Update on gene therapy for hereditary hematological disorders

The past 3 years have been characterized by a number of impressive advances as well as setbacks in gene therapy for genetic disease. Children with X-linked severe combined immunodeficiency disorder (SCID-X1) have shown almost complete reconstitution of their immune system after receiving retrovirally transduced autologous CD34+ hematopoietic stem cells (HSCs). However, two of 11 treated patients subsequently developed a leukemia-like disease probablydue to the undesired activation of an oncogene. Gene transfer to HSCs resulted in substantial correction of immune function and multi-lineage engraftment in two patients with adenosine deaminase (ADA)-SCID. Several Phase I clinical trials for treatment of hemophilia A and B have been initiated or completed. Partial correction of hemophilia A, albeit transient, has been reported by ex vivo gene transfer to autologous fibroblasts. Intramuscular injection of adeno-associated viral (AAV) vector to patients with severe hemophilia B resulted in evidence of Factor IX gene transfer to skeletal muscle and a separate trial based on hepatic infusion of AAV vector is ongoing. Sustained therapeutic levels of coagulation factor expression have been achieved in preclinical models using retroviral, lentiviral, AAV and high capacity adenoviral vectors. Efficient lentiviral gene transfer to HSC in murine models of beta-thalassemia and sickle cell disease demonstrated sustained phenotypic correction.     

Recent progress in gene therapy for hemophilia

Hemophilia A and B are X-linked monogenic disorders caused by deficiencies in coagulation factor VIII (FVIII) and factor IX (FIX), respectively. Current treatment for hemophilia involves intravenous infusion of clotting factor concentrates. However, this does not constitute a cure, and the development of gene-based therapies for hemophilia to achieve prolonged high level expression of clotting factors to correct the bleeding diathesis are warranted. Different types of viral and nonviral gene delivery systems and a wide range of different target cells, including hepatocytes, skeletal muscle cells, hematopoietic stem cells (HSCs), and endothelial cells, have been explored for hemophilia gene therapy. Adeno-associated virus (AAV)-based and lentiviral vectors are among the most promising vectors for hemophilia gene therapy. Stable correction of the bleeding phenotypes in hemophilia A and B was achieved in murine and canine models, and these promising preclinical studies prompted clinical trials in patients suffering from severe hemophilia. These studies recently resulted in the first demonstration that long-term expression of therapeutic FIX levels could be achieved in patients undergoing gene therapy. Despite this progress, there are still a number of hurdles that need to be overcome. In particular, the FIX levels obtained were insufficient to prevent bleeding induced by trauma or injury. Moreover, the gene-modified cells in these patients can become potential targets for immune destruction by effector T cells, specific for the AAV vector antigens. Consequently, more efficacious approaches are needed to achieve full hemostatic correction and to ultimately establish a cure for hemophilia A and B.     

Gene therapy for the hemophilias

Summary.  Significant progress has recently been made in the development of gene therapy for the treatment of hemophilia A and B. These advances parallel the development of improved gene delivery systems. Long-term therapeutic levels of factor (F) VIII and FIX can be achieved in adult FVIII- and FIX-deficient mice and in adult hemophiliac dogs using adeno-associated viral (AAV) vectors, high-capacity adenoviral vectors (HC-Ad) and lentiviral vectors. In mouse models, some of the highest FVIII or FIX expression levels were achieved using HC-Ad vectors with no or only limited adverse effects. Encouraging preclinical data have been obtained using AAV vectors, yielding long-term FIX levels above 10% in primates and in hemophilia B dogs, which prevented spontaneous bleeding. Non-viral ex vivo gene therapy approaches have also led to long-term therapeutic levels of coagulation factors in animal models. Nevertheless, the induction of neutralizing antibodies (inhibitors) to FVIII or FIX sometimes precludes stable phenotypic correction following gene therapy. The risk of inhibitor formation varies depending on the type of vector, vector serotype, vector dose, expression levels and promoter used, route of administration, transduced cell type and the underlying mutation in the hemophilia model. Some studies suggest that continuous expression of clotting factors may induce immune tolerance, particularly when expressed by the liver. Several gene therapy phase I clinical trials have been initiated in patients suffering from severe hemophilia A or B. Some subjects report fewer bleeding episodes and occasionally have low levels of clotting factor activity detected. Further improvement of the various gene delivery systems is warranted to bring a permanent cure for hemophilia one step closer to reality.     

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.     

Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/cDNA-containing lentiviral vector and nonlethal irradiation

Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked primary immunodeficiency characterized by infections, hemorrhages, autoimmune disorders, and lymphomas. Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could represent an alternative treatment to allogeneic HSC transplantation, the latter being often associated with severe complications. We used WAS-/- mice to test the efficacy of a gene therapy approach based on nonlethal irradiation followed by transplantation of WAS-/- HSCs transduced with lentiviral vectors encoding the WAS protein (WASP) from either the ubiquitous PGK promoter or the tissue- specific WAS promoter. The procedure resulted in significant levels of engraftment of WASP-expressing T cells, B cells, platelets, and myeloid cells. T cells harbored one or two vector copies and displayed partial to full correction of T cell receptor-driven interleukin-2 production and proliferation. In addition, polymerization of F-actin and localization of WASP at the site of the immunological synapse were restored. The treatment was well tolerated and no pathology was detected by systematic blood analysis and autopsy. The efficacy of WAS gene transfer into HSCs, using the WAS promoter-containing lentiviral vector, combined with nonlethal irradiation provides a strong rationale for the development of gene therapy for WAS patients.     

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.     

Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector

Extensive studies in animal models of the X-linked bleeding disorder hemophilia B (deficiency in functional coagulation factor IX, F.IX) have shown that muscle-directed adeno-associated (AAV)-mediated F.IX gene transfer can be used to treat this disease. However, large vector doses of AAV-2 vector are required for therapeutic levels of expression, and the number of vector doses that can be injected per intramuscular site is limited. Several studies have shown that some of these limitations can be overcome by use of AAV serotype 1 vector. Here, we demonstrate levels of F.IX transgene expression from a synthetic muscle-specific promoter (C5-12) that were higher than from the cytomegalovirus (CMV) immediate-early enhancer-promoter in cultured muscle cells in vitro and approximately 50% of CMV-driven expression in vivo in murine skeletal muscle after AAV-1 gene transfer. These data show for the first time that a tissue-specific promoter can be used to achieve therapeutic levels of muscle-derived F.IX expression in the context of viral gene transfer. However, use of a muscle-specific promoter did not prevent antibody formation in response to a murine F.IX transgene product in mice with F.IX gene deletion, indicating that the risk of humoral immune responses remains in the context of an immunologically unfavorable mutation.     

Induction of Immune Tolerance to FIX Following Muscular AAV Gene Transfer Is AAV-dose/FIX-level Dependent

Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1–human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1–hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV–hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Role of Vector in Activation of T Cell Subsets in Immune Responses against the Secreted Transgene Product Factor IX

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4⁺ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.     

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.     

Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice

Immune responses leading to antibody-mediated elimination of the transgenic protein are a concern in gene replacement for congenital protein deficiencies, for which hemophilia is an important model. Although most hemophilia B patients have circulating non-functional but immunologically crossreactive factor IX (FIX) protein (CRM+ phenotype), inciting factors for FIX neutralizing antibody (inhibitor) development have been studied in crossreactive material-negative (CRM-) animal models. For this study, determinants of FIX inhibitor development were compared in hemophilia B mice, in which circulating FIX protein is absent (CRM- factor IX knockout (FIXKO) model) or present (CRM+ missense R333Q-hFIX model) modeling multiple potential therapies. The investigations compare for the first time different serotypes of adeno-associated virus (AAV) vectors (AAV2 and AAV1), each at multiple doses, in the setting of two different FIX mutations. The comparisons demonstrate in the FIXKO background (CRM- phenotype) that neither vector serotype nor vector particle number independently determine the inhibitor trigger, which is influenced primarily by the level and kinetics of transgene expression. In the CRM+ missense background, inhibitor development was never stimulated by AAV gene therapy or protein therapy, despite the persistence of lymphocytes capable of responding to FIX with non-inhibitory antibodies. This genotype/phenotype is strongly protective against antibody formation in response to FIX therapy.     

Update on hematopoietic stem cell gene transfer using non-human primate models

Gene transfer into hematopoietic stem cells (HSCs) using integrating vectors is an attractive treatment strategy for many genetic and hematological diseases. The preclinical testing of gene transfer approaches in non-human primates and other large animal models will be invaluable in order to assess toxicity and efficacy, as their HSC biology is much more closely related to humans than murine models. Gene transfer studies targeting HSCs in non-human primates have focused on optimizing gene transfer efficiency, and significant advances have been achieved using standard retroviral vectors. Utilization of lentiviral and other alternative vector system are still very preliminary in large animal models. Further development of post-transduction selection and/or expansion strategies using drug-resistance or amplifier genes will most likely be necessary for clinical applications.     

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration–approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10¹³ vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.     

Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B.

In a phase I study, administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and FIX expression for at least 10 months after vector injection, the last time point assessed by muscle biopsy. In hemophilia B dogs we have demonstrated FIX in both muscle biopsies and circulation >4 years following AAV2-FIX injection. Because circulating FIX levels remained less than 1% of normal in human subjects from the study, the duration of AAV2-mediated transgene expression in humans is unknown. We sought to determine if FIX gene transfer and expression persisted locally at injection sites. Muscle biopsies were obtained from one subject 3.7 years following treatment and revealed transgene FIX DNA and protein by quantitative PCR, DNA fluorescence in situ hybridization, and immunohistochemistry for FIX. These results demonstrate, for the first time, multiyear FIX expression by AAV2 vector in humans and suggest that improved muscle delivery provides effective treatment for protein deficiencies or muscle-specific diseases.     

Pharmacological Modulation of Humoral Immunity in a Nonhuman Primate Model of AAV Gene Transfer for Hemophilia B

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.     

Gene therapy progress and prospects: gene therapy for the hemophilias

Recent gene transfer trials for hemophilia A and B, bleeding disorders lacking either functional factor VIII or IX, respectively, have produced tantalizing results, suggesting that the potential to correct these bleeding disorders at a molecular level may be at hand. Genetic correction of the hemophilias represents a model system to develop a basic understanding of how gene therapy will be achieved. The goals for hemophilia gene transfer require the long-term therapeutic production of the coagulant protein without stimulating an immune response to the transgene product or the vector. Based on a scientific understanding of the molecular and cellular defects, leading to the bleeding phenotype, impressive strides have been made in the last 2 years.     

Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice

We reported total correction of blood coagulation plasma factor VIII (FVIII) activity, using adeno-associated virus serotype 8 (AAV8) vectors for liver-specific gene transfer in hemophilia A mice. We now show, irrespective of immunosuppression or route of administration, total long-term correction of hemophilia A mice with pseudotyped AAV8 and AAV9 vectors. We delivered two FVIII vectors, one expressing canine heavy chain and the other expressing canine light chain. Interestingly, when these vectors were given by hepatic portal vein to hemophilia A dogs, only modest FVIII levels were seen despite the species-specific transgene. No dogs treated developed FVIII inhibitors. However, of three dogs treated with AAV8 vector, the single male, given 1.25 x 10(13) genome copies per vector per kilogram (GC/vector/kg), maintained a level of >4.5% for more than 2 years. In contrast, the two female dogs expressed only 2% FVIII activity despite receiving higher doses of 1.52 x 10(13) and 3 x 10(13) GC/vector/kg, respectively. On the other hand, a male dog treated with AAV9 vector at a low dose (6 x 10(12) GC/vector/kg) maintained FVIII levels of 2-2.5% of normal without bleeding for 200 days (observation ongoing). Although hemophilia A mice were not predictive of vector efficacy in dogs, the two treated male dogs became symptom-free for long periods. Even so, translation of these robust vectors either in appropriate large animals or human beings remains challenging.     

In vivo gene transfer strategies to achieve partial correction of von Willebrand disease

von Willebrand disease (VWD), the most common hereditary coagulation disorder, results from mutations in the 52-exon gene for von Willebrand factor (VWF), which encodes an 8.4-kB cDNA. Studies with VWF cDNA plasmids have demonstrated that in vivo gene transfer to the liver will correct the coagulation dysfunction in VWF(-/-) mice, but the correction is transient. To develop gene therapy for VWF that would mediate long-term expression of the VWF cDNA in liver, we first evaluated segmental pre-mRNA trans-splicing (SPTS) with two adeno-associated virus (AAV) serotype 8 vectors, each delivering one-half of the VWF cDNA. However, although the two vectors functioned well to generate VWF multimers after infection of cells in vitro, the efficiency of SPTS was insufficient to correct the VWF(-/-) mouse in vivo. As an alternative, we assessed the ability of a lentiviral vector to transfer the intact murine VWF cDNA in vivo directly to the neonatal liver of VWF(-/-) mice, using generation of VWF multimers, bleeding time, and bleeding volume as efficacy parameters. The VWF lentivirus generated VWF multimers and partially or completely corrected the coagulation defect on a persistent basis in 33% of the treated VWF-deficient mice. On the basis of the concept that partial persistent correction with gene transfer could be beneficial in VWD patients, these observations suggest that lentiviral delivery of VWF cDNA should be explored as a candidate for gene therapy in patients with a severe form of VWD.