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75. Protease-Activated Receptor 2 as a Novel Target for Immune Modulation for AAV-Mediated Gene Transfer to Skeletal Muscle
VECTOR AND TRANSGENE IMMUNOLOGY under the control of a liver-specific promoter. Following a delay of 1-2 hours, these mice then received an adoptive transfer of splenocytes harvested from capsid matched AAV-immune mice. Control mice received adoptive transfer of splenocytes from naïve mice. Two months post-infusion, mice that received adoptive transfer from AAV-2 immune mice expressed significantly lower levels of hFIX than mice receiving adoptive transfer of naïve splenocytes (775ng/mL vs. 1771ng/mL, p=.03). Recipients of adoptive transfer from AAV-8 immune mice similarly exhibited reduced levels of hFIX two months post-infusion compared to control mice (606ng/mL vs. 1637ng/mL, p=.04). We conclude from these studies that a cellular immune response specific for AAV capsid can reduce AAV-mediated gene transfer in the liver.
75. Protease-Activated Receptor 2 as a Novel Target for Immune Modulation for AAV-Mediated Gene Transfer to Skeletal Muscle Stefano Baila,1 Christian Furlan Freguia,1 Danielle Dunn,1 Federico Mingozzi,1 Joerg Schuettrumpf,1 Valder R. Arruda.1,2 1 Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA. Intramuscular injection (IM) of adeno-associated viral (AAV)-2 vectors is a safe strategy to achieve long-term transgene expression in humans with hemophilia B. The development of AAV-based on alternative serotypes with higher transduction efficiency than AAV2 is an attractive approach to the use of skeletal muscle as target tissue. However, in a series of studies in large animal models using AAV derived from serotypes (-1,-5,-7,-8), the immune response to the transgene has been a major complication. In mice, we showed that IM injection of AAV1-human-FIX induces antibody formation in all injected animals. However, in mice injected with high doses of AAV-1, the inhibitory antibody eventually disappear overtime. Blood proteases play an important role in modulating inflammatory and immune responses through activation of protease-activated receptors (PARs). PARs family is a member of a larger G protein coupled receptor family. To date, four such cleavable receptors have been described (PAR1-4). Mice lacking PAR-1 (-/-) or PAR-2 (-/-) alleles presented amelioration of immune- or infectious-mediated diseases. Here we sought to determine whether PAR-1 or PAR-2 inactivation would provide a strategy to prevent immune responses following AAV-mediated gene transfer to skeletal muscle. Mice received IM injection of AAV-1-hFIX at doses of 5x10^11vg/kg to 1x10^12vg/kg (low and high-dose cohort, respectively), FIX and antibody levels were monitored weekly. PAR2(-/-) mice (n=8) given the low dose exhibited FIX levels of 582±117ng/ml which remained stable for the duration of the experiment (10 weeks), and no antibody for FIX was detected. Whereas in three PAR-2(-/-) mice a non-neutralizing antibody to FIX was detected. Importantly, inhibitory antibodies to FIX clotting activity, determined by a functional assay (Bethesda Unit;BU), was never found in PAR-2 (-/-) mice. In contrast, all PAR-2(+/+) mice (n=13), FIX expression was transiently detected at week 2 followed by a period of 4 weeks with undetectable levels. This was due to the formation of FIX specific IgG-1 antibody that peaked at week 6, and inhibitors to FIX were detected in 8 of 13 mice (BU titers of 1,1-5.5). In the high-dose group, PAR-2(-/-) mice (n=6) resulted in continuous expression of FIX(1,500±353ng/ml)in the absence of FIX antibody. In contrast, antibody to FIX were detected in all 10 mice of PAR-2(+/-)or PAR-2 (+/+) genotypes. In 3 out 10 mice inhibitory antibodies were detected with levels of ∼1 BU. Upon challenges with hFIX protein in complete Freud’s adjuvant 8 months
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following vector injection, only one mouse developed antibody to hFIX. Interestingly, this animal presented the lowest levels of FIX following IM injection (20 ng/ml). We next injected AAV-1-hFIX into PAR-1 (-/-) or PAR-1 (+/+) mice, and all animals (n=4/genotype) developed antibodies to FIX. Together these data suggest that inhibition of PAR-2, but not PAR-1, offers a novel strategy to prevent inadvertent immune response to the transgene following IM injection of AAV-1 without compromising long term tolerance to hFIX.
76. In Vivo Suppression of Cytotoxic T Cell Responses by CD4+ Regulatory T Cells Activated by Hepatic Gene Transfer Lixin Wang,1 Eric Dobrzynski,2 Julie Fitzgerald,2 Ou Cao,1 Roland W. Herzog.1 1 Pediatrics, University of Florida, Alachua, FL; 2Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA. Treatment of genetic disease such as the bleeding disorder hemophilia B (deficiency in blood coagulation factor IX, F.IX) by gene replacement therapy is complicated by the risk of immune responses to the therapeutic gene product and to the gene transfer vector. Previously, we have shown that adeno-associated viral (AAV) hepatic gene transfer can induce immune tolerance to F.IX. Immune competent mice of two different strains (BALB/c and C3H) were tolerized to human F.IX by this protocol. These animals were subsequently challenged by systemic administration of an E1/E3deleted adenoviral vector (Ad-hF.IX), which is known to induce a cytotoxic T lymphocyte (CTL) response to the transgene product. Immune tolerance prevented CTL activation to F.IX (as determined by in vitro CTL assay) and CD8+ cellular infiltrates in the liver, and also reduced antibody formation to F.IX to low and transient responses. Moreover, a sustained (≥4 months) and substantial increase in hepatic F.IX expression from the adenoviral vector was achieved (increase from ∼0.2 to 1-2 µg/ml plasma and from 3-5% of hepatocytes to 20-50%) despite in vitro T cell responses to adenoviral antigens. This suggested an in vivo suppression mechanism. Interestingly, splenocytes from tolerized mice had increased proportions of CD4+CD62L+ and of CD4+CD25+GITR+ cells, and CD4+ splenocytes had increased levels of FoxP3 mRNA. This suggested a complex regulatory mechanism that included cells with a phenotype similar to naturally occuring regulatory CD4+ T cells. To test our suppression hypothesis, splenocytes were adoptively transferred from tolerized C3H/HeJ mice (6 weeks after hepatic AAV gene transfer) to naive C3H/HeJ mice. The following day, Ad-hF.IX was systemically administered to recipient mice (n=4/ experimental group). Compared to CD4-depleted cells and to splenocytes from naive controls, CD4+ cells from AAV-hF.IX hepatic transduced animals (1x10exp6 per recipient mouse) suppressed anti-hF.IX formation and inflammatory lymphocyte responses to Ad-hF.IX transduced liver. Consequently, prolonged hepatic and systemic hF.IX expression was observed (1-2% hF.IX positive hepatocytes for ≥2 months vs. <0.1% in control groups), and hepatic injury normally caused by the response to adenovirus was limited. CD4- cells failed to suppress these immune responses. Therefore, regulatory CD4+ T cells activated by hepatocyte-derived hF.IX expression can suppress responses to viral antigens expressed in the context of hF.IX. These results indicate that augmentation of regulatory CD4+ T cell activation should provide new means to avoid destructive immune responses to therapeutic and viral antigens in gene transfer.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
75. Protease-Activated Receptor 2 as a Novel Target for Immune Modulation for AAV-Mediated Gene Transfer to Skeletal Muscle Stefano Baila,1 Christian Furlan Freguia,1 Danielle Dunn,1 Federico Mingozzi,1 Joerg Schuettrumpf,1 Valder R. Arruda.1,2 1 Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA. Intramuscular injection (IM) of adeno-associated viral (AAV)-2 vectors is a safe strategy to achieve long-term transgene expression in humans with hemophilia B. The development of AAV-based on alternative serotypes with higher transduction efficiency than AAV2 is an attractive approach to the use of skeletal muscle as target tissue. However, in a series of studies in large animal models using AAV derived from serotypes (-1,-5,-7,-8), the immune response to the transgene has been a major complication. In mice, we showed that IM injection of AAV1-human-FIX induces antibody formation in all injected animals. However, in mice injected with high doses of AAV-1, the inhibitory antibody eventually disappear overtime. Blood proteases play an important role in modulating inflammatory and immune responses through activation of protease-activated receptors (PARs). PARs family is a member of a larger G protein coupled receptor family. To date, four such cleavable receptors have been described (PAR1-4). Mice lacking PAR-1 (-/-) or PAR-2 (-/-) alleles presented amelioration of immune- or infectious-mediated diseases. Here we sought to determine whether PAR-1 or PAR-2 inactivation would provide a strategy to prevent immune responses following AAV-mediated gene transfer to skeletal muscle. Mice received IM injection of AAV-1-hFIX at doses of 5x10^11vg/kg to 1x10^12vg/kg (low and high-dose cohort, respectively), FIX and antibody levels were monitored weekly. PAR2(-/-) mice (n=8) given the low dose exhibited FIX levels of 582±117ng/ml which remained stable for the duration of the experiment (10 weeks), and no antibody for FIX was detected. Whereas in three PAR-2(-/-) mice a non-neutralizing antibody to FIX was detected. Importantly, inhibitory antibodies to FIX clotting activity, determined by a functional assay (Bethesda Unit;BU), was never found in PAR-2 (-/-) mice. In contrast, all PAR-2(+/+) mice (n=13), FIX expression was transiently detected at week 2 followed by a period of 4 weeks with undetectable levels. This was due to the formation of FIX specific IgG-1 antibody that peaked at week 6, and inhibitors to FIX were detected in 8 of 13 mice (BU titers of 1,1-5.5). In the high-dose group, PAR-2(-/-) mice (n=6) resulted in continuous expression of FIX(1,500±353ng/ml)in the absence of FIX antibody. In contrast, antibody to FIX were detected in all 10 mice of PAR-2(+/-)or PAR-2 (+/+) genotypes. In 3 out 10 mice inhibitory antibodies were detected with levels of ∼1 BU. Upon challenges with hFIX protein in complete Freud’s adjuvant 8 months
S32
following vector injection, only one mouse developed antibody to hFIX. Interestingly, this animal presented the lowest levels of FIX following IM injection (20 ng/ml). We next injected AAV-1-hFIX into PAR-1 (-/-) or PAR-1 (+/+) mice, and all animals (n=4/genotype) developed antibodies to FIX. Together these data suggest that inhibition of PAR-2, but not PAR-1, offers a novel strategy to prevent inadvertent immune response to the transgene following IM injection of AAV-1 without compromising long term tolerance to hFIX.
76. In Vivo Suppression of Cytotoxic T Cell Responses by CD4+ Regulatory T Cells Activated by Hepatic Gene Transfer Lixin Wang,1 Eric Dobrzynski,2 Julie Fitzgerald,2 Ou Cao,1 Roland W. Herzog.1 1 Pediatrics, University of Florida, Alachua, FL; 2Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA. Treatment of genetic disease such as the bleeding disorder hemophilia B (deficiency in blood coagulation factor IX, F.IX) by gene replacement therapy is complicated by the risk of immune responses to the therapeutic gene product and to the gene transfer vector. Previously, we have shown that adeno-associated viral (AAV) hepatic gene transfer can induce immune tolerance to F.IX. Immune competent mice of two different strains (BALB/c and C3H) were tolerized to human F.IX by this protocol. These animals were subsequently challenged by systemic administration of an E1/E3deleted adenoviral vector (Ad-hF.IX), which is known to induce a cytotoxic T lymphocyte (CTL) response to the transgene product. Immune tolerance prevented CTL activation to F.IX (as determined by in vitro CTL assay) and CD8+ cellular infiltrates in the liver, and also reduced antibody formation to F.IX to low and transient responses. Moreover, a sustained (≥4 months) and substantial increase in hepatic F.IX expression from the adenoviral vector was achieved (increase from ∼0.2 to 1-2 µg/ml plasma and from 3-5% of hepatocytes to 20-50%) despite in vitro T cell responses to adenoviral antigens. This suggested an in vivo suppression mechanism. Interestingly, splenocytes from tolerized mice had increased proportions of CD4+CD62L+ and of CD4+CD25+GITR+ cells, and CD4+ splenocytes had increased levels of FoxP3 mRNA. This suggested a complex regulatory mechanism that included cells with a phenotype similar to naturally occuring regulatory CD4+ T cells. To test our suppression hypothesis, splenocytes were adoptively transferred from tolerized C3H/HeJ mice (6 weeks after hepatic AAV gene transfer) to naive C3H/HeJ mice. The following day, Ad-hF.IX was systemically administered to recipient mice (n=4/ experimental group). Compared to CD4-depleted cells and to splenocytes from naive controls, CD4+ cells from AAV-hF.IX hepatic transduced animals (1x10exp6 per recipient mouse) suppressed anti-hF.IX formation and inflammatory lymphocyte responses to Ad-hF.IX transduced liver. Consequently, prolonged hepatic and systemic hF.IX expression was observed (1-2% hF.IX positive hepatocytes for ≥2 months vs. <0.1% in control groups), and hepatic injury normally caused by the response to adenovirus was limited. CD4- cells failed to suppress these immune responses. Therefore, regulatory CD4+ T cells activated by hepatocyte-derived hF.IX expression can suppress responses to viral antigens expressed in the context of hF.IX. These results indicate that augmentation of regulatory CD4+ T cell activation should provide new means to avoid destructive immune responses to therapeutic and viral antigens in gene transfer.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy