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.     

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.     

Genetic analysis of the antibody response to AAV2 and factor IX.

We have analyzed the antibody response against either AAV2 or canine F.IX (cF.IX) in parental C57BL/6 (B6) and DBA/2 (D2) and 18 strains of B6 x D2 (BXD) recombinant inbred (RI) strains of mice after iv administration of AAV2-(ApoE)4/hAAT-cF.IX. There was a higher anti-AAV2 capsid response in B6 compared to D2 mice, with IgG2b being the major isotype separating the high and low responders in these two strains. In contrast, the antibody response to cF.IX was lower than the response to the AAV2 capsid and was limited to the IgG1 isotype in both strains. Genetic linkage analysis of the IgG2b anti-AAV2 antibody response in BXD RI strains revealed a significant locus at D4Mit164 (29 cM, LRS=15.3) and two suggestive loci at D6Mit16 (30.5 cM, LRS=11.2) and D17Mit187 (47.4 cM, LRS=13.1). Genetic linkage analysis of the IgG1 anti-cF.IX antibody response revealed a suggestive locus at D1Mit218 (67 cM, LRS=14.1). Significant epistatic interaction was found between two loci (D8Mit45 and D16Mit47; LOD=6.54) for anti-AAV2 and two other loci (D5Mit348 and D15Mit161; LOD=7.66) for anti-cF.IX. These results indicate that multiple genetic loci independently regulate the isotype-specific antibody response to the AAV2 capsid and the cF.IX transgene.     

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.     

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.     

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.     

Hyperactive Sleeping Beauty Transposase Enables Persistent Phenotypic Correction in Mice and a Canine Model for Hemophilia B

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.     

Technology evaluation: AAV factor IX gene therapy, Avigen Inc

Avigen has developed a recombinant adeno-associated viral vector expressing human blood-coagulation Factor IX (F.IX) for the potential treatment of hemophilia B. In a phase I clinical trial being conducted at The Children's Hospital of Philadelphia and Stanford University Medical Center, the vector, AAV-CMV-hF.IX (Coagulin-B), was injected at a low dose into three patients with severe hemophilia B. No evidence of toxicity, germline transmission of vector sequences, or formation of inhibitory antibodies against F.IX was observed, and in two of the three patients there was an indication of a modest clinical response.     

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.     

Efficacy and Safety of Long-term Prophylaxis in Severe Hemophilia A Dogs Following Liver Gene Therapy Using AAV Vectors

Developing adeno-associated viral (AAV)–mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose–dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.     

Suppression of the Immune Response to FVIII in Hemophilia A Mice by Transgene Modified Tolerogenic Dendritic Cells

Current methods for eradicating clinically significant inhibitory antibodies to human factor VIII (hFVIII) in patients with hemophilia A rely on repeated delivery of high doses of factor concentrates for a minimum of many months. We hypothesize that tolerance can be induced more efficiently and reliably through hFVIII antigen presentation by tolerogenic dendritic cells (tDCs). In this study, we generated tDCs from hemophilia A mice and modified them with a foamy virus vector expressing a bioengineered hFVIII transgene. Naive and preimmunized mice infused with hFVIII expressing tDCs showed suppression of the T cell and inhibitor responses to recombinant hFVIII (rhFVIII). Treatment with hFVIII expressing tDCs was also associated with a higher percentage of splenocytes demonstrating a regulatory T cell phenotype in immunized mice. Furthermore, CD4⁺ T cells harvested from recipients of hFVIII expression vector-modified tDCs were able to mediate antigen-specific immune suppression in naive secondary recipients. We also demonstrated a trend for improved suppression of inhibitor formation by coexpressing interleukin-10 (IL-10) and hFVIII from a bicistronic vector. These preclinical results demonstrate the potential for employing vector modified ex vivo generated tDCs to treat high titer inhibitors in patients with hemophilia A.     

Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose-response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 10(2), and maximum expression at 10(6), transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS.     

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.     

Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates

Systemic intravascular delivery of adenoviral (Ad) vectors for liver-directed gene therapy has been widely employed because of its simplicity, noninvasiveness, and potential for high transduction. For first-generation Ad vectors (FGAd), this results in high but transient levels of transgene expression and long-term hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, high doses also result in an acute innate inflammatory response with potentially lethal consequences. Unlike FGAd, helper-dependent Ad vectors (HDAd) contain no viral genes and can provide sustained, high-level transgene expression with negligible long-term toxicity. However, whether the absence of viral gene expression leads to any decrease of acute toxicity in nonhuman primates has yet to be determined. To address this, we injected one baboon with 5.6 x 10(12) HDAd viral particles (VP)/kg and a second with 1.1 x 10(13) VP/kg. Approximately 50% hepatocyte transduction, accompanied by mild and transient acute toxicity, was observed in the animal receiving the lower dose. In the animal receiving the higher dose, 100% hepatocyte transduction, accompanied by lethal acute toxicity, was observed. These results indicate that systemic delivery of HDAd, like FGAd, results in acute toxicity in baboons consistent with activation of the innate inflammatory response, the severity of which is dose dependent, and confirm the hypothesis that Ad-mediated acute toxicity is independent of viral gene expression.     

Adenovirus vector-mediated gene transfer to regional lymph nodes

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.     

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.     

Readministration of helper-dependent adenoviral vectors to mouse airway mediated via transient immunosuppression

The efficacy of adenovirus-mediated gene therapy is attenuated by the host immune responses to both vector and transgene products. Even for helper-dependent adenoviral (HD-Ad) vectors, which have all viral-coding sequences deleted, the viral capsid proteins still cause immune reactions. In order to improve the efficiency in transgene expression during HD-Ad vector readministration, we administered cyclophosphamide to transiently modulate the mouse immune system. We delivered a high dose (5 × 10(10) vector particles (vp) per mouse) of empty HD-Ad to the mouse airway to induce an initial immune response. After 4 weeks, the mice were readministered with an HD-Ad vector containing either the reporter gene, LacZ, or the gene for the human cystic fibrosis transmembrane conductance regulator (CFTR) (1.5 × 10(10) vp per mouse). We found that the expression of both transgenes was greatly improved by the administration of cyclophosphamide when compared with the expression in mice without the immunosuppressing drug. We also found that the high dose of the empty HD-Ad vector administered intranasally does not induce an acute systemic immune response, but it does elicit an acute local response of proinflammatory cytokine production. Antibodies against Ad vector, including the neutralizing antibodies, were greatly reduced by the presence of cyclophosphamide in vector readministratiton. Moreover, cyclophosphamide reduced the infiltration of inflammatory cells, including total leukocytes, lymphocytes, CD4+ and CD8+T cells. These results indicate that transient administration of immunosuppressive agent can be used to extend transgene expression as well as attenuating immunogenicity to HD-Ad vectors in airway readministration.     

Augmentation of antitumor activity of a recombinant adeno-associated virus carcinoembryonic antigen vaccine with plasmid adjuvant

Recombinant adeno-associated virus 2 (rAAV) vectors have been successfully used for sustained expression of therapeutic genes. The potential of using rAAV as a cancer vaccine vector and the impact of a bacterial plasmid adjuvant on this activity were investigated. C57BL/6 mice received a single intramuscular injection of rAAV expressing the human tumor-associated antigen, carcinoembryonic antigen (CEA). Three weeks later, when CEA expression was optimal, a bacterial plasmid containing methylated DNA motifs was injected into the same muscle. Mice were challenged 1 week later with syngeneic MC38 tumor cells stably expressing CEA. Immunization with rAAV-CEA alone resulted in sustained transgene expression and the elicitation of a humoral immune response to CEA. Cellular immune response, however, was weak, and tumor protection was not significant. In contrast, immunization with rAAV-CEA and the plasmid adjuvant resulted in stronger cellular immune response to CEA and tumor protection. The addition of plasmid adjuvant increased both myeloid dendritic cell recruitment in situ and CEA-specific T-helper-1-associated immune response. These data indicate that robust rAAV transgene expression of a tumor antigen followed by transient plasmid delivery to recruit and activate dendritic cells is an effective method of eliciting antitumor cellular immune responses.     

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.