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.     

The effect of CpG sequences on the B cell response to a viral glycoprotein encoded by a plasmid vector.

The effect of palindromic CpG sequences on the B cell response to plasmid vectors expressing a highly immunogenic viral glycoprotein was investigated. Methylation of the CpG sequences of bacterial expression vectors abolished their ability to induce an antibody response to the transgene product in mice. The antibody response could be rescued by concomitant injection of oligonucleotides carrying immunostimulatory sequences. The B cell response to two plasmid vectors, both expressing the same viral glycoprotein but containing a different content of the highly stimulatory AACGTT motif, was compared. Comparable B cell responses were induced to the two constructs given at an optimal vaccine dose while the vector containing additional palindromic sequences resulted in higher antibody titers at a suboptimal dose. These data indicate that deletion of CpG motifs or methylation of such sequences in plasmid DNA can abrogate the immune response to the vector encoded antigen and might thus enhance their usefulness as gene therapy vehicles.     

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.     

Tolerance induction by viral in vivo gene transfer

Treatment of genetic disease by protein or gene replacement therapy is hampered by immune responses to the therapeutic protein. An excellent example is formation of inhibitory antibodies to coagulation factors in treatment of the X-linked bleeding disorder hemophilia. Experiments in murine and canine models of hemophilia B (deficiency in factor IX) have demonstrated sustained therapeutic levels of factor IX transgene expression following hepatic adeno-associated viral gene transfer in animals with deletion and nonsense mutations in the factor IX gene. This article reviews experimental evidence for induction of immune tolerance to the factor IX transgene product by hepatic adeno-associated viral gene transfer, which has been shown to limit T helper cell responses and to substantially reduce the risk of antibody responses. Tolerance induction is associated with activation of regulatory CD4(+) T cells capable of suppressing antibody formation to factor IX protein. Hepatic administration of adeno-associated viral vector expressing ovalbumin in mice transgenic for a T cell receptor specific for this antigen provided direct evidence for induction of CD4(+) T cell tolerance, including T cell anergy and clonal deletion. Taken together, these data indicate the potential for viral in vivo gene transfer not only to provide sustained systemic expression, but moreover to induce immunological hypo-responsiveness to the therapeutic gene product.     

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 regulatory T cells in tolerance to coagulation factors

Summary.  The immune response to coagulation factors VIII or IX, in particular formation of inhibitory antibodies, complicates treatment of hemophilia. Therefore, a number of recent studies in animal models have explored novel approaches toward induction of immune tolerance in protein or gene replacement therapy. Strong evidence has emerged that regulatory T cells (Treg) are an important component of the mechanism by which tolerance is maintained and inhibitor formation, a T help dependent response, is prevented. Limited data in patients also support this concept. In particular, CD4⁺ CD25⁺ FoxP3⁺ Treg, whether naturally occurring or induced, have been invoked in suppression of antibody and of cytotoxic T lymphocyte responses to the therapeutic clotting factor. This review summarizes the data on this emerging concept of Treg-mediated regulation of the immune response in treatment of hemophilia, strategies and mechanisms of Treg induction and function, and the implications for development of immune tolerance protocols.     

B cell–activating factor modulates the factor VIII immune response in hemophilia A

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell–activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody–mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.     

白细胞介素-1受体相关激酶1蛋白过表达对细菌脂蛋白耐受的影响

目的 探讨细菌脂蛋白(BLP)信号转导分子Toll样受体2(TLR2)和白细胞介素-1受体相关激酶1(IRAK-1)与BLP耐受发生的关系.方法 在人胚肾293(HEK293)细胞中过表达TLR2以及IRAK-1蛋白,利用蛋白质免疫印迹法和双荧光素酶报告基因检测实验观察其对BLP耐受的影响.结果 以BLP刺激稳定转染并表达TLR2的HEK293细胞可以剂量依赖性地诱导NF-κB活化,而且BLP预处理HEK-TLR2细胞可诱导BLP耐受的发生.在HEK-TLR2细胞中过表达IRAK-1可以剂量依赖性地增加NF-κB的活化并逆转BLP耐受:转染前BLP活化组与耐受组的NF-κB活化程度分别为0.329±0.010和0.168±0.010;转染0.02μg IRAK-1质粒后活化组与耐受组的NF-κB活化程度分别为0.493±0.010和0.427±0.035,均比转染前明显升高,差异均有统计学意义(均P＜0.01).结论 HEK293细胞中过表达TLR2不能阻止BLP耐受的诱导,但是在HEK-TLR2细胞中过表达IRAK-1可部分逆转BLP耐受,提示IRAK-1蛋白表达水平的变化在BLP耐受发生中起关键作用;IRAK-1可作为细菌感染和脓毒症治疗的重要靶点.     

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.     

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.     

Clinical implications of hepatitis B virus envelope protein variation

Summary The envelope proteins of hepatitis B virus are highly conserved. They are involved in viral attachment to hepatocytes, virion assembly and secretion and induction of humoral and T-cell immune responses. The major neutralisation epitope is located within the S protein and is conformation dependent, probably due to the formation of disulphide bridges. Variation in the envelope proteins of hepatitis B virus has been described in vaccinees and in patients receiving monoclonal and polyclonal antibody therapy. Thes arise through point mutations in the surface gene and appear to be selected by the immune response. The emergence of such variants has clinical relevance, in particular with regard to vaccine failure and diagnostic difficulties.     

Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine pompe disease

Pompe disease can be treated effectively, if immune tolerance to enzyme replacement therapy (ERT) with acid α-glucosidase (GAA) is present. An adeno-associated viral (AAV) vector carrying a liver-specific regulatory cassette to drive GAA expression (AAV-LSPhGAA) established immune tolerance in GAA knockout (KO) mice, whereas ubiquitous expression with AAV-CBhGAA provoked immune responses. Therefore, we investigated the hypothesis that immune tolerance induced by hepatic-restricted expression was dominant. AAV-LSPhGAA and AAV-CBhGAA were administered singly or in combination to groups of adult GAA-KO mice, and AAV-LSPhGAA induced immune tolerance even in combination with AAV-CBhGAA. The dual vector approach to GAA expression improved biochemical correction of GAA deficiency and glycogen accumulations at 18 weeks, and improved motor function testing including wire-hang and grip-strength testing. The greatest efficacy was demonstrated by dual vector administration, when both vectors were pseudotyped as AAV8. T cells from mice injected with AAV-LSPhGAA failed to proliferate at all after an immune challenge with GAA and adjuvant, whereas mock-treated GAA-KO mice mounted vigorous T cell proliferation. Unlike AAV-LSPhGAA, AAV-CBhGAA induced selective cytokine and chemokine expression in liver and spleen after the immune challenge. AAV-CBhGAA transduced dendritic cells and expressed high-level GAA, whereas AAV-LSPhGAA failed to express GAA in dendritic cells. The level of transduction in liver was much higher after dual AAV8 vector administration at 18 weeks, in comparison with either vector alone. Dual vector administration failed to provoke antibody formation in response to GAA expression with AAV-CBhGAA; however, hepatic-restricted expression from dual vector expression did not prevent antibody formation after a strong immune challenge with GAA and adjuvant. The relevance of immune tolerance to gene therapy in Pompe disease indicates that hepatic expression might best be combined with nonhepatic expression, achieving the benefits of ubiquitous expression in addition to evading deleterious immune responses.     

High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses

Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses. HMGN1 induced DC maturation via TLR4 (Toll-like receptor 4), recruitment of APCs at sites of injection, and activation of NF-κB and multiple mitogen-activated protein kinases in DCs. HMGN1 promoted antigen-specific immune response upon co-administration with antigens, and Hmgn1(-/-) mice developed greatly reduced antigen-specific antibody and T cell responses when immunized with antigens in the presence of lipopolysaccharide (LPS). The impaired ability of Hmgn1(-/-) mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from nonleukocytes was critical for the induction of antigen-specific antibody and T cell responses. Thus, extracellular HMGN1 acts as a novel alarmin critical for LPS-induced development of innate and adaptive immune responses.     

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.     

The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.     

Progress in drug development of immunostimulatory CpG oligodeoxynucleotide ligands for TLR9

Oligodeoxynucleotides (ODNs) with unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are recognised by Toll-like receptor (TLR)9 expressed in specialised cell subsets of the human immune system, B cells and plasmacytoid dendritic cells. TLR9-mediated stimulation of the immune system leads to a plethora of directed effects linking innate to adaptive immune responses. This allows the use of TLR9 agonists as highly effective targeted immunomodulatory drugs with broad potential applications as vaccine adjuvants, stand-alone therapy or in combination with other therapies in cancer, infectious diseases or asthma and allergy. TLR9 agonists represent a new class of small (8-30 bases long), easily synthesised, non-antisense ODN pharmaceuticals.     

B cell receptor revision diminishes the autoreactive B cell response after antigen activation in mice

Autoreactive B cells are regulated in the BM during development through mechanisms, including editing of the B cell receptor (BCR), clonal deletion, and anergy. Peripheral B cell tolerance is also important for protection from autoimmune damage, although the mechanisms are less well defined. Here we demonstrated, using a mouse model of SLE-like serology, that during an autoimmune response, RAG was reinduced in antigen-activated early memory or preplasma B cells. Expression of RAG was specific to antigen-reactive B cells, required the function of the IL-7 receptor (IL-7R), and contributed to maintenance of humoral tolerance. We also showed that soluble antigen could diminish a non-autoreactive antibody response through induction of BCR revision. These data suggest that tolerance induction operates in B cells at a postactivation checkpoint and that BCR revision helps regulate autoreactivity generated during an ongoing immune response.     

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.     

High expression reduces an antibody response after neonatal gene therapy with B domain-deleted human factor VIII in mice

BACKGROUND: Gene therapy could prevent bleeding in patients with hemophilia A, but might induce antibodies that block factor VIII (FVIII) function. OBJECTIVES: To test the efficacy of gene therapy in the newborn period for preventing a response to human FVIII (hFVIII) because of immaturity of the immune system. METHODS: Varying doses of a retroviral vector (RV) expressing a B domain-deleted hFVIII cDNA were injected i.v. into newborn hemophilia A C57BL/6 or normal C3H mice. Mice were evaluated for hFVIII expression, hemostasis, and development of anti-hFVIII antibodies with inhibitory activity. RESULTS AND CONCLUSIONS: Injection of a high RV dose [10(10) transducing units (TU) kg(-1)] into newborn hemophilia A or C3H mice resulted in 61% and 13% of normal hFVIII antigen in plasma, respectively; most mice did not produce anti-hFVIII antibodies, and hemophilia A mice did not bleed. Furthermore, most mice with >20 ng mL(-1) of hFVIII in plasma (10% normal, 1 x 10(-10) m) were tolerant to hFVIII, as an antibody response was markedly reduced after challenge with hFVIII with or without adjuvant. However, most RV-treated animals with lower antigen levels developed antibodies before or after challenge. Thus, initiation of a gene therapy trial with low RV doses might increase inhibitor formation. Furthermore, frequent hFVIII infusions in newborns with hemophilia A might reduce inhibitor formation. Finally, difficulties in achieving tolerance after gene therapy for hemophilia A as compared to hemophilia B may relate to lower expression of FVIII than FIX, as high antigen levels are most effective at inducing tolerance.     

The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8⁺ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.