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.     

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.     

Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy

Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.     

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.     

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.     

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.     

Immune Tolerance Induction to Factor IX through B Cell Gene Transfer: TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors. In a murine model of this immune complication, retrovirally transduced primary B cells expressing FIX antigen fused with immunoglobulin-G heavy chain prevented antibody formation to FIX and was also highly effective in desensitizing animals with preexisting response. In contrast, transplant of B cells that received the identical expression cassette via nucleofection of plasmid vector substantially heightened antibody formation against FIX, a response that could be blocked by toll-like receptor 9 (TLR9) inhibition. While innate responses to TLR4 activation or to retrovirus were minimal in B cells, plasmid DNA activated TLR9, resulting in CpG-dependent NF-κB activation/IL-6 expression and adaptor protein 3 dependent, CpG-independent induction of IFN-I. Neither response was seen in TLR9-deficient B cells. Therefore, TLR9 signaling in B cells, in particular in response to plasmid vector, is highly immunogenic and has to be avoided in design of tolerance protocols.     

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.     

Blockade of the costimulatory CD28‐B7 family signal axis enables repeated application of AAV8 gene vectors

Adeno‐associated virus serotype 8 (AAV8) gene therapy has shown efficacy in several clinical trials and is considered a highly promising technology to treat monogenic diseases such as hemophilia A and B. However, a major drawback of AAV8 gene therapy is that it can be applied only once because anti‐AAV8 immunity develops after the first treatment. Readministration may be required in patients who are expected to need redosing, eg, due to organ growth, or to boost suboptimal expression levels, but no redosing protocol has been established. We have developed a preventive immune‐suppressive protocol for a human factor IX (FIX) vector with an intended dose of ~5 × 10¹¹ vg/kg that inhibits the development of anti‐AAV8 neutralizing‐antibody (NAb) responses and anti‐AAV8 T‐cell responses using CTLA4‐IgG (abatacept). In a preclinical model, transient treatment with abatacept during initial human FIX gene therapy efficiently inhibited the generation of AAV8‐specific cellular and humoral responses, and thus permitted redosing of FIX. Furthermore, our data suggest that by suppression of anti‐AAV8 NAb responses after the second higher dose (4 × 10¹² vg/kg) this protocol can be used to enable redosing up to such high doses. An additional advantage of CTLA4‐IgG blocking CD28‐mediated signals is its potential suppression of AAV8‐specific cytotoxic CD8 T‐cell responses, which are believed to kill transduced hepatocytes and might interfere with a successful readministration. Redosing protocols using approved drugs would be beneficial for patients because they could effortlessly be applied in clinical trials and enable safe and efficient treatment options for patients undergoing AAV8 gene therapy.     

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.     

Generation of a novel factor IX with augmented clotting activities in vitro and in vivo

Summary. Background: Hemophilia B is an X‐linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma‐derived or recombinant FIX. Objective: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Methods: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. Results: One variant containing three substitutions (V86A/E277A/R338A, FIX‐Triple) exhibited 13‐fold higher specific clotting activity and a 10‐fold increased affinity for human FVIIIa compared with FIX‐wild‐type (FIX‐WT) and was thus investigated systematically in vivo. Liver‐specific FIX‐Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5‐fold higher specific clotting activity compared with FIX‐WT. Human FIX‐Triple and FIX‐WT knock‐in mice were generated and it was confirmed that FIX‐Triple has 7‐fold higher specific clotting activity than FIX‐WT under normal physiological conditions. Protein infusion of FIX‐Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX‐WT. Moreover, tail‐vein administration of a serotype 8 recombinant Adeno‐associated vector (AAV8) expressing either FIX‐WT or FIX‐Triple in hemophilia B mice demonstrated a 7‐fold higher specific clotting activity of FIX‐Triple than FIX‐WT. Conclusions: Our results indicate that the FIX‐Triple variant exhibits significantly enhanced clotting activity relative to FIX‐WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX‐Triple is a good candidate for further evaluation in protein replacement therapy as well as gene‐based therapeutic strategies.     

Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.

The application of AAV2 or AAV8 vectors for delivery of human coagulation factor IX (hF.IX) is a promising gene therapy for hemophilia B. One major limitation of this therapy is the development of antibodies and a cytotoxic T lymphocyte (CTL) response against both the vector capsid and the transgene. We determined the class I and class II MHC peptide epitopes for AAV2, AAV8, and hF.IX after administration of AAV-2-hF.IX or AAV8-hF.IX in H2(b) (C57BL/6), H2(d) (BALB/c), or H2(k) (C3H) strains of mice. The results indicate that the AAV2 capsid peptide AA(373-381), the AAV8 capsid peptide AA(50-58), and the hF.IX transgene peptide AA(311-319) can elicit a CTL response as indicated by an IFN-gamma ELISPOT assay and an in vivo CTL assay. Furthermore, a strong H2(k) MHC II-restricted Th1 response can be elicited in C3H mice by the AAV8 capsid peptide AA(126-140) and the hF.IX peptide AA(108-122), whereas a strong Th2 response can be elicited by the AAV2 peptide AA(475-489). These results show that specific CTL responses are generated to both AAV capsid epitopes and hF.IX epitopes after injection of AAV-hF.IX, and MHC class II epitopes derived from AAV-hF.IX promote development of either Th1 or Th2 cells.     

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.     

Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes.

The goal of these studies was to test whether adeno-associated virus (AAV) capsid-specific CD8(+) T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8(+) T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells' effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8(+) T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and na?ve animals. These results suggest that, in mice, pre-existing CD8(+) T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer.     

Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8⁺ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm⁻ nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.     

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.     

Specific detection of human coagulation factor IX in cynomolgus macaques

Summary.  After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg⁻¹; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 ± 37.6 ng mL⁻¹ at 1 h after the injection and gradually decreased to 1.79 ± 1.1 ng mL⁻¹ by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.