Nine hemophilia A dogs were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9%−11.3% of normal FVIII levels. In two of nine dogs, FVIII activity increased gradually starting about four years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of these integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A while emphasizing the importance of long-term monitoring for potential genotoxicity.

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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. (Blood. 2010;116(26): 5842-5848) IntroductionThe development of neutralizing antibodies to replacement protein is a major complication of protein and enzyme replacement therapies for several genetic diseases. Hemophilia A is an X-linked bleeding disorder characterized by deficiency in the activity of factor VIII (FVIII), a key component of the coagulation cascade. The disease occurs in approximately 1 in 10 000 live births worldwide, and Ͼ 40% of these patients have severe disease, with FVIII activity Ͻ 1% of normal. 1 Infusion of plasma-derived or recombinant FVIII is the standard treatment. Alloantibodies (inhibitors) that neutralize the protein-replacement therapy develop in 20% to 30% of young patients with severe and moderate hemophilia A, resulting in high morbidity and mortality, 2,3 and this is a growing problem for adults as well. 4,5 Risk factors for inhibitor formation include both genetic and environmental factors. Underlying mutations in the FVIII gene, such as large gene deletions, nonsense mutations, and the most common mutation in severe hemophilia A patients, the inversion of intron 22, are all associated with inhibitor formation; however, it is not possible to predict with certainty which patients will develop inhibitors. For this reason, preventive strategies are not currently feasible. [6][7][8] Patients with high titers of inhibitors, defined as Ͼ 5 Bethesda units (BU), cannot usually be treated with FVIII replacement, necessitating the use of products that bypass the procoagulant effect of FVIII and are extremely expensive. 1 Thus, strategies for the eradication of inhibitors are of fundamental clinical relevance.Currently, the only pro...

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.

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 ϳ 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. (Blood. 2012;119(13):3024-3030) IntroductionHemophilia A is an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII) activity. 1 The disease is characterized by spontaneous hemorrhage and excessive bleeding after trauma. Over time, repeated bleeding into muscles and joints, which begins in early childhood, results in hemophilic arthropathy and irreversible joint damage. This damage is progressive and leads to pronounced musculoskeletal morbidity. 1 Prophylaxis significantly reduces joint damage and long-term sequelae, and improves quality of life in comparison to on-demand treatment. 2,3 However, the short half-life (10-12 hours) of FVIII necessitates dosing every other day or 3 times per week by IV injection for full prophylaxis. 2,4,5 Therefore, a longer-acting FVIII would represent a key advancement in the management of hemophilia A.We have developed a recombinant factor VIII-Fc (rFVIIIFc) fusion protein to extend the half-life of FVIII by leveraging a naturally occurring biologic pathway. rFVIIIFc is a heterodimeric protein comprising a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 (IgG1). The Fc domain enables binding to the neonatal Fc receptor (FcRn), which is responsible for protection of IgG from degradation and facilitates its recycling, 6,7 resulting in a half-life for IgG of ϳ 3 weeks in humans. The Fc domain of IgG1 has been fused to growth factors, cytokines, enzymes, and ligand-binding regions of receptors [8][9][10] ; several of these fusion proteins have been approved as therapeutics (eg, etanercept, abatacept, belatacept, alefacept, rilonacept, aflibercept, and romiplostim). However, traditional dimeric Fc fusions, created through the fusion of the monomeric effector p...

We investigated the safety and efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B, and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We show that gene therapy using lentiviral vectors targeting expression of a canine factor IX transgene to hepatocytes was well-tolerated and provided stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we show that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be useful for the treatment of hemophilia.

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 (nϭ36) 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 10 9 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 10 6 to 10 7 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 gingivalischallenged 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. Key Words: coronary and aortic atherosclerosis Ⅲ Porphyromonas gingivalis Ⅲ periodontitis Ⅲ pig model Ⅲ bacteremia P eriodontitis has been associated with cardiovascular disease in many, but not all, epidemiological studies. 1-15 Mild forms of periodontal disease affect 75% of the population, and more severe forms affect 20% to 30% of adults in the United States. 8 The mechanism by which periodontitis could contribute to cardiovascular disease is unknown but likely relates to the associated intense local and systemic inflammation and the virulence properties of the causative organisms. 16 -21 Porphyromonas gingivalis has been strongly associated with adult periodontitis. This bacteria is a Gramnegative, nonmotile, obligate anaerobe that can invade and infect epithelium, endothelium, and vascular smooth muscle cells 22-29 and appears to alter endothelial function. 30 The capacity for invasion is in part mediated by fimbriae, 31,32 and the virulence of P gingivalis may vary among individual strains. 33 Recurrent bacteremias with P gingivalis and a mixture of other oral bacteria can occur in periodontitis patients during simple mastication. 34 -36 P gingivalis has been detected by immunostaining and polymerase chain reaction (PCR) in some human carotid and coronary atheromas. 27,37 In addition, P gingivalis produces arginine-specific cysteine proteases, called gingipain Rs, that can activate both the cellular and humoral coagulation systems. These gingipain Rs activate protease-activated receptors 1 and 4 (PAR-1 and PAR-4) on platelets to a degree sufficient to support ex vivo aggregation. 38 -42 Moreover, gingipain Rs can activate coagulation factors I...

Both acute coronary occlusion and reperfusion of an infarct-related artery lead to significant myocardial cell death. Recent evidence has been presented that activation of the transcription factor nuclear factor-κB (NF-κB) plays a critical role in reperfusion injury. NF-κB is usually bound to its inhibitor, IκB, and classic activation of NF-κB occurs when the 20S proteasome degrades IκB that has been phosphorylated and ubiquitinated. In this study, activation of NF-κB was inhibited by systemic administration of a 20S proteasome inhibitor (PS-519) in a porcine model of myocardial reperfusion injury. The experimental protocol induced myocardial ischemia in the distribution of the left anterior descending coronary artery for 1 h with subsequent reperfusion for 3 h. A single systemic treatment with PS-519 reduced 20S proteasome activity; blocked activation of NF-κB induced by reperfusion; reduced creatine kinase, creatine kinase-muscle-brain fraction, and troponin I release from the myocardium; preserved regional myocardial function measured by segmental shortening; significantly reduced the size of myocardial infarction; and exhibited no acute toxicity. These data show that myocardial reperfusion injury can be inhibited by using proteasome inhibitors, which likely function through the inhibition of NF-κB activation.