A. Disclosure of potential conflicts of interest is found at the end of this article.

Ex Vivo Gene Therapy for Haemophilia A: FVIII-Overexpressing Mesenchymal Stromal Cells Fail To Produce Sustained Therapeutic Levels of Circulating FVIII In Vivo.

The use of ex vivo transfected autologous fibroblasts was explored for the delivery of coagulation factor VIII (FVIII) to treat haemophilia A patients, albeit with mitigated results (Roth et al., NEJM 2001). A limiting factor to this approach is the limited expansion potential of somatic cells and cellular dose manufactured for therapeutic use. The use of adult stem or progenitor cells offers a possible remedy to this specific challenge because of their high proliferative capacities, allowing the generation of large number of cells ex vivo. Mesenchymal stromal cells (MSCs) can easily be isolated by bone marrow aspiration and extensively expanded ex vivo using standard tissue culture procedures. They also possess endogenous wound healing properties and are relatively easy to gene engineer. When engineered to secrete therapeutic plasma-soluble proteins, they could be injected with a supporting matrix - as a subcutaneous plug - and used to treat a variety of haematological disorders responsive to secreted proteins such as haemophilia A and B and anaemia for instance. The retrievability of the implanted cells is desirable if unexpected side effects were to occur. The aim of this project was to test a gene engineered MSC platform to treat a clinically relevant model disease, haemophilia A. Canine MSCs (cMSCs) isolated from normal and haemophilia A dogs were genetically modified using a 3rd generation lentiviral vector carrying the B-domain deleted canine FVIII (cFVIII) gene. In vitro production of cFVIII was assessed by ELISA and found to be about 2,4 U/106 cells/24h. 107 of those cells were injected intraperitoneally in NOD-SCID mice and transient expression (8–10 days) of therapeutic levels of cFVIII (up to 10%) could be detected using an ELISA capable of discriminating cFVIII from murine FVIII. However, when these cells were embedded in an injectable human compatible collagen based scaffolding material (Contigen) and implanted subcutaneously into NOD-SCID mice no cFVIII could be detected above background in any animals. Because we previously demonstrated that erythropoietin gene-modified MSCs implanted as described led to an increase in hematocrit for an extended period (Eliopoulos et al. Mol Ther 2004), we believe that the failure of the present approach is attributable to idiosyncrasies of the FVIII system. Of particular relevance is the requirement for the presence of von Willebrand factor (vWF) in stabilizing the FVIII protein and protect it from degradation. We already have evidence that ex vivo transduced endothelial progenitor cells known to produce endogenous vWF can serve as better delivery vehicles for FVIII as part of a matrix embedded subcutaneous plug and lead to prolonged circulating levels of FVIII in mice (Matsui et al., submitted). These data support the hypothesis that FVIII delivery by engineered somatic and stem cells likely requires co-expression of key cofactors, such as vWF, for durable and clinically relevant effects in vivo.

Highly Conserved Antigenic Structure of the Factor VIII C2 Domain in Some Mammals

To elucidate differences in the antigenic structure of factor VIII (FVIII) among mammals, we evaluated cross-reactivities of well-defined antihuman FVIII antibodies with canine and other mammalian FVIII proteins. Monoclonal antibodies against human FVIII recognizing the A1 domain in the heavy chain and the A3 domain in the light chain showed 2.7% and 0% cross-reactivities, respectively, with canine FVIII. The cross-reactivities of 2 alloantibodies and a monoclonal antibody that recognized the A2 domain in the heavy chain were 10%, 38%, and 0%, respectively. On the other hand, 2 kinds of alloantibodies and a monoclonal antibody recognizing the C2 domain in the light chain showed 160%, 390%, and 130% cross-reactivity, respectively, with canine FVIII. The anti-C2 monoclonal antibody (NMC-VIII/5) showed a type 2 inactivating property when tested with canine and human plasma. Moreover, cross-reactivities of NMC-VIII/5 with simian and feline FVIII were 54.5% and 82.8%, respectively, while the cross-reactivities of the anti-A2 monoclonal antibody (JR8) with simian and feline FVIII were 1.3% and 0%, respectively. These findings suggest that the antigenic structure of the C2 epitope has remained relatively conserved throughout mammalian evolution in contrast to the A2 epitope and that a canine model of hemophilia A is useful for FVIII inhibitor experiments.

Phenotype Correction of Hemophilia A Mice with Adeno-Associated Virus (AAV) Vectors Carrying the B Domain Deleted Canine Factor VIII Gene.

Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x1012 gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x1012 gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

DDAVP-Induced Increase of Factor VIII Activity in Blood Is Likely Due To Release of Factor VIII That Is Synthesized by Endothelial Cells.

Desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) is commonly used as a nonreplacement therapy for mild von Willebrand disease (VWD) and hemophilia A. In humans, IV injection of 0.3 μg/kg of DDAVP induces a rapid 2 to 5-fold increase in plasma levels of both von Willebrand factor (VWF) and Factor VIII (FVIII) within 30–60 minutes, which is due to release from Wiebel-Palade bodies (WPBs) in endothelial cells. The stored FVIII may be synthesized by endothelial cells, which express FVIII in vitro. However, hepatoma cells can also express FVIII in vitro, and liver transplantation can correct hemophilia A. Thus, the liver may be the major site of production of FVIII in vivo, thus, an alternative explanation is that endothelial cells take up FVIII from blood and store it in WPBs with VWF, which can be released after DDAVP. DDAVP is effective in humans and dogs, but not in mice. In this study, we tested the effect of DDAVP on hemophilia A dogs after neonatal hepatic gene therapy with a retroviral vector (RV) expressing canine FVIII (cFVIII). With this gene therapy approach, canine hepatocytes express high levels of a reporter gene from an RV, but no expression is observed in endothelial cells. Thus, the major site of FVIII synthesis is the hepatocyte in this model. Our hypothesis is that if DDAVP increases FVIII levels in this dog model, it would indicate that the FVIII increase is due to uptake from blood by endothelial cells. Alternatively, if no increase in FVIII occurs after DDAVP stimulation, it would suggest that the increase in normal dogs is due to synthesis of FVIII by endothelial cells. An RV that contains the liver-specific human α1-antitrypsin promoter and the canine B-domain deleted FVIII cDNA was generated. RV was given IV to two hemophilia A dogs at 8x109 transducing units (TU)/kg at 3 days after birth. The whole blood clotting time (WBCT) and APTT time in both dogs have been normalized, and the plasma cFVIII COATEST activity has been maintained at 100–200% of normal for 11 months to date. DDAVP was injected IV at 0.5 μg/kg into RV-treated hemophilia A dogs at 7 months of age. Two separate doses of DDAVP were given with an interval of one week. The same dose of DDAVP was given to normal dogs as controls (N=4). In normal dogs, both VWF and FVIII levels increased 40% and 50% between 15 to 60 minutes after DDAVP, respectively. However, FVIII levels were not changed in RV-treated dogs, although VWF levels increased 150% or 60%. Thus, our data suggest that the normal FVIII increase after DDAVP administration is due to release of FVIII that is synthesized by endothelial cells. These data also demonstrate that DDAVP will not be effective at increasing FVIII activity in patients that receive liver-directed gene therapy and only achieve partial correction. Such patients would need to be treated with factor replacement if bleeding episodes occur.

The canine factor VIII 3'-untranslated region and a concatemeric hepatocyte nuclear factor 1 regulatory element enhance factor VIII transgene expression in vivo.

If gene therapy is to be an effective treatment modality for hemophilia A, therapeutic levels and tissue-restricted expression of factor VIII (FVIII) must be achieved through optimization of transgene expression. To this end, we incorporated three types of sequence elements into a canine B domain-deleted FVIII transgene cassette and individually evaluated their effect on FVIII transgene expression. Functional FVIII activity was initially assessed in vitro and hydrodynamic injection of the different transgene constructs into mice was subsequently used as a model to compare in vivo expression of the various modified transgenes. Our results demonstrate that in vitro transgene expression is, in these studies, not a good predictor of in vivo transgene performance. In vivo analysis of a hybrid tissue-restricted promoter element, consisting of a concatemer of five hepatocyte nuclear factor 1 (HNF-1) consensus-binding motifs juxtaposed to the human FVIII proximal promoter, indicates that it is as efficient at mediating expression of the FVIII protein as the cytomegalovirus promoter. Addition of the full-length canine FVIII 3'-UTR also enhances transgene expression of FVIII in vivo. Sequence analysis of the canine FVIII 3'-UTR and human FVIII 3'-UTR indicates that the former lacks instability sequences and may therefore be more effective in stabilizing FVIII mRNA. Subsequent inclusion of FVIII introns 16 and 17 into the natural locations of the transgene disrupted mRNA processing and abolished expression of the FVIII protein. Introduction of intron 17 proximal to the FVIII cDNA did not enhance in vivo expression of canine FVIII from the transgene.

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs

AbstractCanine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.