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.     

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.     

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.     

Muscle‐directed gene therapy for hemophilia B with more efficient and less immunogenic AAV vectors

Summary. Background: Adeno‐associated viral vector (AAV)‐mediated and muscle‐directed gene therapy is a safe and non‐invasive approach to treatment of hemophilia B and other genetic diseases. However, low efficiency of transduction, inhibitor formation and high prevalence of pre‐existing immunity to the AAV capsid in humans remain as main challenges for AAV2‐based vectors using this strategy. Vectors packaged with AAV7, 8 and 9 serotypes have improved gene transfer efficiencies and may provide potential alternatives to overcome these problems. Objective: To compare the long‐term expression of canine factor IX (cFIX) levels and anti‐cFIX antibody responses following intramuscular injection of vectors packaged with AAV1, 2, 5, 7, 8 and 9 capsid in immunocompetent hemophilia B mice. Results: Highest expression was detected in mice injected with AAV2/8 vector (28% of normal), followed by AAV2/9 (15%) and AAV2/7 (10%). cFIX expression by AAV2/1 only ranged from 0 to 5% of normal levels. High incidences of anti‐cFIX inhibitor (IgG) were detected in mice injected with AAV2 and 2/5 vectors, followed by AAV2/1. None of the mice treated with AAV2/7, 2/8 and 2/9 developed inhibitors or capsid T cells. Conclusions: AAV7, 8 and 9 are more efficient and safer vectors for muscle‐directed gene therapy with high levels of transgene expression and absence of inhibitor formation. The absence of antibody response to transgene by AAV7, 8 and 9 is independent of vector dose but may be due to the fact that these three serotypes are associated with high level distribution to, and transduction of, hepatocytes following i.m. injection.     

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.     

Efficient AAV1-AAV2 hybrid vector for gene therapy of hemophilia

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1x10(11) vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This "hybrid vector" retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.     

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.     

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.     

Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8⁺ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.     

Long-term phenotypic correction in factor IX knockout mice by using ΦC31 integrase-mediated gene therapy

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ΦC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with ΦC31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding ΦC31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when ΦC31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in ΦC31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.     

Enhancing transduction of the liver by adeno-associated viral vectors

A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 x 10(7) vg per mouse (4 x 10(8) vg kg(-1)) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4+/-0.6 to 9+/-2 microg ml(-1) in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10-20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.     

Muscle as a target for supplementary factor IX gene transfer

Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.     

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.     

High-efficiency Transduction and Correction of Murine Hemophilia B Using AAV2 Vectors Devoid of Multiple Surface-exposed Tyrosines

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30–80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic 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.     

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.     

Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid–derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.     

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.