Elizabeth P. Merricks

Prolonged activity of factor IX as a monomeric Fc fusion protein

Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.

Porphyromonas gingivalis Bacteremia Induces Coronary and Aortic Atherosclerosis in Normocholesterolemic and Hypercholesterolemic Pigs

Objectives—The aim of this study was to determine whether recurrent intravenous injections with Porphyromonas gingivalis (P gingivalis), mimicking periodontitis-associated bacteremia, promotes coronary artery and aortic atherosclerosis in pigs. Methods and Results—Pigs (n=36) fed low- or high-fat chow were divided into P gingivalis–sensitized and P gingivalis–challenged groups or P gingivalis–sensitized controls and saline-treated controls. Pigs were sensitized with 109 killed P gingivalis subcutaneously. Four weeks later all sensitized pigs in the group to be challenged started intravenous injections thrice weekly for 5 months with 106 to 107 colony forming units of P gingivalis while controls received saline. Anti–P gingivalis antibody, serum cholesterol, and complete blood counts were assayed monthly. Pigs were euthanized 2 weeks after the last injection, and coronary arteries and aortas were analyzed by histomorphometry and immunohistochemistry. Anti–P gingivalis antibody was increased by P gingivalis exposure. P gingivalis–challenged pigs developed a significantly greater amount of coronary and aortic atherosclerosis than controls in the normocholesterolemic group and nearly significant in the hypercholesterolemic group. P gingivalis was detected by polymerase chain reaction in arteries from most (94%, 16 of 17) P gingivalis–challenged pigs but not controls. Conclusions—Recurrent P gingivalis bacteremia induces aortic and coronary lesions consistent with atherosclerosis in normocholesterolemic pigs and increases aortic and coronary atherosclerosis in hypercholesterolemic pigs.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.

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

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

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.

The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

Studies on gene therapy for hemophilia B (HB) using adeno-associated viral (AAV) vectors showed that the safety of a given strategy is directly related to the vector dose. To overcome this limitation, we sought to test the efficacy and the risk of immunogenicity of a novel factor IX (FIX) R338L associated with ∼ 8-fold increased specific activity. Muscle-directed expression of canine FIX-R338L by AAV vectors was carried out in HB dogs. Therapeutic levels of circulating canine FIX activity (3.5%-8%) showed 8- to 9-fold increased specific activity, similar to humans with FIX-R338L. Phenotypic improvement was documented by the lack of bleeding episodes for a cumulative 5-year observation. No antibody formation and T-cell responses to FIX-R338L were observed, even on challenges with FIX wild-type protein. Moreover, no adverse vascular thrombotic complications were noted. Thus, FIX-R338L provides an attractive strategy to safely enhance the efficacy of gene therapy for HB.

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor-dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Hyperactive sleeping beauty transposase enables persistent phenotypic correction in mice and a canine model for hemophilia B.

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

ARFI imaging for noninvasive material characterization of atherosclerosis. Part II: toward in vivo characterization.

Seventy percent of cardiovascular disease (CVD) deaths are attributed to atherosclerosis. Despite their clinical significance, nonstenotic atherosclerotic plaques are not effectively detected by conventional atherosclerosis imaging methods. Moreover, conventional imaging methods are insufficient for describing plaque composition, which is relevant to cardiovascular risk assessment. Atherosclerosis imaging technologies capable of improving plaque detection and stratifying cardiovascular risk are needed. Acoustic radiation force impulse (ARFI) ultrasound, a novel imaging method for noninvasively differentiating the mechanical properties of tissue, is demonstrated for in vivo detection of nonstenotic plaques and plaque material assessment in this pilot investigation. In vivo ARFI imaging was performed on four iliac arteries: (1) of a normocholesterolemic pig with no atherosclerosis as a control, (2) of a familial hypercholesterolemic pig with diffuse atherosclerosis, (3) of a normocholesterolemic pig fed a high-fat diet with early atherosclerotic plaques and (4) of a familial hypercholesterolemic pig with diffuse atherosclerosis and a small, minimally occlusive plaque. ARFI results were compared with spatially matched immunohistochemistry, showing correlations between elastin and collagen content and ARFI-derived peak displacement and recovery time parameters. Faster recoveries from ARFI-induced peak displacements and smaller peak displacements were observed in areas of higher elastin and collagen content. Importantly, spatial correlations between tissue content and ARFI results were consistent and observable in large and highly evolved as well as small plaques. ARFI imaging successfully distinguished nonstenotic plaques, while conventional B-mode ultrasound did not. This work validates the potential relevance of ARFI imaging as a noninvasive imaging technology for in vivo detection and material assessment of atherosclerotic plaques.