AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 x 10(11) vector genomes (vg)/kg to 1.8 x 10(12) vg/kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Pre-existing high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals.     

Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression. Stable and persistent human FIX (hFIX) expression (> 22 weeks) was observed from 4 vectors after injection into the portal circulation of immunodeficient mice. The level of expression was dependent on promoter with the highest expression, 10% of physiologic levels, observed with a vector containing the cytomegalovirus (CMV) enhancer/beta-actin promoter complex (CAGG). The kinetics of expression after injection of vector particles into muscle, tail vein, or portal vein were similar with hFIX detectable at 2 weeks and reaching a plateau by 8 weeks. For a given dose, intraportal administration of rAAV CAGG-FIX resulted in a 1.5-fold or 4-fold higher level of hFIX compared to tail vein or intramuscular injections, respectively. Polymerase chain reaction analysis demonstrated predominant localization of the rAAV FIX genome in liver and spleen after tail vein injection with a higher proportion in liver after portal vein injection. Therapeutic levels of hFIX were detected in the majority of immunocompetent mice (21 of 22) following intravenous administration of rAAV vector without the development of anti-hFIX antibodies, but hFIX was not detected in 14 immunocompetent mice following intramuscular administration, irrespective of strain. Instead, neutralizing anti-hFIX antibodies were detected in all the mice. These observations may have important implications for hemophilia B gene therapy with rAAV vectors.     

Adeno-associated virus-mediated gene transfer for hemophilia B

Hemophilia is the bleeding diathesis caused by mutations in the gene encoding factor VIII (hemophilia A) or factor IX (hemophilia B). Currently, the disease is treated by intravenous infusion of the missing purified clotting factor. The goal of gene transfer for treating hemophilia is to achieve sustained expression of factor VIII or factor IX at levels high enough to improve the symptoms of the disease. Hemophilia has proven to be an attractive model for those interested in gene transfer, and multiple gene-transfer strategies are currently being investigated for the hemophilias. The most promising preclinical studies have been with adeno-associated viral vectors (AAV); introduction of AAV vectors expressing factor IX into skeletal muscle or liver in hemophilic dogs has resulted in the long-term expression of factor IX at levels that are adequate to improve disease symptoms. Efforts to translate these findings into the clinical arena have proceeded slowly because of the lack of prior clinical experience with parenteral administration of AAV. In a staged approach, AAV-factor IX (AAV-F.IX) was first administered at doses of up to 1.8 x 10(12) vector genomes/kg (vg/kg) into the skeletal muscles of men with hemophilia B. This trial established the safety of parenteral administration and also showed that general characteristics of AAV transduction were similar in mice, dogs, and humans. In an ongoing trial, AAV-F.IX is being administered into the hepatic circulation of men with severe hemophilia B. The goal of these studies is to identify a safe dose that reliably yields circulating levels of factor IX >2% of normal levels in all subjects. This goal has already been achieved in the hemophilia B dog model; the ongoing study will determine whether a similar result can be achieved in humans with hemophilia B.     

Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX

The present study sought to determine the impact of the route of administration of an adeno-associated virus (AAV) vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX. Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice, which typically demonstrate significant immune tolerance to hFIX. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. At all dose levels tested, delivery of AAV-hFIX by an intramuscular (IM) route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals even at doses of 2 x 10(11) DNA viral particles (vp) of AAV-hFIX. This was in stark contrast to the mice that received AAV-hFIX by intraportal vein (IPV) administration. No anti-hFIX inhibitors were observed in any of these mice and therapeutic levels of hFIX were detected in the serum of all mice that received doses of 2 x 10(10) vp AAV-hFIX and higher. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. These results suggest that the threshold of inducing an immune response against a secreted transgene product, in this case the xenoprotein hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV. (Blood. 2001;97:3733-3737)     

Neonatal gene transfer with a retroviral vector results in tolerance to human factor IX in mice and dogs

The effect of neonatal gene transfer on antibody formation was determined using a retroviral vector (RV) expressing human factor IX (hFIX). Normal mice from different strains injected intravenously with RV as newborns achieved therapeutic levels of hFIX without antibody production and were tolerant as adults to challenge with hFIX. Neonatal hemophilia B mice that received different amounts of RV achieved stable and dose-related expression of hFIX without anti-hFIX antibody formation. After protein challenge, antibody formation was markedly reduced for animals that expressed hFIX at levels higher than 14 ng/mL (0.3% of normal). However, antibodies developed for animals that received the lowest dose of RV and expressed hFIX at approximately 2 ng/mL before protein challenge. In dogs, neonatal injection of a high dose of RV resulted in 500 ng/mL hFIX in plasma without antibody formation. We conclude that neonatal gene transfer with RV does not induce antibody responses to hFIX in mice or dogs and that mice achieving levels greater than 3 x 10-10 M hFIX are usually tolerant to protein injection as adults. Low-dose gene therapy or frequent protein injections in the neonatal period might induce tolerance to subsequent injections of protein with a low risk for adverse effects.     

Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice

Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.     

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.     

Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates

The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1x10(12) vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1+/-0.5 microg/mL, or 22% of normal) that were comparable to those achieved after direct delivery of the same vector dose into the portal circulation (1.3+/-0.3 microg/mL, or 26% of normal). Importantly, the pattern of vector biodistribution after systemic and portal vein administration of scAAV-LP1-hFIXco was almost identical. Additionally, comparable levels of gene transfer were achieved in macaques with preexisting immunity to AAV8 following peripheral vein administration of 1x10(12) vg/kg AAV5-pseudotyped scAAV-LP1-hFIXco. This confirms that alternative serotypes can circumvent preexisting naturally acquired immunity to AAV. Thus, peripheral venous administration of AAV5 and AAV8 vectors is safe and as effective at transducing the liver in nonhuman primates as direct vector administration into the portal circulation. These results should make vector administration to patients, especially those with a severe bleeding diathesis, significantly easier and safer.     

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.     

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.     

Pathobiology of hemophilic synovitis I: overexpression of mdm2 oncogene

Hemophilia is a genetic disease caused by a deficiency of blood coagulation factor VIII or IX. Bleeding into joints is the most frequent manifestation of hemophilia. Hemarthrosis results in an inflammatory and proliferative disorder termed hemophilic synovitis (HS). In time, a debilitating, crippling arthritis, hemophilic arthropathy, develops. Although the clinical sequence of events from joint bleeding to synovitis to arthropathy is well documented, the component or components in blood and the molecular changes responsible for hemophilic synovitis are not known. Iron has long been suspected to be the culprit but direct evidence has been lacking. Previously, we showed that iron increased human synovial cell proliferation and induced c-myc expression. Here we show that bleeding into a joint in vivo and iron in vitro result in increased expression of the p53-binding protein, mdm2. Iron induced the expression of mdm2 by normal human synovial cells approximately 8-fold. In a murine model of human hemophilia A, hemarthrosis resulted in pathologic changes observed in human hemophilic synovitis and a marked increase in synovial cell proliferation. Iron, in vitro, induced the expression of mdm2. The molecular changes induced by iron in the blood may be the basis of the increase in cell proliferation and the development of hemophilic synovitis.     

Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 x 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.     

Prenatal diagnosis of hemophilia B by an immunoradiometric assay of factor IX

An immunoradiometric assay of factor IX was developed based on homologous antibodies that arose in a hemophilic patient. With this assay, 11 of 12 patients with severe hemophilia B had factor IX antigen levels below 1 U/dl and 6 patients with mild hemophilia B had various levels. Factor IX antigen in 8 fetuses (16th-20th gestational week) aborted for therapeutic reasons ranged from 1.8 to 10.0 U/dl. Six amniotic fluids contained 0.28-1.2 U/dl factor IX antigen. Using the immunoradiometric assay, we could diagnose hemophilia B prenatally in one fetus at risk. No factor IX antigen (< 0.2 U/dl) was detectable in the fetoscopic sample. After termination of the pregnancy, analysis of blood from the abortus confirmed the diagnosis of severe hemophilia B. We conclude that very sensitive immunologic assays, such as the one described here, will prove useful in prenatal diagnosis of severe hemophilia B, since determination of factor IX activity in fetoscopic samples is unrealiable because of possible contamination with thromboplastic material.     

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.     

Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver

Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1alpha enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P < 0.005) in serum hFIX of up to 350 ng/mL compared with the nonhepatectomized counterparts. The expression of plasma hFVIII reached 30 ng/mL (15% of normal) but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies. However, hFVIII levels were persistent in immunodeficient C57Bl/6 scid mice, suggesting humoral immunity-limited gene expression in immunocompetent mice. This study demonstrates that lentiviral vectors can produce therapeutic levels of coagulation factors in vivo, which can be enhanced with hepatocellular proliferation.     

Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Hemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus-based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 microg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.     

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.     

Future aspects of hemophilia research and care

Key issues in the management of patients with hemophilia include a thorough understanding of the mechanisms of blood coagulation and the complications that follow recurrent joint bleeding. Monoclonal antibodies are powerful tools for dissecting the intrinsic coagulation pathway and deriving reagents that could lead, on the long term, to the identification of molecules that enhance, or perhaps even replace factor (F) VIII concentrates in the management of hemophilia A. In recent in vitro experiments, it was demonstrated that plasmatic thrombin generation and intrinsic FX activation was enhanced by each of two FIXa-specific monoclonal antibodies, one of which had FIXa-agonistic activity only, whereas the other enhanced the activity of the intrinsic FX-activating complex (FVIIIa/FIXa) by at least two distinct mechanisms. Hemophilic synovitis, an inflammatory and proliferative disorder in patients with hemophilia, is the result of bleeding into joints and can lead to debilitating arthritis and chronic arthropathy. A major causative factor in the development of hemophilic synovitis is blood-derived iron deposited in joints. FVIII-deficient knockout mice with trauma-induced hemarthrosis serve as a model system for hemophilic synovitis, reproducing the histological features observed in patients. In addition, this animal model recapitulates the observations made in vitro with synovial cell cultures stimulated by iron. These in vitro experiments suggested a role for iron as an agent capable of inducing proliferation and oncogene expression by human and murine synovial fibroblasts. A better understanding of iron-regulated pathways and oncogene expression may lay the groundwork for targeted molecular interventions in hemophilic synovitis.