Immunomodulatory Gene Therapy Prevents Antibody Formation and Lethal Hypersensitivity Reactions in Murine Pompe Disease
Authors: Baodong Sun, Michael D Kulis, Sarah P Young, Amy C Hobeika, Songtao Li, Andrew Bird, Haoyue Zhang, Yifan Li, Timothy M Clay, Wesley Burks, Priya S Kishnani, Dwight D Koeberl
Journal: Molecular Therapy (2009)

Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 × 1010 particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement. Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 × 1010 particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement. IntroductionInfantile-onset glycogen storage disease type II (Pompe disease; MIM 232300) caused death early in childhood from cardiorespiratory failure related to an underlying hypertrophic cardiomyopathy, prior to the availability of enzyme-replacement therapy (ERT).1Hirschhorn R Reuser AJJ Glycogen storage disease type II: acid α-glucosidase (acid maltase) deficiency.in: Scriver CR Beaudet AL Sly WS Valle D The Metabolic and Molecular Basis for Inherited Disease. 8th edn. McGraw-Hill, New York2001Google Scholar Pilot studies of ERT with recombinant human acid α-glucosidase (rhGAA) (purified from Chinese hamster ovary cell cultures2Amalfitano A Bengur AR Morse RP Majure JM Case LE Veerling DL et al.Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial.Genet Med. 2001; 3: 132-138Abstract Full Text Full Text PDF PubMed Google Scholar or transgenic rabbit milk3Van den Hout JM Kamphoven JH Winkel LP Arts WF De Klerk JB Loonen MC et al.Long term intravenous treatment of Pompe disease with recombinant human alpha-glucosidase from milk.Pediatrics. 2004; 113: E448-E457Crossref PubMed Scopus (314) Google Scholar) resolved or improved cardiomyopathy and prolonged the survival of all subjects beyond 1 year. Pompe disease patients who lacked any residual GAA protein are deemed crossreacting immune material negative (CRIM-negative). CRIM-negative Pompe disease subjects produced very high anti-hGAA antibodies and demonstrated markedly reduced efficacy from ERT. In the first pilot study of ERT in Pompe disease using Chinese hamster ovary cell–derived recombinant hGAA, the two patients who were CRIM-negative produced higher titers of anti-hGAA antibodies than the third patient who was CRIM-positive.2Amalfitano A Bengur AR Morse RP Majure JM Case LE Veerling DL et al.Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial.Genet Med. 2001; 3: 132-138Abstract Full Text Full Text PDF PubMed Google Scholar Poor outcomes were associated with CRIM-negative status in the pivotal clinical trials that led to marketing approval for rhGAA.4Kishnani PS Nicolino M Voit T Rogers RC Tsai AC Waterson J et al.Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset Pompe disease.J Pediatr. 2006; 149: 89-97Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar,5Kishnani PS Corzo D Nicolino M Byrne B Mandel H Hwu WL et al.Recombinant human acid [α]-glucosidase: major clinical benefits in infantile-onset Pompe disease.Neurology. 2007; 68: 99-109Crossref PubMed Scopus (613) Google Scholar CRIM-negative Pompe disease subjects in these clinical trials formed very high, sustained anti-hGAA antibodies and demonstrated markedly reduced efficacy from ERT.2Amalfitano A Bengur AR Morse RP Majure JM Case LE Veerling DL et al.Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial.Genet Med. 2001; 3: 132-138Abstract Full Text Full Text PDF PubMed Google Scholar,4Kishnani PS Nicolino M Voit T Rogers RC Tsai AC Waterson J et al.Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset Pompe disease.J Pediatr. 2006; 149: 89-97Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar,5Kishnani PS Corzo D Nicolino M Byrne B Mandel H Hwu WL et al.Recombinant human acid [α]-glucosidase: major clinical benefits in infantile-onset Pompe disease.Neurology. 2007; 68: 99-109Crossref PubMed Scopus (613) Google ScholarThe antibody response to ERT in Pompe disease has been remarkably similar to inhibitory antibody formation in hemophilia.6DiMichele D Inhibitor development in haemophilia B: an orphan disease in need of attention.Br J Haematol. 2007; 138: 305-315Crossref PubMed Scopus (191) Google Scholar Hemophilia B is similar to Pompe disease, in that CRIM-negative patients frequently mounted high-titer IgG antibody responses to protein replacement therapy with coagulation factor IX (FIX) that interfere with efficacy. Taken together, these data suggest that immune tolerance to ERT is absent in CRIM-negative patients, and that high-titer antibody formation reduced any clinical benefit from ERT.Tolerization therapy, including administration of high-dose rhGAA with immune suppressant drugs, failed to improve the clinical response to ERT in CRIM-negative subjects. Indeed, high-dose hGAA therapy precipitated nephrotic syndrome in one of the CRIM-negative subjects, possibly related to effects of antibody complexes upon the glomerular basement membrane.7Hunley TE Corzo D Dudek M Kishnani P Amalfitano A Chen YT et al.Nephrotic syndrome complicating alpha-glucosidase replacement therapy for Pompe disease.Pediatrics. 2004; 114: e532-e535Crossref PubMed Scopus (85) Google Scholar At present there is no successful immune modulation or tolerization protocol for patients that maintained the efficacy of ERT following the formation of anti-GAA antibodies.The potential advantages of gene therapy over ERT have become clear in experiments with Pompe disease mice. The availability of novel adeno-associated virus (AAV) serotypes, including AAV8, advanced gene therapy by improving the tropism of vectors for target tissues.8Gao GP Alvira MR Wang L Calcedo R Johnston J Wilson JM Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1212) Google Scholar AAV2 vectors pseudotyped with AAV8 (AAV2/8) delivered genes to the liver ~100-fold more efficiently in mice, including GAA-knockout (KO) mice, in comparison with traditional AAV2 vectors.8Gao GP Alvira MR Wang L Calcedo R Johnston J Wilson JM Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1212) Google Scholar,9Sun B Zhang H Franco LM Young SP Schneider A Bird A et al.Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II.Mol Ther. 2005; 11: 57-65Abstract Full Text Full Text PDF PubMed Scopus (120) Google ScholarLiver-restricted expression of GAA with an AAV vector prevented the formation of anti-hGAA antibodies in GAA-KO mice. A single administration of a low dose AAV2/8 vector containing a liver-specific regulatory cassette substantially corrected glycogen storage in the diaphragm and heart of GAA-KO mice [3 × 1010 vector particles (vp), equivalent to 1 × 1012 vp/kg], whereas an even lower dose prevented anti-GAA antibody formation without achieving biochemical correction.10Sun B Zhang H Benjamin DK Brown T Bird A Young SP et al.Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II.Mol Ther. 2006; 14: 822-830Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Another AAV vector containing a liver-specific regulatory cassette expressed high-level hGAA in the liver of adult GAA-KO mice for over 12 weeks without provoking a detected anti-hGAA IgG response.11Franco LM Sun B Yang X Bird A Zhang H Schneider A et al.Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II.Mol Ther. 2005; 12: 876-884Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Increasing plasma hGAA was detected between 1 and 14 days and sustained for >12 weeks following AAV-LSPhGAApA administration.11Franco LM Sun B Yang X Bird A Zhang H Schneider A et al.Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II.Mol Ther. 2005; 12: 876-884Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar The aforementioned AAV vectors contained a liver-specific regulatory cassette that drove therapeutically relevant coagulation FIX expression and diminished antibody responses in hemophilia B mice and dogs.12Wang L Takabe K Bidlingmaier SM Ill CR Verma IM Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.Proc Natl Acad Sci USA. 1999; 96: 3906-3910Crossref PubMed Scopus (196) Google Scholar,13Wang L Nichols TC Read MS Bellinger DA Verma IM Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver.Mol Ther. 2000; 1: 154-158Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar These data suggested that liver-restricted, high-level expression of hGAA induced immune tolerance in Pompe disease mice, similarly to the effect of liver-specific expression of therapeutic proteins in hemophilia mice12Wang L Takabe K Bidlingmaier SM Ill CR Verma IM Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.Proc Natl Acad Sci USA. 1999; 96: 3906-3910Crossref PubMed Scopus (196) Google Scholar,14Sarkar R Tetreault R Gao G Wang L Bell P Chandler R et al.Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype.Blood. 2004; 103: 1253-1260Crossref PubMed Scopus (170) Google Scholar and in Fabry disease mice.15Ziegler RJ Lonning SM Armentano D Li C Souza DW Cherry M et al.AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice.Mol Ther. 2004; 9: 231-240Abstract Full Text Full Text PDF PubMed Scopus (113) Google ScholarLike CRIM-negative patients with Pompe disease, GAA-KO mice lack immune tolerance to hGAA and ERT has no efficacy, even provoking fatal anaphylaxis.16Raben N Danon M Gilbert AL Dwivedi S Collins B Thurberg BL et al.Enzyme replacement therapy in the mouse model of Pompe disease.Mol Genet Metab. 2003; 80: 159-169Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar A strategy for immunomodulatory gene therapy was developed in GAA-KO mice by administering a low number of AAV-LSPhGAApA particles prior to the initiation of ERT, thereby inducing immune tolerance to rhGAA and enhancing the efficacy of ERT.17Sun B Bird A Young SP Kishnani PS Chen YT Koeberl DD Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.Am J Hum Genet. 2007; 81: 1042-1049Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar This approach to immunomodulatory gene therapy was apparently limited by the need to administer the vector prior to initiation of ERT, which seemed to be required to prevent antibody formation. Currently, we have evaluated the kinetics and mechanism for immunomodulatory gene therapy in GAA-KO mice to better understand the potential clinical application of this strategy.ResultsVector-mediated immune modulation prevented mortality in Pompe disease miceThe tolerogenic AAV vector (AAV-LSPhGAApA) previously induced immune tolerance to hGAA, by preventing the anti-GAA antibody response observed in naive GAA-KO mice.17Sun B Bird A Young SP Kishnani PS Chen YT Koeberl DD Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.Am J Hum Genet. 2007; 81: 1042-1049Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The timing of AAV vector administration relative to the immune challenge with hGAA was further evaluated in adult GAA-KO mice (Table 1; Figure 1a). AAV-LSPhGAApA administration (2 × 1010 vp intravenously) significantly prolonged survival in groups (G) of mice, if administered either prior to (G1; P = 0.05) or following (G2; P = 0.05) initial rhGAA injection (Figure 1b). AAV-LSPhGAApA administration enhanced the efficacy of ERT, as reflected by the increased time that vector-treated GAA-KO mice could run on the Rotarod (Figure 1c; G1-3). Decreased Rotarod time indicates progressive loss of muscle function in GAA-KO mice, which can be prevented by reducing the glycogen content of striated muscle.11Franco LM Sun B Yang X Bird A Zhang H Schneider A et al.Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II.Mol Ther. 2005; 12: 876-884Abstract Full Text Full Text PDF PubMed Scopus (137) Google ScholarTable 1Immune challenges prior to ERTGroupWeek 0Week 3G1AAV-LSPhGAApArhGAAG2rhGAAAAV-LSPhGAApAG3AAV-LSPhGAApA + rhGAArhGAAG4rhGAArhGAAG5PBSrhGAAG6AAV-CBhGAApAPBSG7aNo ERT was administered to group G7 providing mock-treated GAA-KO mice.PBSPBSAbbreviations: AAV, adeno-associated virus; ERT, enzyme-replacement therapy; PBS, phosphate-buffered saline; rhGAA, recombinant human acid α-glucosidase. Week 6, rhGAA injection. Week 12-22, rhGAA (20 mg/kg) every other week.a No ERT was administered to group G7 providing mock-treated GAA-KO mice. Open table in a new tab Urinary Hex4, a biomarker that was decreased in correlation with biochemical correction in Pompe disease mice, was reduced following AAV-LSPhGAApA administration, in comparison with GAA-KO mice that received rhGAA injections only (Figure 1d; G1-3). The negative impact of pre-existing anti-GAA antibodies was demonstrated by the persistent elevations of urinary Hex4 following antibody formation. GAA-KO mice that were immunized with two injections of rhGAA demonstrated elevated urinary Hex4 at week 10, in comparison with a group of phosphate-buffered saline–injected GAA-KO mice that had not yet formed anti-GAA antibodies (Figures 1d and 2a; G5 versus G4).Figure 2Antibody responses to hGAA. (a) IgG responses detected by anti-GAA ELISA. (b) Inverse titer of anti-hGAA IgG at week 10. (c) IgG1 responses at week 10. (d) IgE responses at week 10. G1, G2, G3, and G5, n = 5; G4, n = 4 (mean ± SD shown). *P < 0.05, **P < 0.01, and ***P < 0.001, indicated for each group in comparison with G4 (homoscedastic t-test). ELISA, enzyme-linked immunosorbent assay; G, group; hGAA, human acid α-glucosidase; IgG, immunoglonin G.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Formation of anti-GAA antibodies associated with hypersensitivity reactions to rhGAA challengeThe formation of anti-GAA antibodies occurred uniformly, if mice were not treated with AAV-LSPhGAApA (Figure 2a, G4 and G5). In contrast, tolerogenic AAV vector administration suppressed IgG titers, even when vector administration followed the initial rhGAA injection by 3 weeks (Figure 2b, G2). IgG titers reached 1:8,000 in response to four injections of rhGAA in mock-treated mice (G4), whereas groups of vector-treated mice had significantly reduced titers at week 10 (Figure 2b). The primary antibody response was IgG1 (Figure 2c), although IgE was significantly elevated in G4, in comparison with groups treated with AAV-LSPhGAApA (Figure 2d). One-way analysis of variance confirmed significant differences in the antibody response at 6 weeks (P = 0.0006) in vector-treated mice (G1, G2, G3), in comparison with mice treated with ERT only (G4, G5).Decreased body temperature and increased signs of hypersensitivity, quantified by an allergy score, have correlated with hypersensitivity reactions in mice.18Li XM Srivastava K Grishin A Huang CK Schofield B Burks W et al.Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy.J Allergy Clin Immunol. 2003; 112: 159-167Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Hypersensitivity was demonstrated by decreased body temperature (Figure 3a; G4 and G5) and allergic reactions in mice that were not immune tolerant to rhGAA. The nontolerant mice became immobile and died within 30 minutes of the rhGAA challenge (right panel, Figure 3b). The allergy score18Li XM Srivastava K Grishin A Huang CK Schofield B Burks W et al.Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy.J Allergy Clin Immunol. 2003; 112: 159-167Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar was adopted to quantify symptoms ranging from eye edema (score = 2) to death (score = 5) in nonvector-treated mice (Figure 3c, G4 and G5). Simultaneous administration of the vector and rhGAA (G3) failed to completely prevent changes in both body temperature (Figure 3a) and allergy scores (Figure 3c), in comparison with untreated GAA-KO mice (G7); however, the changes in body temperature and allergy scores were significantly reduced in the simultaneous administration group (G3), in comparison with the rhGAA only groups (G4, G5).Figure 3Hypersensitivity reactions associated with elevated MMCP-1 in nonvector-treated GAA-KO mice. (a) Temperature measured 30 minutes following rhGAA+ adjuvant challenge. G1 and G2, n = 5; G4 and G5 and G6 and G7, n = 4. (b) GAA-KO mouse prior to and 10 minutes following rhGAA administration, illustrating an allergy score of four. (c) Allergy score, G1, G2, and G6, n = 5; G3, n = 3; G4 and G5 and G7, n = 4. (d) MMCP-1 levels, each measured 30 minutes following rhGAA+ adjuvant challenge. G1 and G2, n = 5; G4 and G5 and G6 and G7, n = 4. Results for G4 and G5 were combined (G4 and G5), due to mortality. Mean ± SD shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (homoscedastic t-test). Lines overhead indicate the values being compared, either with G3 or with untreated GAA-KO mice (G7). KO, knockout; MMCP-1, mouse mast cell protease-1; rhGAA, recombinant human acid α-glucosidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hypersensitivity reactions were associated with elevated mouse mast cell protease-1 (MMCP-1) in nonvector-treated GAA-KO mice, in comparison with untreated GAA-KO mice (Figure 3d; G4 and G5 versus G7). MMCP-1 was previously increased markedly during anaphylaxis in mice sensitized to a strong antigen, ovalbumin, in association with elevated IgG and IgE.19Pemberton AD Wright SH Knight PA Miller HR Anaphylactic release of mucosal mast cell granule proteases: role of serpins in the differential clearance of mouse mast cell proteases-1 and -2.J Immunol. 2006; 176: 899-904Crossref PubMed Scopus (21) Google Scholar,20Vaali K Puumalainen TJ Lehto M Wolff H Rita H Alenius H et al.Murine model of food allergy after epicutaneous sensitization: role of mucosal mast cell protease-1.Scand J Gastroenterol. 2006; 41: 1405-1413Crossref PubMed Scopus (26) Google Scholar Cytokines associated with hypersensitivity, including interferon-γ, interleukin (IL)-4, and IL-5,18Li XM Srivastava K Grishin A Huang CK Schofield B Burks W et al.Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy.J Allergy Clin Immunol. 2003; 112: 159-167Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar were not elevated in mice exhibiting hypersensitivity reactions (data not shown). Similarly, cytokines associated with suppression, including IL-10 and transforming growth factor-β, were not elevated in vector-treated mice immediately following rhGAA administration (data not shown).21Faria AM Weiner HL Oral tolerance.Immunol Rev. 2005; 206: 232-259Crossref PubMed Scopus (600) Google Scholar One-way analysis of variance confirmed significant differences in the allergy score (<0.0001), body temperature (<0.0001), and MMCP-1 levels (<0.0001) in AAV-LSPhGAApA-treated mice (G1, G2, G3) following the immune challenge with rhGAA, in comparison with phosphate-buffered saline-treated mice (G4, G5). In contrast, administration of an immunogenic AAV vector (2 × 1010 vp) containing a ubiquitously active regulatory cassette (AAV-CBhGAApA)11Franco LM Sun B Yang X Bird A Zhang H Schneider A et al.Evasion of immune responses to introduced human acid alpha-glucosidase by liver-restricted expression in glycogen storage disease type II.Mol Ther. 2005; 12: 876-884Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar provoked lethal hypersensitivity reactions associated with decreased body temperature and increased allergy scores (Figure 3a,c; G6). When AAV-CBhGAApA was administered to 3-month-old wild-type mice to sensitize GAA sufficient animals to rhGAA, anti-GAA IgG1 was detected in vector-treated wild-type mice 6 weeks later (data not shown).A lack of efficacy from ERT was anticipated in GAA-KO mice that exhibited lethal hypersensitivity responses (Figure 3). The efficacy of ERT was evaluated by GAA analysis and glycogen quantification of the heart and skeletal muscles after ERT (Table 1). GAA activity was elevated in the muscles of all vector-treated mice (Figure 4a; G1, G2, G3, and G6). However, the relevance of lacking immune tolerance was demonstrated in GAA-KO mice that received the immunogenic vector, which had elevated glycogen content despite the presence of supraphysiologic GAA activity (Figure 4b; G6). One-way analysis of variance revealed that the glycogen content in the heart was significantly reduced only by AAV-LSPhGAApA administration (P < 0.0001), in comparison with ERT only. As reported, the efficacy of ERT was reduced in skeletal muscle, in comparison with the heart, even following the induction of immune tolerance.17Sun B Bird A Young SP Kishnani PS Chen YT Koeberl DD Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance.Am J Hum Genet. 2007; 81: 1042-1049Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Only when AAV-LSPhGAApA administration preceded rhGAA injection by 3 weeks did ERT significantly reduce glycogen content in the gastrocnemius, in comparison with sham-treated GAA-KO mice (Figure 4b; G1).Figure 4Efficacy of ERT following induction of immune tolerance in GAA-KO mice. (a) GAA activity, and (b) glycogen content of heart and skeletal muscles in GAA-KO mice (Table 1). G1 and G2, n = 5; G3 and G6, n = 3; G7, n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 (homoscedastic t-test), in comparison with untreated GAA-KO mice (G7). ERT, enzyme-replacement therapy; G, group; GAA, α-glucosidase; Gastroc, gastrocnemius; KO, knockout; Quad, quadriceps.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The vector particle number should be as low as possible to enhance safety from immunomodulatory gene therapy, while maintaining efficacy. The number of vector particles administered simultaneously with rhGAA, as in G3 above, was varied threefold higher and lower to model the likely scenario of immunomodulatory gene therapy and ERT starting together (Table 2). GAA-KO mice were challenged with rhGAA (20 mg/kg) 8 weeks following the initial vector and rhGAA administration and mortality was significantly decreased by the higher dose (6 × 1010 vp/mouse; P < 0.05), in comparison with mice treated with rhGAA alone (G1 versus G3, Figure 5a). The decrease in mortality approached significance following administration of the lowest dose (6 × 109 vp/mouse; G2; P = 0.07); moreover, the lowest and highest vector doses had a comparable effect, because only one out five mice developed a lethal hypersensitivity reaction in G2 versus one out of six mice in G1 (Figure 5a). Antibody formation was significantly suppressed by vector administration at either dose by 3 weeks, but not at 6 weeks (Figure 5b), which was unexplained. The allergy score was similarly significantly reduced only by the highest vector dose (Figure 5c; G1), as was the change in body temperature following rhGAA administration (data not shown). Therefore, a threefold reduced vector dose significantly suppressed antibody formation only at 3 weeks following vector administration, indicating a waning effect of the lower vector dose.Table 2Vector dosage titrationGroupWeek 0Week 6G1AAV-LSPhGAApA (6 × 1010 vp/mouse) + rhGAA (20 mg/kg)rhGAA (20 mg/kg)G2AAV-LSPhGAApA (6 × 109 vp/mouse) + rhGAA (20 mg/kg)rhGAA (20 mg/kg)G3rhGAA (20 mg/kg)rhGAA (20 mg/kg)G4PBSrhGAA (20 mg/kg)Abbreviations: AAV, adeno-associated virus; PBS, phosphate-buffered saline; rhGAA, recombinant human acid α-glucosidase; vp, vector particles.Week 6, rhGAA injection, followed by allergy scoring, temperature measurement, serum and tissue collection. Open table in a new tab Figure 5Effect of varying vector dose at the time of initiation of ERT upon prevention of hypersensitivity and mortality. (a) ELISA detection of anti-hGAA IgG1. (b) Allergy score 30 minutes following rhGAA (20 mg/kg) administration at 8 weeks. G1, n = 6; other groups, n = 5. ELISA, enzyme-linked immunosorbent assay; ERT, enzyme-replacement therapy; IgG, immunoglobin G; rhGAA, recombinant human acid α-glucosidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Regulatory T cell–mediated immune tolerance to rhGAA and desensitization in GAA-KO miceThe role of regulatory T cell (Treg) in immune tolerance has recently been elucidated and is currently an area of intense investigation.22Li W Carper K Liang Y Zheng XX Kuhr CS Reyes JD et al.Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance.Transplant Proc. 2006; 38: 3207-3208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,23van Wijk F Wehrens EJ Nierkens S Boon L Kasran A Pieters R et al.CD4+CD25+ T cells regulate the intensity of hypersensitivity responses to peanut, but are not decisive in the induction of oral sensitization.Clin Exp Allergy. 2007; 37: 572-581Crossref PubMed Scopus (47) Google Scholar,24Morse MA Hobeika AC Osada T Serra D Niedzwiecki D Lyerly HK et al.Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines.Blood. 2008; 112: 610-618Crossref PubMed Scopus (257) Google Scholar The depletion of Treg with anti-CD25 mouse monoclonal PC61 has been characterized as a strategy to investigate the role of Treg in immune tolerance.22Li W Carper K Liang Y Zheng XX Kuhr CS Reyes JD et al.Anti-CD25 mAb admin