Gene therapy for hemophilia

PURPOSE OF REVIEW: This review will highlight the progress achieved in the past 2 years on using gene therapy to treat hemophilia in animals and humans. RECENT FINDINGS: There has been substantial progress in using gene therapy to treat animals with hemophilia. Novel approaches for hemophilia A in mice include expression of Factor VIII in blood cells or platelets derived from ex-vivo transduced hematopoietic stem cells, or in-vivo transfer of transposons expressing Factor VIII into endothelial cells or hepatocytes. Advances in large-animal models include the demonstration that neonatal administration of a retroviral vector expressing canine Factor VIII completely corrected hemophilia A in dogs, and that double-stranded adeno-associated virus vectors resulted in expression of Factor IX that is 28-fold that obtained using single-stranded adeno-associated virus vectors. In humans, one hemophilia B patient achieved 10% of normal activity after liver-directed gene therapy with a single-stranded adeno-associated virus vector expressing human Factor IX. Expression fell at 1 month, however, which was likely due to an immune response to the modified cells. SUMMARY: Gene therapy has been successful in a patient with hemophilia B, but expression was unstable due to an immune response. Abrogating immune responses is the next major hurdle for achieving long-lasting gene therapy.     

Immune deviation by mucosal antigen administration suppresses gene-transfer-induced inhibitor formation to factor IX

Formation of inhibitory antibodies is a serious complication of protein or gene replacement therapy for hemophilias, congenital X-linked bleeding disorders. In hemophilia B (coagulation factor IX [F.IX] deficiency), lack of endogenous F.IX antigen expression and other genetic factors may increase the risk of antibody formation to functional F.IX. Here, we developed a protocol for reducing inhibitor formation in gene therapy by prior mucosal (intranasal) administration of a peptide representing a human F.IX-specific CD4(+) T-cell epitope in hemophilia B mice. C3H/HeJ mice with a F.IX gene deletion produced inhibitory IgG to human F.IX after hepatic gene transfer with an adeno-associated viral vector. These animals subsequently lost systemic F.IX expression. In contrast, repeated intranasal administration of the specific peptide resulted in reduced inhibitor formation, sustained circulating F.IX levels, and sustained partial correction of coagulation following hepatic gene transfer. This was achieved through immune deviation to a T-helper-cell response with increased IL-10 and TGF-beta production and activation of regulatory CD4(+)CD25(+) T cells.     

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.     

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.     

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome. Whereas onco-retroviral vectors can only transduce dividing cells, lentiviral vectors can transduce a broad variety of cell types irrespective of cell division. Several preclinical and clinical studies have explored the use of onco-retroviral and, more recently, lentiviral vectors for gene therapy of hemophilia A or B. Both ex vivo and in vivo gene therapy approaches have been evaluated, resulting in therapeutic FVIII or FIX levels in preclinical animal models. Whereas in vivo gene therapy using onco-retroviral or lentiviral vectors often led to long-term FVIII or FIX expression from transduced hepatocytes, ex vivo approaches were generally hampered by either low or transient expression of FVIII or FIX levels in vivo and/or inefficient engraftment. Furthermore, immune responses against the transgene product remain a major issue that must be resolved before the full potential of these vectors eventually can be exploited clinically. Nevertheless, the continued progress in vector design combined with a better understanding of vector biology may ultimately yield more effective gene therapy approaches using these integrating vectors.     

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.     

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.     

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.     

Nonviral gene therapy approaches to hemophilia

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.     

Gene transfer for hemophilia and viral infection

The goal of our work has been to establish an experimental basis for gene transfer as a method of treating hemophilia, an inherited bleeding disorder that results from the absence of functional Factor VIII or Factor IX. We have carried out pre-clinical and clinical studies of AAV-mediated gene transfer of the Factor IX gene in animal models and in human subjects with severe hemophilia B. Target tissues in humans have included both skeletal muscle (via direct intramuscular injection) and liver (via hepatic artery infusion). Our preclinical efficacy studies have demonstrated a life-long correction of the bleeding diathesis in hemophilia B in mice, and long-term therapeutic reconstitution of canine factor IX in deficient dogs after AAV-mediated gene transfer into the liver. To date, hemophilia B dogs have been followed for over 4 years and still maintain their initial levels of factor IX following the administration of AAV-2. As a result, a number of preclinical studies of AAV into the liver of various species were performed to generate the appropriate safety data to support a Phase I liver-based clinical trial. The comparative results from the two clinical trials, the correlative results of the preclinical studies from the animal models, and the potential advantages/disadvantages of the two approaches will be discussed. Hepatitis C Virus (HCV) infection is a major health problem world-wide. We have recently evaluated different gene transfer approaches for targeted disruption of the viral life cycle. RNA interference (RNAi) is a recently discovered RNA surveillance mechanism used in lower animals and plants to silence specific genes. We have recently demonstrated that RNAi can function in adult mammals. By adapting this to a gene transfer strategy, we have established selective suppression of a HCV-reporter chimeric gene in mouse liver. RNAi has potential for use as a gene therapeutic to treat HCV infection as well as a large number of other acquired and genetic disorders.     

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.     

Gene therapy of hemophilia

Hemophilia A and B are X-linked bleeding disorders caused by mutations within the factor VIII and factor IX genes, respectively. Although both disorders can be easily treated by substitution with concentrates of functional factor VIII and factor IX, considerable effort has been undertaken to develop a gene therapy for hemophilia in order to improve patients' life quality and reduce high costs of therapy. The principle of gene therapy is the introduction of an intact copy of the factor VIII/factor IX gene in somatic cells, compensating for the defective gene. To do this, retroviral, adenoviral, and adeno-associated virus (AAV) vector systems, among others, were used. Encouraged by the results of preliminary experiments using preponderant mouse and canine models, three clinical phase I studies on hemophilia A and B patients have been initiated, one of which has been preliminarily reported successful.     

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.     

Immune implications of gene therapy for hemophilia

Similar to any novel treatment strategy for hemophilia, gene therapy faces the question of the risk of formation of inhibitory antibodies to the therapeutic factor VIII or factor IX protein. Activation of CD4 (+) or CD8 (+) T cells could lead to antibody formation or cytotoxic T lymphocyte responses to transgene-expressing cells. Preclinical studies in animal models of hemophilia A and B with different mutations in the dysfunctional gene shed light on the risk for such immune responses and point toward strategies to avoid immune activation or even promote tolerance induction. The impacts of variables such as choice and design of gene transfer vector, underlying gene mutation, route of vector administration, and transient immune suppression are discussed. Maintenance of immunological hyporesponsiveness to the therapeutic gene product is critical for successful gene therapy. Recent studies provide evidence for tolerance induction to coagulation factor antigens by viral hepatic or neonatal in vivo gene transfer, by in utero gene delivery, and by oral or nasal administration of protein or peptides.     

Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy

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.     

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.     

Phenotypic correction of factor IX deficiency in skin fibroblasts of hemophilic dogs.

Primary skin fibroblasts from hemophilic dogs were transduced by recombinant retrovirus (LNCdF9L) containing a canine factor IX cDNA. High levels of biologically active canine factor IX (1.0 micrograms per 10(6) cells per 24 hr) were secreted in the medium. The level of factor IX produced increased substantially if the cells were stimulated by basic fibroblast growth factor during infection. Additionally, we also report that endothelial cells transduced by this virus can produce high levels of biologically active factor IX. We propose that skin fibroblasts and endothelial cells from hemophilia B dogs may serve as potential venues for the development and testing of models for treatment of hemophilia B by retrovirally mediated gene replacement therapy.     

Preclinical gene therapy studies for hemophilia using adeno-associated virus (AAV) vectors

Gene therapy offers a potential cure for hemophilia and several gene transfer vectors have been evaluated for their ability to treat this disease. This article reviews the studies that have been performed to evaluate the ability of recombinant adeno-associated virus (AAV) vectors to achieve safely the sustained expression of clotting factors following intramuscular, intravenous, and intrahepatic delivery to several animal models. These routes of administration are all effective in providing sustained and therapeutic levels of factor IX (FIX), although the levels vary. Intrahepatic delivery is more efficacious than intravenous administration, which is superior to intramuscular delivery. The recent development of efficient factor VIII (FVIII) expression cassettes has made AAV-based gene therapy for hemophilia A also within reach. Although no acute toxicity has been observed with any route of administration, an increased risk of antibody formation against FIX has been noted following intramuscular delivery. Biodistribution studies concluded that the vector disseminates to most tissues in a dose-dependent and time-dependent manner, but the majority of the vector resides in the targeted tissue. In addition, the risk of germline transmission has been shown to be low or absent. The relatively recent isolation of new AAV serotypes has resulted in the identification of vectors that have enhanced tropism for certain tissues. This combined with the potential of these new vectors to evade the immune response to AAV2, makes them attractive candidates for gene therapy. Although much progress has been made using AAV to treat hemophilia, there are several outstanding issues that need to be addressed. Delivery of AAV to large animals has not been reproducible, which could be due to nonoptimized delivery and/or immune responses to the vector or transgene product. In addition, a complete understanding of the biology of these vectors is required to assess their long-term safety. Solving these issues will lead to the development of a successful gene therapy product.     

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.