(b) Allergy score 30 minutes following rhGAA (20?mg/kg) administration at 8 weeks. to an increase in GAA-specific IgG (< 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: < 0.01; symptoms: < 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. Introduction Infantile-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).1 Pilot studies of ERT with recombinant human acid -glucosidase (rhGAA) (purified from Chinese hamster ovary cell cultures2 or transgenic rabbit milk3) 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 unfavorable (CRIM-negative). CRIM-negative Pompe disease subjects produced very high anti-hGAA antibodies and exhibited markedly reduced efficacy from ERT. In the first pilot study of ERT Anxa5 in Pompe disease using Chinese hamster ovary cellCderived recombinant hGAA, the two patients who were CRIM-negative produced higher titers of anti-hGAA antibodies than the third patient who was CRIM-positive.2 Poor outcomes were associated with CRIM-negative status in the pivotal clinical trials that led to marketing approval for rhGAA.4,5 CRIM-negative Pompe disease subjects in these clinical trials formed very high, sustained anti-hGAA antibodies and exhibited markedly reduced efficacy from ERT.2,4,5 The antibody response to ERT in Pompe disease has been remarkably similar to inhibitory antibody formation in hemophilia.6 Hemophilia B is similar to Pompe disease, in that CRIM-negative patients frequently mounted high-titer IgG antibody responses to protein alternative therapy with coagulation factor IX (FIX) that interfere with efficacy. Taken together, these data suggest that immune tolerance to ERT is usually 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.7 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.8 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.8,9 Liver-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 made up of a liver-specific regulatory cassette substantially corrected glycogen storage in the diaphragm and heart of GAA-KO cAMPS-Sp, triethylammonium salt 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.10 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.11 Increasing plasma hGAA was detected between cAMPS-Sp, triethylammonium salt 1 and 14 days and sustained for >12 weeks following AAV-LSPhGAApA administration.11 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.12,13 These data suggested that liver-restricted, high-level expression of hGAA induced immune tolerance in Pompe disease mice, cAMPS-Sp, triethylammonium salt similarly to the effect of liver-specific expression of therapeutic proteins in hemophilia mice12,14 and in Fabry disease mice.15 Like CRIM-negative patients with Pompe disease, GAA-KO.