[Show abstract][Hide abstract] ABSTRACT: Dogs with hemophilia A, hemophilia B, von Willebrand disease (VWD), and factor VII deficiency faithfully recapitulate the severe bleeding phenotype that occurs in humans with these disorders. The first rational approach to diagnosing these bleeding disorders became possible with the development of reliable assays in the 1940s through research that used these dogs. For the next 60 years, treatment consisted of replacement of the associated missing or dysfunctional protein, first with plasma-derived products and subsequently with recombinant products. Research has consistently shown that replacement products that are safe and efficacious in these dogs prove to be safe and efficacious in humans. But these highly effective products require repeated administration and are limited in supply and expensive; in addition, plasma-derived products have transmitted bloodborne pathogens. Recombinant proteins have all but eliminated inadvertent transmission of bloodborne pathogens, but the other limitations persist. Thus, gene therapy is an attractive alternative strategy in these monogenic disorders and has been actively pursued since the early 1990s. To date, several modalities of gene transfer in canine hemophilia have proven to be safe, produced easily detectable levels of transgene products in plasma that have persisted for years in association with reduced bleeding, and correctly predicted the vector dose required in a human hemophilia B liver-based trial. Very recently, however, researchers have identified an immune response to adeno-associated viral gene transfer vector capsid proteins in a human liver-based trial that was not present in preclinical testing in rodents, dogs, or nonhuman primates. This article provides a review of the strengths and limitations of canine hemophilia, VWD, and factor VII deficiency models and of their historical and current role in the development of improved therapy for humans with these inherited bleeding disorders.
ILAR journal / National Research Council, Institute of Laboratory Animal Resources 02/2009; 50(2):144-67. · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion and is decreased in von Willebrand disease (VWD). 1-8 deamino-d-arginine vasopressin (DDAVP), the most common treatment for VWD, is limited by tachyphylaxis and inconvenience, and in 20% of the patients, unresponsiveness. Recombinant human interleukin-11 (rhIL-11), a gp-130 signalling cytokine with haematopoietic and anti-inflammatory activity, increases VWF antigen and its activity in heterozygous VWF(+/-) mice and dogs. To determine the biological efficacy and safety of rhIL-11 in non-bleeding human subjects with mild VWD, we conducted a phase II prospective open-label trial of rhIL-11 at 10, 25 and 50 mug kg(-1) subcutaneously (s.c.), given daily for 7 days in nine subjects with mild VWD. VWF and factor VIII (FVIII) levels increased gradually and progressively after s.c. rhIL-11, which was sustained through 7 days of dosing to 1.5- to 3-fold over baseline. Following intravenous DDAVP, 0.3 mug kg(-1), on day 7 there was a further boost in VWF and FVIII levels, suggesting that the mechanism of rhIL-11 differs from that of DDAVP. Platelet VWF mRNA expression measured by quantitative PCR increased from two- to eightfold over baseline, suggesting that the mechanism of rhIL-11 effect may be upregulation of VWF mRNA. VWF and FVIII levels returned to baseline by day 14. rhIL-11 was well tolerated with less than grade-1 hypertension, hypokalaemia and fluid retention. Recombinant IL-11 increases VWF levels in humans with mild VWD, justifying future clinical trials to determine its potential in preventing or reducing bleeding in this patient population.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy could prevent bleeding in hemophilia. However, antibodies could inhibit coagulation, while cytotoxic T lymphocytes could destroy modified cells. The immaturity of the newborn immune system might prevent these immune responses from occurring after neonatal gene therapy.
Newborn dogs, cats, or mice were injected intravenously with a retroviral vector expressing human Factor IX. Plasma was evaluated for antigen and anti-human Factor IX antibodies. Cytotoxic T lymphocyte responses were evaluated indirectly by analysis of retroviral vector RNA in liver. Lymphocytes were evaluated for cytokine secretion and the ability to suppress an immune response to human Factor IX in mice.
Hemophilia B dogs that achieved 942+/-500 ng/ml (19% normal) or 5+/-0.4 ng/ml (0.1% normal) of human Factor IX in plasma only bled 0 or 1.2 times per year, respectively, and were tolerant to infusion of human Factor IX. Normal cats expressed human Factor IX at 118+/-29 ng/ml (2% normal) in plasma without antibody formation. However, plasma human Factor IX disappeared at late times in 1 of 4 cats, which was probably due to a cytotoxic T lymphocyte response that destroyed cells with high expression. C3H mice were tolerant to human Factor IX after neonatal gene therapy, which may involve clonal deletion of human Factor IX-responsive cells. These data demonstrate that neonatal gene therapy does not induce antibodies to human Factor IX in dogs, cats, or mice. The putative cytotoxic T lymphocyte response in one cat requires further study.
Thrombosis Research 02/2007; 120(2):269-80. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences offer high transduction efficiencies but were hampered by transient phenotypic correction at a non-toxic dose. For persistent hemostatic correction in hemophilia B dogs, our current study utilized a novel gene-deleted adenoviral vector system which combines adenoviral vectors for high transduction efficiencies and for the first time in a larger animal the Sleeping Beauty (SB) transposase for somatic integration and stable transgene expression. We generated the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 in which a transposon with a canine factor IX (cFIX) expression cassette is flanked by FRT sites. In the presence of Flp recombinase and the recently developed hyperactive transposase HSB5 (Yant et al., unpublished), the transgene undergoes Flp mediated excision followed by HSB5 mediated somatic integration.To analyze transgene persistence in vivo C57Bl/6 mice were co-transfused with 2×10e9 transducing units (TU) of the vector FTC/TcFIX/attB/(FRT)2 and 7×10e8 TU of a second HD vector which encodes HSB5 and Flp. Control mice received an inactive version of SB, respectively. Rapid cell cycling of mouse hepatocytes was induced by performing a two-thirds partial hepatectomy and by injecting CCl(4). Seventy-five days post-injection we detected serum levels of cFIX of up to 2000 ng/ml in the active SB group and no cFIX in the control mice. This indicated that integration of the transgene from the episomal adenoviral vector genome into the host genome occurred.To test for persistence of hemostatic correction of the bleeding diathesis in hemophila B dogs we have co-injected 5.4×10e11 TU of the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 and 2.6×10e11 TU of the HSB5 and Flp encoding vector. This equals a total dose of 1.6×10e13 viral particles (vps) and 1×10e12 vps/kg body weight. We measured plasma cFIX levels of up to 3900ng/ml (normal level = 5000ng/ml) and observed complete phenotypic correction of the factor IX deficiency. The whole blot clotting time (WBCT) was reduced from 60min to 18.5min on day 35, the length of the study to date. This was in sharp contrast to our previous studies in hemophilia B dogs in which we used a non-integrating HD vector. In that study we observed a 3-fold increase of the WBCT 35 days post-injection which indicates that in our current study transposition stabilized phenotypic correction. In contrast to previous studies using early generation adenoviral vectors and keeping in mind that for the first time the in vivo performance of the SB transposase in a larger animal was evaluated, we observed no vector-related elevation of liver enzymes and no fall in platelet counts. Taken together, this study demonstrates that this adeno-transposon hybrid vector system will be important for treating genetic diseases.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown successful correction of hemophilia B in dogs by muscle-directed gene delivery of canine F.IX (cF.IX) using an adeno-associated virus (AAV) serotype 2. Intravascular delivery of the cF.IX gene to skeletal muscle allowed long-term expression of cF.IX of 4-20%, thereby correcting the disease phenotype. However, concerns over the risk of immune responses against the transgene and viral vector antigens have focused recent work on further characterizing the immunogenicity of both AAV capsid and F.IX in a gene therapy setting.Six HB dogs from the Chapel Hill colony received three different doses of an AAV2-cF.IX vector (1x1012 vg/kg, n=3; 3 × 1012 vg/kg, n=2; 8 × 1012 vg/kg, n=1) by intravascular delivery to skeletal muscle, in addition to an immunosuppression (IS) regimen (6 weekly infusions of cyclophosphamide beginning at the time of vector administration). T cell responses were measured on PBMCs isolated from whole blood drawn prior to vector infusion, during IS and after discontinuing IS. PBMCs were used for ELISpot assays to measure IL-10 and IFN-γ secretion in response to both cF.IX and AAV-2 antigens. This approach utilized a peptide library consisting of 15- mers overlapping by 10 amino acids, spanning the entire antigen sequence, arranged in a matrix such that each peptide was contained in two orthogonal pools. All HB dogs in this study exhibited a unique Th2 response consisting of IL-10 secretion in response to a common epitope corresponding to peptide 68 in the cF.IX library. This epitope was also found in normal dogs, which have the natural form of the protein, suggesting a role in tolerance to the cF.IX transgene rather than immunity. Interestingly, the dog that received the highest vector dose (1.5 μg cF.IX/ml, 30%) at day 15 post- injection, developed a non-neutralizing antibody response to cF.IX, as well as a T cell response to an epitope corresponding to peptide 84. This peptide maps to the region containing the missense mutation responsible for the disease phenotype (Chapel Hill mutation, Glu379→︀Gly). No IFN-γ secretion was detected following AAV2-cF.IX gene transfer to muscle, contrasting the Th1 response previously seen in intramuscularly delivered cF.IX using an AAV-1 vector. While all animals that received an AAV2-cF.IX vector developed B cell responses against the viral capsid antigen, T cell responses to the capsid were undetectable in PBMCs even after in vitro expansion with the antigen.In conclusion, intravascular delivery of AAV2-cF.IX to skeletal muscle elicited a Th2 T cell response characterized by the secretion of IL-10 in response to a specific cF.IX peptide. An additional immunodominant peptide was detected in the presence of a non- neutralizing immune response to the cF.IX transgene at the highest dose. These results suggest that while route of administration and/ or AAV serotype influence the type of immune response elicited, transgene level may impose additional safety limitations in terms of T cell response.
[Show abstract][Hide abstract] ABSTRACT: Adeno-associated virus 8 (AAV8), a new member of the AAV family isolated from nonhuman primates, is an attractive candidate for hepatic gene transfer applications because of 10- to 100-fold improved transduction efficiency in mouse liver models. Additionally, AAV8 has lesser frequency of pre-existing immunity in humans. These properties could solve some of the problems associated with AAV2 vectors. The benefits of AAV8 demonstrated in mouse models, however, have not been confirmed in larger animals. In this study, we evaluate the efficacy and safety of AAV2/8 vector in both naive and AAV2-pretreated hemophilia B dogs. Two naive hemophilia B dogs that received a single intraportal administration of AAV2/8 vector have achieved sustained expression of 10% and 26% of normal levels of canine factor IX (cFIX) for more than a year. In an AAV2-pretreated hemophilia B dog, cFIX expression increased from less than 1% to 16% of normal levels when treated with an AAV2/8 vector, and a high level of expression has lasted for more than 2 years. No significant liver toxicity or cFIX-specific antibodies have been detected in these animals. Studies here have demonstrated the safety and improved efficacy of AAV2/8 vector in large-animal models for liver-directed gene therapy.
[Show abstract][Hide abstract] ABSTRACT: Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity.