Hemophilic Dogs Research Articles

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.

Novel, high incidence exercise‐induced muscle bleeding model in hemophilia B mice: rationale, development and prophylactic intervention

IntroductionMuscle hematomas are the second most common complication of hemophilia and insufficient treatment may result in serious and even life-threatening complications. Hemophilic dogs and rats do experience spontaneous muscle bleeding, but currently, no experimental animal model is available specifically investigating spontaneous muscle bleeds in a hemophilic setting.AimThe objective of this study was to develop a model of spontaneous muscle bleeds in hemophilia B mice. We hypothesized that treadmill exercise would induce muscle bleeds in hemophilia B mice but not in normal non-hemophilic mice and that treatment with recombinant factor IX (rFIX) before treadmill exercise could prevent the occurrence of pathology.MethodsA total of 203 mice (123 F9-KO and 80 C57BL/6NTac) were included in three separate studies: (i) the model implementation study investigating the bleeding pattern in hemophilia B mice after treadmill exercise; (ii) a study evaluating the pharmacokinetics of recombinant FIX (rFIX) in hemophilia B mice and based on these data; (iii) the treatment study, which tested therapeutic intervention with rFIX. At termination of the treadmill studies the presence of bleeds was evaluated.ResultsTreadmill exercise resulted in a high incidence of muscle bleeds in F9-KO mice but not in C57BL/6NTac mice. Treating hemophilia B mice with rFIX before treadmill exercise prevented muscle bleeds.ConclusionA novel model of muscle bleeds in hemophilia B mice, responsive to rFIX, has been developed.

VKORc1 Is Under-Expressed in Skeletal Muscle of Humans, Dogs and Mice: Potential Implications for Ectopic Coagulation Factor Expression in Pre-Clinical and Therapeutic Applications

Post-translational modifications of coagulation factors in the liver are essential for function. The vitamin K dependent coagulation proteins (VKCPs) require vitamin K to undergo gamma carboxylation of the glutamic residues in their Gla domain by gamma-glutamyl carboxylase [GGCX]. The vitamin K is then recycled by the action of epoxide reductase [VKORc1] and/or quinone reductase [NQO1]. The hemostatic importance of the vitamin K “cycle” is evidenced by patients who may suffer bleeding complications when anticoagulated with warfarin, which targets the vitamin K cycle. Moreover, the ability of a variety of VKCPs to secrete a biologically active product depends on the removal of their propeptide by the action of the intracellular endoprotease furin [FURIN gene]. Previous in vitro work on recombinant coagulation Factor IX, which is used for hemophilia B treatment, has connected these two processing steps by showing that endogenous VKORc1 as well as FURIN can be limiting factors in high-yield expression systems. In vivo, skeletal muscle (in contrast to liver) has been utilized to express low levels of coagulation Factor IX in the first hemophilia B gene therapy clinical trial. However, our experiments in mice demonstrated that the specific activity of muscle-synthesized Factor IX via gene transfer decreased at the high levels of FIX expression by a limited muscle area (Schuettrumpf J. et al., Blood 2005). These results suggest that in vitro and in vivo expression of biologically-active VKCPs outside the liver may be limited by the host cell post-translational modification machinery. Here, we performed a systematic study to determine the expression profiles of the vitamin K cycle and furin endoprotease genes in human liver and muscle, compared to the mouse. We also established these profiles in two hemophilic dogs, given the extensive use of this animal model in gene-based hemophilia therapies. RNA from liver and skeletal muscle was used as a template for reverse transcription and the subsequent relative quantification of the GGCX, VKORc1, NQO1, and FURIN genes by qPCR in each tissue using a housekeeping reporter gene. For this, a variety of housekeeping genes were investigated in all three species to identify ones with similar transcript levels in both liver and muscle tissue. We identified the housekeeping genes HPRT1, beta actin, and 18s rRNA as equivalently expressed in the liver and skeletal muscle of human, mouse, and dog, respectively. The relative mRNA transcript quantification of the vitamin K cycle genes in humans showed that the transcript levels of GGCX were similar in liver and muscle. In contrast, both VKORc1 and NQO1 were under-expressed in muscle vs. liver (69.5 ± 4.9% and 67.8 ± 12.5%, respectively, P<0.01). In the mouse, VKORc1 transcript levels in the muscle were reduced to 73.8 ± 9.9% vs. liver (P<0.05), while GGCX and NQO1 exhibited similar transcript levels in both tissues. In the dog, we observed a dramatic reduction in VKORc1 and GGCX transcript levels in the muscle vs. liver (11.8 ± 4.2% and 29.5 ± 15.8%, respectively, P<0.01). Surprisingly, NQO1 transcript levels were 253.8 ± 156.7% higher in muscle than liver (P<0.05). Lastly, in all three species tested, transcript levels for FURIN were similar in both muscle and liver. Our results indicate that VKORc1, a key enzyme in the vitamin K cycle, is consistently under-expressed in the skeletal muscle of humans as well as in mice and hemophilic dogs. In contrast, FURIN transcripts are similarly abundant in the liver and muscle of all three species tested. These suggest that the vitamin K cycle but not propeptide processing by furin can be a limiting factor in the secretion of biologically active muscle-expressed VKCPs. As a result, our observations provide (1) a plausible explanation for the inverse relationship between specific activity and Factor IX expression levels in mice following Factor IX gene transfer, and (2) further support for the mouse and dog as useful models for therapies that depend on the muscle-derived expression of VKCPs. DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics and pharmacodynamics of turoctocog alfa and N8-GP in haemophilia A dogs.

The objective of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the new recombinant FVIII compound turoctocog alfa and a Glyco-PEGylated FVIII derivative thereof (N8-GP) in Haemophilia A dogs. Six haemophilic dogs divided into two groups were included in the study. Each dog was administered a dose of 125 U kg(-1) , blood samples were collected at predetermined time points for both pharmacokinetic (FVIII measured by one-stage aPTT assay) and pharmacodynamic [whole blood clotting time (WBCT)] evaluations. After intravenous administration to haemophilic dogs, the plasma concentration at the first sampling point was comparable for turoctocog alfa and N8-GP, and the clearance was estimated to be 6.5 and 3.9 mL h(-1) kg(-1) for turoctocog alfa and N8-GP respectively. Both turoctocog alfa and N8-GP were able to reduce the WBCT time to normal levels (<20 min), however, the reduced clearance was reflected in the WBCT, which returned to baseline at a later time point for N8-GP as compared with dogs dosed with turoctocog alfa. The clearance was 40% reduced for N8-GP as compared with turoctocog alfa. Simulations of a multiple dosing regimen in dogs, suggest that to maintain WBCT <20 min N8-GP can be dosed at reduced intervals, e.g. with 4 days between doses, whereas turoctocog alfa will have to be dosed with 2½ day between doses. Data thereby supports N8-GP as an alternative to standard rFVIII replacement therapy, with a more convenient dosing regimen.

Porcine factor VIII

Provided are porcine factor VIII nucleotide sequences and hybrid sequences encoding human/porcine chimeric factor VIII-type procoagulant proteins. DNAs encoding such chimeric factor VIII-type procoagulant proteins, pharmaceutical compositions containing such chimeric factor VIII-type procoagulant proteins, and methods of treating hemophilia using such chimeric factor VIII-type procoagulant proteins are also provided.

Solulin increases clot stability in whole blood from humans and dogs with hemophilia

Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)-deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor-initiated fibrin formation and tissue-plasminogen activator-induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/μM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses.

Gene-based continuous expression of FVIIa for the treatment of hemophilia

Qualitative or quantitative defects in the genes for coagulation factors VIII (FVIII) or IX (FIX) result in a life-threatening, bleeding phenotype (hemophilia A (HA) or B (HB), respectively). Although hemophilia treatment by clotting factor replacement is effective, a proportion of patients develop neutralizing antibodies (inhibitors) to the infused factor that complicate the disease management. For inhibitor patients, recombinant human activated coagulation Factor VII (rhFVIIa), when administered at therapeutic doses, has been shown to bypass the deficiency in FVIII or FIX and result in hemostasis. As an alternative to this protein infusion therapy, a gene-based approach for the treatment of hemophilia with inhibitors has been developed, using continuous expression of a transgene coding for FVIIa following viral-mediated delivery. This approach was validated in hemophilic mice and, notably, in dogs as a model that closely resembles the human disease. In particular, liver-directed FVIIa gene delivery in hemophilic dogs resulted in multi-year transgene expression that ameliorated the bleeding phenotype, without thrombotic complications. These data support the gene-based FVIIa expression as a novel bypass therapy for hemophilia with inhibitors.

Gene-based continuous expression of FVIIa for the treatment of hemophilia

Qualitative or quantitative defects in the genes for coagulation factors VIII (FVIII) or IX (FIX) result in a life-threatening, bleeding phenotype (hemophilia A (HA) or B (HB), respectively). Although hemophilia treatment by clotting factor replacement is effective, a proportion of patients develop neutralizing antibodies (inhibitors) to the infused factor that complicate the disease management. For inhibitor patients, recombinant human activated coagulation Factor VII (rhFVIIa), when administered at therapeutic doses, has been shown to bypass the deficiency in FVIII or FIX and result in hemostasis. As an alternative to this protein infusion therapy, a gene-based approach for the treatment of hemophilia with inhibitors has been developed, using continuous expression of a transgene coding for FVIIa following viral-mediated delivery. This approach was validated in hemophilic mice and, notably, in dogs as a model that closely resembles the human disease. In particular, liver-directed FVIIa gene delivery in hemophilic dogs resulted in multi-year transgene expression that ameliorated the bleeding phenotype, without thrombotic complications. These data support the gene-based FVIIa expression as a novel bypass therapy for hemophilia with inhibitors.

Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy

Human blood outgrowth endothelial cells (hBOECs) may be useful delivery cells for gene therapy. hBOECs have high expansion capacity and a stable phenotype. If incorporated into blood vessels, hBOECs could release therapeutic agents directly into the bloodstream. However, little is known about the lodging and homing of hBOECs in vivo. We examined the homing patterns of hBOECs in mice and explored extending cell-based factor VIII (FVIII) gene therapy from mice to larger animals. hBOECs were injected into NOD/SCID mice to determine where they localize, how localization changes over time, and if there were toxic effects on host organs. The presence of hBOECs in mouse organs was determined by quantitative polymerase chain reaction (qPCR) and immunofluorescence microscopy. hBOECs lodged most notably in mouse lungs at 3 h, but by 24 h, no differences were observed among 9 organs. The longevity of hBOECs was assessed up to 7 months in vivo. hBOECs expanded well and then reached a plateau in vivo. hBOECs from older cultures expanded equally well in vivo as younger hBOECs. hBOECs caused no noticeable organ toxicity up to 3 days after injection. When mice were pretreated with antibodies to E-selectin, P-selectin, or anti-alpha4 integrin prior to hBOEC injection, the number of hBOECs in lungs at 3 h was decreased. Preliminary studies that infused hemophilic dogs with autologous canine BOECs that overexpressed FVIII (B-domain deleted) showed improvement in whole blood clotting times (WBCTs). In conclusion, the survivability, expandability, and lack of toxicity of BOECs in vivo indicate that they may be valuable host cells for gene therapy.

Successful and Safe Treatment of Canine Hemophilia by Continuous Expression of Canine FVIIa: a Model for FVIII/FIX Gene-Based Bypassing Agents

Abstract Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. However, the short plasma half-life and need for repeated infusions are amongst the limiting factors for its use in prophylaxis. In an attempt to overcome these, we have designed a gene transfer approach using continuous expression of a modified FVII transgene that is secreted in the active form, FVIIa. In proof-of-concept experiments, we previously demonstrated that Adeno-associated virus (AAV)–mediated gene transfer of the modified Factor VII gene can result in long-term phenotypic correction in hemophilia B mice. However, a prerequisite to a human application is the demonstration of efficacy as well as safety in a large animal model of hemophilia. Hence, we utilized the canine hemophilia model that closely resembles the human disease and has been shown to be an excellent predictor of efficacy of treatments for hemophilia in humans. As we previously reported (Blood 110 (11), p65a. 2007), we generated a canine FVII transgene that is expressed and secreted in a two-chain, activated form (cFVIIa). An AAV serotype 8 vector directing expression of this transgene was delivered via the portal vein into 4 hemophilia dogs, at vector doses of 2.06 E13 (one hemophilia B dog) − 1.25 E14 (three hemophilia A dogs) vector genomes (vg)/kg. The current abstract presents a rigorous characterization of the clotting parameters in the treated dogs, clinical safety and efficacy results of a substantially longer follow-up, and a laboratory safety profile of continuous expression of canine FVIIa. Gene expression in the treated dogs was followed by antigen and clotting assays (prothrombin time [PT], whole blood clotting time [WBCT]) as well as rotational thromboelastography (TEG). The initial dose of 2.06 E13 vg/kg resulted in a modest and transient reduction in the PT, WBCT and TEG parameters over a period of &gt;31 months, accompanied by marginal changes in cFVIIa antigen levels. However, a considerable and sustained reduction in PT (supra-physiologic), WBCT (partial correction) and TEG parameters (near-normalization) was observed following administration of 6.25 E13 – 1.25 E14 vg/kg in three hemophilia A dogs in a cumulative total of 42 months of observation. Antigen levels in the treated hemophilia A dogs were in the range of 1–2.5 μg/ml. As untreated hemophilic dogs exhibit 5–6 spontaneous bleeds per year, we utilized this as an important efficacy endpoint. None of the treated dogs (including the lowest dose hemophilia B dog) exhibited any spontaneous bleeding episodes (34 expected within the observation period) in contrast to concurrently observed controls, which exhibited clinically detectable bleeding episodes at the predicted rate. Given the procoagulant nature of cFVIIa, the safety profile of this approach was thoroughly investigated. We did not observe any abnormal changes in serum chemistries, platelet counts, levels of D-dimer, fibrinogen or thrombin-antithrombin, arguing against a procoagulant state following cFVIIa-gene transfer. In summary, our results demonstrate for the first time that AAV8 vector doses of up to 1.25 E14 vg/kg (2.6 E15 vg total) are well-tolerated in hemophilic dogs; continuous expression of FVIIa at levels of 1–2.5 μg/ml is safe in hemophilic dogs, and can result in a sustained, measurable improvement in the clinical bleeding course of these animals. These data suggest that this model approach of a gene-based Factor VIII/Factor IX bypassing agent has therapeutic potential for hemophilia as has been shown for bolus infusion of rhFVIIa

AAV-mediated gene transfer for the treatment of hemophilia B: problems and prospects

Adeno-associated viral vector-mediated gene transfer of coagulation factor IX to the skeletal muscle or to liver has resulted in sustained correction of hemophilia B in mice and dogs. The two initial phase I/II AAV clinical trials for hemophilia B, delivering a factor IX cDNA to skeletal muscle or liver, showed no serious adverse events. Although the muscle trial failed to achieve a therapeutic level of factor IX in the circulation, long-term expression of clotting factor was demonstrated on muscle biopsies taken up to 3 years after vector injection. Administration of vector to liver via the hepatic artery identified a therapeutic dose, which agreed closely with the doses predicted by studies in hemophilic dogs. However, expression in human subjects lasted for only a period of weeks, followed by a gradual decline in factor IX levels accompanied by a self-limited, asymptomatic rise and fall in liver enzymes. Immune responses to vector capsid may account for this difference in outcome between humans and other species. Here we review the results from both preclinical and clinical studies of adeno-associated viral vector gene transfer for hemophilia B, and the problems that have been identified and that must be overcome to achieve successful transduction and sustained expression.

A Murine FVIIa Variant with Increased Tissue Factor-Independent Intrinsic Activity Corrects the Murine Hemophilia B Phenotype Following AAV Administration: Implications for Therapeutic AAV Vector Doses.

We have previously designed a gene transfer approach using a modified FVII transgene that is cleaved intracellularly, secreted in the active form (FVIIa) and results in phenotypic correction of hemophilia B mice, following adeno-associated virus (AAV)-mediated, liver-directed gene delivery. Research into FVIIa variants with increased catalytic efficiency will not only provide information on the mechanism of action of high-dose FVIIa [tissue factor (TF)-dependent or independent], but may also lower the vector doses required in gene therapy settings. However, the use of such variants in an animal model of hemophilia is lacking. To address this, based on published observations using human FVIIa, we have generated a murine FVIIa variant (L305V/A314E/K337A/I374Y, mFVIIa-VEAY) with substitutions located in the catalytic domain of murine FVIIa. Purified mFVIIa and mFVIIa-VEAY from conditioned medium were almost exclusively in the activated form. Mouse FVII zymogen was purified as a single chain, indicating the lack of autoactivation during purification. Using clotting-based assays for the extrinsic (PT) and intrinsic pathway (aPTT), we found that variant VEAY had almost identical extrinsic activity to unsubstituted mFVIIa but substantially increased intrinsic activity (between 3–7 fold) using FVIII, FIX or FVII deficient plasmas, suggesting that this variant has TF-independent clotting activity. From in vitro kinetic assays, binding to human or murine soluble TF was identical for both mFVIIa and mFVIIa-VEAY (2.0 and 3.2 nM for murine TF, respectively; 86.7 and 71.7 nM for human TF, respectively). Similarly, using the physiological substrate (human FX) at saturating TF conditions, FXa generation was identical for both mFVIIa and mFVIIa-VEAY. However, in the absence of TF, mFVIIa-VEAY exhibited substantial proteolytic activity towards FX (6.6 fold), when compared to mFVIIa (which results in a minimal rate of FXa generation). In rotational thromboelastometry with citrated hemophilia B whole mouse blood and mFVIIa-VEAY or mFVIIa added at a dose of 1μg/ml (corresponding to the clinically effective dose of 90μg/kg), mFVIIa-VEAY exhibited a reduction in the clot time (CT) relative to both mFVIIa-treated hemophilia B blood or wild-type blood [61 ± 3 sec vs. 79 ± 13 sec (mFVIIa-treated) and 106 ± 13 sec (wild-type)]. Lastly, we administered 1.2 × 10^12 vector genomes of AAV8-mFVIIa or AAV-mFVIIa-VEAY into the hepatic circulation of hemophilia B mice. At 2 weeks post transduction, AAV-mFVIIa-VEAY treated mice exhibited superphysiological correction of aPTT and PT, considerably below that of wild type animals (aPTT: 17.4 vs. 28.2 sec; PT: 20.8 vs. 39.7, respectively). In contrast, AAV-mFVIIa treated mice exhibited near normalization of phenotype, indicating that phenotypic correction of the hemophilia B phenotype may be achieved using lower AAV-mFVIIa-VEAY vector doses, compared to mFVIIa. The effect of mFVIIa-VEAY in TF-dependent mouse injury models as well as a low TF mouse background is currently under investigation. In conclusion, we describe a murine FVIIa variant with increased TF-independent activity in vitro; we demonstrate that the variant also shows increased activity in vivo. This variant mFVIIa has potential for lowering the AAV dose in vivo for use in gene transfer applications and may allow for the extension of this approach to a large animal model of hemophilia, since high doses of vector are required to treat hemophilic dogs using the wild-type molecule.

Improved Coagulation in Bleeding Disorders by NASP Treatment.

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, and severe von Willebrand disease. A novel treatment approach which may benefit such clotting disorders has been identified that, parodoxically, involves heparin-like sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). Candidate NASPs were shown in clotting assays to demonstrate at least ten-fold lower anticoagulant activity as compared to heparin. Moreover, a subset of NASPs, including pentosan polysulfate (PPS) and fucoidan, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced FVII levels when tested at concentrations ranging from 4–500 nM in dilute prothrombin time (dPT) assays. A broad window of efficacy was evident with both PPS and fucoidan. A mechanism involving potent blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI), was implicated from modified dPT clotting tests. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Studies in hemophilic dogs are in progress. Accordingly, administration of select NASPs may represent a unique approach for treating multiple bleeding disorders.

Gene transfer for hemophilia: can therapeutic efficacy in large animals be safely translated to patients?

Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid rather than a protein or small molecule. As early as 1997, investigators reported long-term expression of therapeutic levels of factor IX using gene transfer techniques in hemophilia B mice, and similar data were thereafter reported in mice with hemophilia A. Efforts to translate these results to hemophilic dog models at first yielded only marginally therapeutic levels (1%-2% normal circulating levels), but within the past few years have achieved levels in the range of 10%-20% through multiple different gene transfer strategies. Early phase clinical testing has revealed that many aspects of gene transfer in humans were accurately predicted by studies in hemophilic dogs, but that other aspects were not, and were only appreciated as a result of clinical testing. Studies in the next few years will determine whether the problems identified in preclinical and early phase clinical testing can be solved to develop a therapeutic gene transfer approach to hemophilia.

397. AAV-Mediated Gene Transfer to Endothelial Cells

Adeno-associated virus (AAV) vectors have been evaluated in clinical studies of gene transfer for hemophilia by two different strategies, one using liver as the target tissue and the other using skeletal muscle. Both routes have resulted in long-term cures in hemophilic dogs. In humans, delivery to skeletal muscle resulted in long-term expression but at sub-therapeutic levels, while the liver-targeted study raised expression to therapeutic levels at the highest dose, although duration of expression was only short-lived (<10 weeks). Moreover, the high prevalence of hepatitis in the adult hemophilia population limits the utility of liver as a target tissue in practice. Therefore we investigated the possibility of targeting other cell types. Endothelial cells (ECs) are of interest because of their easy accessibility and the direct secretion of transgene product into the circulation.

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

AbstractIn earlier work, we showed that adeno-associated virus–mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.

Human Immune Responses to AAV-2 Capsid May Limit Duration of Expression in Liver-Directed Gene Transfer in Humans with Hemophilia B.

Based on studies in mice, hemophilic dogs, and non-human primates demonstrating long-term (>5 yrs) expression of Factor IX (FIX) after infusion of an AAV vector expressing FIX into the portal vein or the hepatic artery, we undertook a Phase I dose escalation study of AAV-FIX in humans with severe hemophilia B. The first two doses, 2x1011 vg/kg, and 1x1012 vg/kg, were safe but subtherapeutic. Two subjects treated at a dose of 5x1012 vg/kg showed detectable circulating levels of FIX (up to 11.8% and 3% respectively), but expression was transient and accompanied in one case (subject E) by a transient asymptomatic transaminitis. There was never evidence of a FIX inhibitor. Two differences between the large animal models and humans with the disease were hypothesized to contribute to the difference in duration of expression; long-term in hemophilic dogs, short-term in hemophilic humans. First was pre-existing immunity to wild-type AAV-2, which infects humans, but not dogs; and the other was prior exposure to viral hepatitis , found in humans but not in animals. To further assess the roles of viral hepatitis and of the immune response to AAV-2, we treated an additional subject (subject G) at a dose of 1x1012 vg/kg. This subject was 20 yrs. of age and had never been infected with hepatitis. Nevertheless, his transaminases began to rise 3 weeks after vector injection, peaked 6 weeks after injection, and resolved spontaneously as had been seen in subject E. In subject G, magnitude of the peak ALT response was 5-fold less than that found in subject E (5-fold higher dose). Both subjects had similar and low baseline anti-AAV antibody titers. Immune response to AAV-2 was assessed by ELISpot at serial time points before and after vector injection in subject G. The subject's PBMCs were incubated with a peptide library arrayed in a matrix of 24 pools, each containing 12 peptides of 15-mers overlapping by 10 and spanning the entire VP-1 protein. There was no detectable IFN- γ secretion in response to AAV-2 peptides at baseline, although there was a strong IFN- γ response to PHA. Two weeks after vector infusion, three pools elicited IFN- γ secretion from the subject's PBMCs. Response to the same pools of peptides, but not to other pools, was repeatedly detected over the next 6 weeks. By week 12, IFN- γ responses were no longer detectable. The matrix array allowed identification of two specific AAV-2 capsid peptides as the T cell immunoreactive epitopes. These peptides are highly conserved in AAV serotypes 1–8. Similar experiments were conducted with a FIX peptide library and demonstrated no response. These data are consistent with a model in which a T cell response to AAV capsid epitopes results in elimination of the transduced cells. This response is only briefly detectable in PBMCs, and hepatitis is not an important risk factor. These immune responses may limit use of standard serotypes of AAV for gene transfer into human liver. Transient immunomodulation may prevent these responses.

Preclinical gene therapy studies for hemophilia using adenoviral vectors.

Hemophilia A and B are hereditary coagulation defects resulting from a deficiency of factor VIII (FVIII) and factor IX (FIX), respectively. Introducing a functional FVIII or FIX gene could potentially provide a cure for these bleeding disorders. Adenoviral vectors have been used as tools to introduce potentially therapeutic genes into mammalian cells and are by far the most efficient vectors for hepatic gene delivery. Long-term expression of both FVIII and FIX has been achieved in preclinical (hemophilic) mouse models using adenoviral vectors. Therapeutic levels of FVIII and FIX also have been achieved in hemophilic dogs using adenoviral vectors and in some cases expression was long-term. The performance of earlier generation adenoviral vectors, which retained residual viral genes, was compromised by potent acute and chronic inflammatory responses that contributed to significant toxicity and morbidity and short-term expression of FVIII and FIX. The development of improved adenoviral vectors devoid of viral genes (gutless or high-capacity adenoviral vectors) was therefore warranted, which led to a significant reduction in acute and chronic toxicity and more prolonged expression of FVIII and FIX. Strategies aimed at making these vectors safer and less immunogenic and their implications for hemophilia gene therapy are discussed in this review.

Preclinical animal models for hemophilia gene therapy: predictive value and limitations.

Hemophilia A and B are excellent candidate disorders for the application of somatic cell gene therapy. One of the major advantages in the preclinical development of hemophilia gene therapy strategies has been the availability of several animal models for both hemophilia A and B. These models recapitulate many of the phenotypic aspects of human hemophilia and have proven to be very informative in exploring the efficacy and safety of gene therapy. Considerable progress has been made in the design of gene therapy protocols, and over the last 5 years it has been shown that long-term phenotypic correction, with sustained therapeutic levels of factor VIII (FVIII) and factor IX (FIX), can be attained in FVIII- and FIX-deficient mice and dogs using various viral vector-mediated gene therapy approaches. These animal models also have elucidated potential complications of gene therapy protocols, including acute vector-associated toxicities and the induction of neutralizing antibodies to the FVIII and FIX transgene products. Nevertheless, although the preclinical paradigm of hemophilic mouse followed by hemophilic dog studies has proven to be extremely helpful in evaluating the efficacy and safety of potential clinical gene therapy protocols, several limitations to these animal models still exist. This review presents a summary of the animal models available for hemophilia gene therapy, and highlights the various strengths and weaknesses of these models.