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.     

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.     

Serologic evidence that factor IX inhibitor in the plasma of hemophilia B patients detects factor IX on normal red cells

BACKGROUND: Patients with hemophilia B lack factor IX (F IX). These patients may become alloimmunized after the transfusion of F IX concentrates and may develop F IX inhibitors, which have been characterized as polyclonal IgG4 alloantibodies. Two cases in which F IX inhibitors caused difficulty in compatibility testing and antibody identification were encountered. It was hypothesized that, because F IX is present in normal plasma, it might be adsorbed by red cells in vivo and then be detected during antibody screening tests with serum containing F IX inhibitors. CASE REPORT: Sera from two African American half-brothers with hemophilia B were incompatible with all common and rare red cell phenotypes tested in the anti-human globulin test, but did not react with each other's red cells. The brothers' red cell antibodies were neutralized with both normal plasma and a commercially available F IX concentrate, which indicated that the red cell incompatibility was most probably caused by their F IX inhibitors. Red cells from an unrelated patient with hemophilia B and a very low titer of F IX inhibitor were tested against the half-brothers' sera and did not react. The compatible red cells from one of the half-brothers and the unrelated patient with hemophilia B adsorbed F IX from normal plasma or F IX concentrate after 37 degrees C incubation; this rendered them incompatible with the plasma containing F IX inhibitor from the other half-brother. CONCLUSION: F IX appears to be present on normal red cells and may be detected during compatibility and antibody identification procedures when serum or plasma containing F IX inhibitors is tested.     

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.     

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.     

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.     

Heterogeneity of the factor IX locus in nine hemophilia B inhibitor patients.

DNA from nine hemophilia B patients who produce anti-factor IX inhibitors (antibodies), including two brothers, was analyzed by the Southern blotting method and hybridization with factor IX cDNA, intragenomic, and 3'-flanking probes. Two inhibitor patients were shown to have total deletions of the factor IX gene. Two other inhibitor patients, the brothers, were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is of approximately 5.0 kb and removes exon e. The second deletion is between 9 and 29 kb and removes exons g and h but leaves exon f intact. An abnormal Taq I fragment at one end of the deletion junctions acted as a marker for the inheritance of hemophilia B in the patients' family. Five other inhibitor patients have a structurally intact factor IX gene as detected by this method. Our studies indicate that whereas large structural factor IX gene defects predispose hemophilia B patients to developing an anti-factor IX inhibitor, the development of an inhibitor can be associated with other defects of the factor IX gene.     

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.     

成肌细胞介导的hFⅨ蛋白在小鼠体内的持续表达

目的：以小鼠C2C12成肌细胞为对象开展血友病乙基因治疗研究。方法：首先将构建好的带有MCK增强手和hCMV启动子的反转录病毒载体G1NaMCⅨ转染PA317细胞，并用其上情感染C2C12细胞，所得克隆细胞在离体分别进行了Southern、Northern、Western鉴定。进一步用带有hFⅨcDNA的C2C12细胞进行C3H小鼠活体表达研究。结果：hFⅨ活体表达量与细胞注射数量、细胞离体表达量呈正相关，使用免疫抑制剂可提高hFⅨ活体表达量并延长表达时间；较长时间间隔后进行重复注射亦可延长hFⅨ表达时间。结论：同种异体成肌细胞存在免疫排斥现象，较长时间间隔重复注射可提高移植细胞存活率，从而有助于实现成肌细胞介导的血友病乙基因治疗。     

Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer

Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4(+) T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.     

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.     

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.     

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.     

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.     

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.     

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 β-thalassemia and sickle cell disease demonstrated sustained phenotypic correction.     

Pharmacokinetic analysis of plasma-derived and recombinant F IX concentrates in previously treated patients with moderate or severe hemophilia B

BACKGROUND: Hemophilia B is an X-linked bleeding disorder that affects approximately 1 in 25,000 males. Therapy for acute bleeding episodes consists of transfusions of plasma-derived (pd-F IX) or recombinant (r-F IX) concentrates. STUDY DESIGN AND METHODS: A double-blind, two-period crossover study was initiated to assess the pharmacokinetics of pd-F IX and r-F IX and to address patient-specific variables that might influence in vivo recovery. Study product was administered by a single bolus infusion (50 IU/kg) to 43 previously treated patients in the nonbleeding state, and F IX:C levels were measured over a period of 48 hours after infusion. RESULTS: The mean in vivo recovery in the pd-F IX group was 1.71 +/- 0.73 IU per dL per IU per kg compared with 0.86 +/- 0.31 IU per dL per IU per kg with r-F IX (p 相似文献   

Sustained Expression of Therapeutic Level of Factor IX in Hemophilia B Dogs by AAV-Mediated Gene Therapy in Liver

We demonstrate that a single intraportal vein injection of a recombinant adeno-associated virus (rAAV) vector encoding canine factor IX (cFIX) cDNA under the control of a liver-specific enhancer/promoter leads to a long-term correction of the bleeding disorder in hemophilia B dogs. Stable expression of the therapeutic level of cFIX (5% of normal level) was detected in the plasma of a dog injected with an AAV vector at a dose of 4.6 × 10¹² particles/kg for over 7 months. Both whole-blood clotting time (WBCT) and activated partial thromboplastin time (aPTT) of the treated dogs have been greatly decreased since the treatment. No anti-canine factor IX antibodies have been detected in the treated animals. Importantly, no bleeding has been observed in the dog that expresses a therapeutic level of cFIX for 7 months following vector administration. Moreover, no persistent significant hepatic enzyme abnormalities were detected in the treated dogs. Thus, a single intraportal injection of a rAAV vector expressing cFIX successfully corrected the bleeding disorder of hemophilia B dogs, supporting the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.     

Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice.

The major complication associated with protein replacement therapy currently used in the treatment of hemophilia B (HB) is the development of antibodies to the infused human Factor IX (hF.IX). We hypothesized that vector-mediated expression of hF.IX, either at a prenatal stage or early in life may lead to tolerance to hF.IX and long-term transgene expression. Fetal, neonatal, and adult F.IX-deficient mice were injected with AAV-1-hF.IX, and the hF.IX levels as well as antibodies to hF.IX in the circulation were assayed. In utero injection followed by postnatal re-administration of adeno-associated virus 1 (AAV-1) vector achieved persistent expression of hF.IX in all animals, with no cellular or humoral immune response to F.IX. Similar results were seen after initial injection in neonatal mice followed by re-administration, whereas all mice injected at the adult stage developed antibodies to hF.IX. In contrast, after administration of AAV-2-hF.IX in the neonatal period, antibodies to hF.IX were formed in all the injected animals. We conclude that in utero or neonatal-stage injection of AAV-1-hF.IX can lead to long-term expression and absence of immune response. The differences in immune response between the AAV-1 and AAV-2 groups suggests that tolerance may be related to differences in bio-distribution, timing of expression, and/or the initial levels of hF.IX expression. This supports the concept of a narrow "window of opportunity" for tolerance induction.