Optimizing treatment outcomes: immune tolerance induction in Pompe disease patients undergoing enzyme replacement therapy.

Introduction: Pompe disease, a lysosomal storage disorder, is characterized by acid α-glucosidase (GAA) deficiency and categorized into two main subtypes: infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD). The primary treatment, enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA), faces challenges due to immunogenic responses, including the production of anti-drug antibody (ADA), which can diminish therapeutic efficacy. This study aims to assess the effectiveness of immune tolerance induction (ITI) therapy in cross-reactive immunologic material (CRIM)-positive Pompe disease patients with established high ADA levels. Method: In a single-center, open-label prospective study, we assessed ITI therapy's efficacy in Pompe disease patients, both IOPD and LOPD, with persistently elevated ADA titers (≥1:12,800) and clinical decline. The ITI regimen comprised bortezomib, rituximab, methotrexate, and intravenous immunoglobulin. Biochemical data, biomarkers, ADA titers, immune status, and respiratory and motor function were monitored over six months before and after ITI. Results: This study enrolled eight patients (5 IOPD and 3 LOPD). After a 6-month ITI course, median ADA titers significantly decreased from 1:12,800 (range 1:12,800-1:51,200) to 1:1,600 (range 1:400-1:12,800), with sustained immune tolerance persisting up to 4.5 years in some cases. Serum CK levels were mostly stable or decreased, stable urinary glucose tetrasaccharide levels were maintained in four patients, and no notable deterioration in respiratory or ambulatory status was noted. Adverse events included two treatable infection episodes and transient symptoms like numbness and diarrhea. Conclusion: ITI therapy effectively reduces ADA levels in CRIM-positive Pompe disease patients with established high ADA titers, underscoring the importance of ADA monitoring and timely ITI initiation. The findings advocate for personalized immunogenicity risk assessments to enhance clinical outcomes. In some cases, prolonged immune suppression may be necessary, highlighting the need for further studies to optimize ITI strategies for Pompe disease treatment. ClinicalTrials.gov NCT02525172; https://clinicaltrials.gov/study/NCT02525172.

Immune Tolerance-Adjusted Personalized Immunogenicity Prediction for Pompe Disease.

Infantile-onset Pompe disease (IOPD) is a glycogen storage disease caused by a deficiency of acid alpha-glucosidase (GAA). Treatment with recombinant human GAA (rhGAA, alglucosidase alfa) enzyme replacement therapy (ERT) significantly improves clinical outcomes; however, many IOPD children treated with rhGAA develop anti-drug antibodies (ADA) that render the therapy ineffective. Antibodies to rhGAA are driven by T cell responses to sequences in rhGAA that differ from the individuals' native GAA (nGAA). The goal of this study was to develop a tool for personalized immunogenicity risk assessment (PIMA) that quantifies T cell epitopes that differ between nGAA and rhGAA using information about an individual's native GAA gene and their HLA DR haplotype, and to use this information to predict the risk of developing ADA. Four versions of PIMA have been developed. They use EpiMatrix, a computational tool for T cell epitope identification, combined with an HLA-restricted epitope-specific scoring feature (iTEM), to assess ADA risk. One version of PIMA also integrates JanusMatrix, a Treg epitope prediction tool to identify putative immunomodulatory (regulatory) T cell epitopes in self-proteins. Using the JanusMatrix-adjusted version of PIMA in a logistic regression model with data from 48 cross-reactive immunological material (CRIM)-positive IOPD subjects, those with scores greater than 10 were 4-fold more likely to develop ADA (p<0.03) than those that had scores less than 10. We also confirmed the hypothesis that some GAA epitopes are immunomodulatory. Twenty-one epitopes were tested, of which four were determined to have an immunomodulatory effect on T effector response in vitro. The implementation of PIMA V3J on a secure-access website would allow clinicians to input the individual HLA DR haplotype of their IOPD patient and the GAA pathogenic variants associated with each GAA allele to calculate the patient's relative risk of developing ADA, enhancing clinical decision-making prior to initiating treatment with ERT. A better understanding of immunogenicity risk will allow the implementation of targeted immunomodulatory approaches in ERT-naïve settings, especially in CRIM-positive patients, which may in turn improve the overall clinical outcomes by minimizing the development of ADA. The PIMA approach may also be useful for other types of enzyme or factor replacement therapies.

A Race Against Time-Changing the Natural History of CRIM Negative Infantile Pompe Disease.

We report the clinical course of the first prenatally diagnosed cross-reactive immunologic material (CRIM)-negative infantile Pompe disease (IPD) patient [homozygous for c.2560C>T (p.Arg854X) variant in the GAA gene] to undergo prophylactic immune tolerance induction (ITI) and enzyme replacement therapy (ERT) within the first 2 days of life. Both parents were found to be carriers of the c.2560C>T (p.Arg854X) variant through prenatal carrier screening. Fetal echocardiogram at 31 weeks of gestation showed left ventricular hypertrophy. An echocardiogram on the 1st day of life revealed marked biventricular hypertrophy. Physical exam was significant for macroglossia and hypotonia. A short course of Prophylactic ITI with rituximab, methotrexate, and intravenous immunoglobulin (IVIG) in conjunction with ERT at a dose of 20 mg/kg every other week was started on day 2 of life. The patient completed the ITI protocol safely and complete B-cell recovery, based on CD19 count, was noted by 3 months of age. The patient never developed anti-rhGAA IgG antibodies to ERT. Vaccinations were initiated at 9 months of age, with adequate response noted. Complete recovery of cardiac function and left ventricular mass was seen by 11 weeks of age. At 8 months of age, the patient developmentally measured at 75-90% on the Alberta Infant Motor Scale, walked at 11 months and continues to develop age-appropriately at 50 months of age based on the Early Learning Accomplishment Profile. ERT dosing was increased to 40 mg/kg every 2 weeks at 32 months of age and frequency increased to 40 mg/kg every week at 47 months of age. Patient continues to have undetectable antibody titers, most recently at age 50 months and urine Hex4 has remained normal. To our knowledge, this is the first report of successful early ERT and ITI in a prenatally diagnosed CRIM-negative IPD patient and the youngest IPD patient to receive ITI safely. With the addition of Pompe disease to the Recommended Uniform Screening Panel(RUSP) and its addition to multiple state newborn screening programs, our case highlights the benefits of early diagnosis and timely initiation of treatment in babies with Pompe disease, who represent the most severe end of the disease spectrum.

Benefits of Prophylactic Short-Course Immune Tolerance Induction in Patients With Infantile Pompe Disease: Demonstration of Long-Term Safety and Efficacy in an Expanded Cohort.

Immune tolerance induction (ITI) with a short-course of rituximab, methotrexate, and/or IVIG in the enzyme replacement therapy (ERT)-naïve setting has prolonged survival and improved clinical outcomes in patients with infantile Pompe disease (IPD) lacking endogenous acid-alpha glucosidase (GAA), known as cross-reactive immunologic material (CRIM)-negative. In the context of cancer therapy, rituximab administration results in sustained B-cell depletion in 83% of patients for up to 26-39 weeks with B-cell reconstitution beginning at approximately 26 weeks post-treatment. The impact of rituximab on serum immunoglobulin levels is not well studied, available data suggest that rituximab can cause persistently low immunoglobulin levels and adversely impact vaccine responses. Data on a cohort of IPD patients who received a short-course of ITI with rituximab, methotrexate, and IVIG in the ERT-naïve setting and had ≥6 months of follow-up were retrospectively studied. B-cell quantitation, ANC, AST, ALT, immunization history, and vaccine titers after B-cell reconstitution were reviewed. Data were collected for 34 IPD patients (25 CRIM-negative and 9 CRIM-positive) with a median age at ERT initiation of 3.5 months (0.1-11.0 months). B-cell reconstitution, as measured by normalization of CD19%, was seen in all patients (n = 33) at a median time of 17 weeks range (11-55 weeks) post-rituximab. All maintained normal CD19% with the longest follow-up being 248 weeks post-rituximab. 30/34 (88%) maintained negative/low anti-rhGAA antibody titers, even with complete B-cell reconstitution. Infections during immunosuppression were reported in five CRIM-negative IPD patients, all resolved satisfactorily on antibiotics. There were no serious sequelae or deaths. Of the 31 evaluable patients, 27 were up to date on age-appropriate immunizations. Vaccine titers were available for 12 patients after B-cell reconstitution and adequate humoral response was observed in all except an inadequate response to the Pneumococcal vaccine (n = 2). These data show the benefits of short-course prophylactic ITI in IPD both in terms of safety and efficacy. Data presented here are from the youngest cohort of patients treated with rituximab and expands the evidence of its safety in the pediatric population.

The potential impact of timing of IVIG administration on the efficacy of rituximab for immune tolerance induction for patients with Pompe disease.

Immune modulation with rituximab, methotrexate, and intravenous immunoglobulin (IVIG) has shown great success in inducing immune tolerance in a large cohort of enzyme replacement therapy (ERT)-naïve infantile Pompe disease patients. Antibody-dependent cellular cytotoxicity, the principal mechanism by which rituximab depletes B-cells, requires CD20 binding by Fab'2 of rituximab on B-cells and the concomitant binding of its Fc region to Fc receptors on effector cells or to complement. To protect patients against microbial infections when using rituximab, IVIG was added to the immunomodulation regimen used in Pompe disease. Administration of IVIG can saturate neonatal Fc receptors (FcRn), which recycle endogenous as well as administered polyclonal/monoclonal antibodies via the binding of the Fc moiety to FcRn. As such, the administration of IVIG prior to rituximab, a chimeric mouse-human monoclonal antibody, may sharply reduce the half-life of rituximab and in turn, its efficacy. Based on this understanding, it is vital to understand the optimal timing of IVIG administration in relation to rituximab administration for the purposes of inducing immune tolerance.

Effects of higher and more frequent dosing of alglucosidase alfa and immunomodulation on long-term clinical outcome of classic infantile Pompe patients.

The aim of this study was to compare the long-term outcome of classic infantile Pompe patients treated with 20 mg/kg alglucosidase alfa every other week (eow) to those treated with 40 mg/kg/week, and to study the additional effect of immunomodulation. Six patients received 20 mg/kg eow and twelve 40 mg/kg/week. Five patients were cross-reactive immunologic material (CRIM)-negative, two in the 20 mg, three in the 40 mg group. We compared (ventilator-free) survival, motor outcome, infusion associated reactions (IARs), and antibody formation. From 2012 on patients >2 months in the 40 mg group also received immunomodulation with rituximab, methotrexate, and intravenous immunoglobulin (IVIG) in an enzyme replacement therapy (ERT)-naïve setting. Survival was 66% in the 20 mg group and 92% in the 40 mg group. Ventilator-free survival was 50% and 92%. Both CRIM-negative patients in the 20 mg group died, whereas all three are alive in the 40 mg group. In the 20 mg group, 67% learned to walk compared with 92% in the 40 mg group. At the age of 3 years, 33% and 92% were able to walk. Peak antibody titers ranged from 1:1250 to 1:31 250 in the 20 mg group and from 1:250 to 1:800 000 in the 40 mg group. Five patients of the 40 mg group of whom two CRIM-negative also received immunomodulation. B-cell recovery was observed between 5.7 and 7.9 months after the last dose of rituximab. After B-cell recovery titers of patients with and without immunomodulation were similar (ranges 1:6 250-1:800 000 and 1:250-1:781 250). This study shows that classic infantile patients treated with 40 mg/kg/week from the start to end have a better (ventilator-free) survival and motor outcome. Immunomodulation did not prevent antibody formation in our study.

Effects of immunomodulation in classic infantile Pompe patients with high antibody titers.

PURPOSE: To evaluate whether immunomodulation can eliminate high sustained antibody levels, and thereby improve clinical outcome in classic infantile Pompe patients receiving enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA). METHODS: Three patients (two cross-reactive immunologic material (CRIM) negative) with high sustained antibodies received a three-week treatment protocol with Rituximab and Bortezomib, followed by daily Rapamycin and monthly IVIG. Patients received 40 mg/kg/week rhGAA. Antibody titers were measured using ELISA. Neutralizing effects on cellular uptake were determined. Clinical efficacy was measured in terms of (ventilator-free) survival, reduction in left ventricular mass index (LVMI) and improvement in motor function. RESULTS: Before immunomodulation anti-rhGAA antibody titers ranged from 1:156,250 to 1:781,250 and at last assessment from 1:31,250 to 1:156,250. Neutralizing effects of anti-rhGAA antibody titers (observed in two patients) disappeared. Infusion-associated reactions were no longer present. Immunomodulation resulted in substantial increases of aspartate transaminase, alanine transaminase, and creatine kinase levels. The two CRIM-negative patients who could walk at start of immunomodulation maintained their ability to walk; the patient who had lost this ability did not regain it. CONCLUSIONS: To some extent, the immunomodulation protocol used in our study reduced antibody titers, but it did not eliminate them. Overall, there have been few reports on secondary immunomodulation, and various protocols have been applied. Future research should seek to identify the most successful immunomodulation protocol in patients with high sustained titers.

Desensitization of two young patients with infantile-onset Pompe disease and severe reactions to alglucosidase alfa.

Pompe disease is a metabolic myopathy, due to deficiency of alpha glucosidase, with a wide clinical spectrum. Enzyme replacement therapy is the only available treatment to improve morbidity and mortality, especially in infantile-onset form. However, some patients experience infusion-associated reactions, which may restrict their access to this treatment. We report on two patients (respectively 12 and 3 months old) with infantile-onset Pompe disease and severe cardiomyopathy, that presented with severe reactions during infusion of enzyme replacement therapy and were successfully desensitized with a new individualized protocol. Our protocol, using microdilution and a premedication with antihistamines, corticosteroids, and tranexamic acid, seems safe and effective and it may allow the continuation of therapy in Pompe patients resulting in the reduction of morbidity and mortality related to this disease.

An immune tolerance approach using transient low-dose methotrexate in the ERT-naïve setting of patients treated with a therapeutic protein: experience in infantile-onset Pompe disease.

PURPOSE: To investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients. METHODS: Newly diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and <51,200 within 12 months), or low (LT; ≤6400 within 12 months), were compared with those of 37 CRIM-positive IOPD historic comparators receiving rhGAA alone. RESULTS: Fourteen IOPD TLD-MTX recipients at the median age of 3.8 months (range, 0.7-13.5 months) had a median last titer of 150 (range, 0-51,200) at median rhGAA duration ~83 weeks (range, 36-122 weeks). One IOPD patient (7.1%) developed titers in the SIT range and one patient (7.1%) developed titers in the HSAT range. Twelve of the 14 patients (85.7%) that received TLD-MTX remained LT, versus 5/37 HSAT (peak 51,200-409,600), 7/37 SIT (12,800-51,000), and 23/37 LT (200-12,800) among comparators. CONCLUSION: Results of TLD-MTX coinitiated with rhGAA are encouraging and merit a larger longitudinal study.

Phosphatidylserine Is Not Just a Cleanup Crew but Also a Well-Meaning Teacher.

Phosphatidylserine (PS) exposure during apoptosis leads to silent clearance of cells without adverse immune reactions to self-proteins. Given the biological functions of PS in cellular cleanup and global immunosuppression, we hypothesized that administration of PS-protein complexes would reduce immunogenicity. Here, we report that exposing Pompe disease mice to acid alpha glucosidase (rhGAA) with PS or immunosuppressant dexamethasone resulted in lower anti-rhGAA antibodies than in animals receiving rhGAA alone. However, upon rechallenge with rhGAA, only PS-rhGAA pre-exposed mice displayed a durable hyporesponsiveness even after PS administration was ceased. Thus, pre-exposure of antigens administered together with PS were not silently cleared, but the immune system acquired memory about the antigen that averted mounting of a response during rechallenge. In hemophilia A mice, PS hyporesponsiveness toward Factor VIII was reversed by administration of function-blocking antibody against the PS receptor T-cell immunoglobulin and mucin 4, implicating this receptor in PS's effect. Moreover, pre-exposure of myelin oligodendrocyte glycoprotein peptide with PS delayed the onset and reduced the severity of experimental autoimmune encephalomyelitis. These observations suggest that PS's function in apoptosis is not limited to silent antigen clearance without immune responses toward self-proteins but shows that PS reduces immune response during rechallenge to several antigens that also involves initiation of antigen tolerance.

High Sustained Antibody Titers in Patients with Classic Infantile Pompe Disease Following Immunomodulation at Start of Enzyme Replacement Therapy.

OBJECTIVE: To evaluate whether immunomodulation at start of enzyme replacement therapy induces immune tolerance to recombinant human acid alpha-glucosidase (rhGAA) in patients with classic infantile Pompe disease. STUDY DESIGN: Three patients (1 cross reactive immunologic material negative, 2 cross reactive immunologic material positive) were treated with 4 weekly doses of rituximab, weekly methotrexate, and monthly intravenous immunoglobulin and enzyme replacement therapy at 40 mg/kg/week. Antibody titers were measured using enzyme-linked immunosorbent assay. Neutralizing effects on rhGAA activity and cellular uptake were determined and combined with pharmacokinetic analysis. Clinical efficacy was evaluated by (ventilator-free) survival, reduction in left ventricular mass index, and improvement of motor function. RESULTS: Immunomodulation induced B cell depletion that was accompanied by absence of antibody formation in all 3 patients. Upon cessation of rituximab treatment, all 3 patients showed B cell recovery, which was accompanied by formation of very high sustained antibody titers in 2 patients. Neutralizing effects on infused rhGAA were low to mild/moderate. All patients were alive at study end, learned to walk, and showed (near) normalization of left ventricular mass index. CONCLUSIONS: Immunomodulation as recommended in the literature prevented formation of rhGAA antibodies only during B cell depletion but failed to induce immune tolerance in 2 out of 3 patients.

High dose IVIG successfully reduces high rhGAA IgG antibody titers in a CRIM-negative infantile Pompe disease patient.

Alglucosidase alfa (rhGAA) has altered the course of an otherwise fatal outcome in classic infantile Pompe disease (IPD), which presents with cardiomyopathy and severe musculoskeletal involvement. However, the response to therapy is determined by several factors including the development of high and sustained antibody titers (HSAT) to rhGAA. Cross-reactive immunologic material (CRIM) negative patients are at the highest risk for development of HSAT. Immune tolerance induction (ITI) with methotrexate, rituximab, and intravenous immunoglobulin (IVIG) has been largely successful in preventing the immune response and in achieving tolerance when done in conjunction with enzyme replacement therapy (ERT) initiation. Reducing antibody titers in cases with an entrenched immune response remains a challenge in the field despite the use of multiple immunomodulatory agents. Success has been shown with addition of bortezomib to the ITI regimen, yet the prolonged course and potential risks with the use of such agents' demands caution. We present here a 7-year-old CRIM-negative IPD patient who was not successfully tolerized by an ITI regimen with rituximab, methotrexate, and IVIG due to intolerability to the regimen and recurrent infections. She went on to develop HSAT, with significant clinical decline, loss of all motor abilities, and a fragile medical state, which made it challenging to institute the bortezomib based regimen to reduce HSAT. She had severe developmental delay, respiratory failure with invasive ventilation and tracheostomy, persistent hypotonia, ptosis of eyelids, diffuse severe osteopenia, contractures, and was completely G-tube fed. As a rescue mechanism, we treated her with high dose and high frequency IVIG in an attempt to reduce rhGAA IgG antibody titers (antibody titers; titers). Her titers saw a steady decline on weekly IVIG doses at 1g/kg for 20weeks. Subsequently when the IVIG regimen was altered to 1g/kg every month, rising titers were detected and therefore the regimen was changed to a biweekly regimen. High dose IVIG resulted in an eightfold decrease in antibody titers. Clinically, she showed improvement with partial recovery of previously lost motor abilities, especially hand movements and better head and neck control than before. The regimen was safely tolerated with no hospitalizations. The effectiveness of IVIG as a single agent, in this case with multiple comorbidities and fragile clinical status, was lifesaving and may represent an effective, perhaps lifesaving rescue approach to reduce antibody titers.

Pompe disease in adulthood: effects of antibody formation on enzyme replacement therapy.

PURPOSE: To determine the effect of antibodies against recombinant human acid α-glucosidase (rhGAA) on treatment efficacy and safety, and to test whether the GAA genotype is involved in antibody formation. METHODS: We used enzyme-linked immunosorbent assay (ELISA) to determine anti-rhGAA antibody titers at baseline and at 6, 12, and 36 months of rhGAA treatment. We measured the capacity of antibodies to neutralize rhGAA enzymatic activity or cellular uptake and the effects on infusion-associated reactions (IARs), muscle strength, and pulmonary function. RESULTS: Of 73 patients, 45 developed antibodies. Maximal titers were high (≥1:31,250) in 22% of patients, intermediate (1:1,250-1:31,250) in 40%, and none or low (0-1:1,250) in 38%. The common IVS1/delex18 GAA genotype was absent only in the high-titer group. The height of the titer positively correlated with the occurrence and number of IARs (P ≤ 0.001). On the group level, antibody titers did not correlate with treatment efficacy. Eight patients (11%) developed very high maximal titers (≥156,250), but only one patient showed high sustained neutralizing antibodies that probably interfered with treatment efficacy. CONCLUSIONS: In adults with Pompe disease, antibody formation does not interfere with rhGAA efficacy in the majority of patients, is associated with IARs, and may be attenuated by the IVS1/delex18 GAA genotype.Genet Med 19 1, 90-97.

Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients.

Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

Phosphatidylserine Converts Immunogenic Recombinant Human Acid Alpha-Glucosidase to a Tolerogenic Form in a Mouse Model of Pompe Disease.

Development of unwanted immune responses against therapeutic proteins is a major clinical complication. Recently, we have shown that exposure of Factor VIII in the presence of phosphatidylserine (PS) induces antigen-specific hyporesponsiveness to Factor VIII rechallenge, suggesting that PS is not immune suppressive, but rather immune regulatory in that PS converts an immunogen to a tolerogen. Since PS is exposed in the outer leaflet during apoptosis, we hypothesize that PS imparts tolerogenic activity to this natural process. Thus, immunization with PS containing liposomes would mimic this natural process. Here, we investigate the immune regulatory effects of PS in inducing tolerance toward recombinant human acid alpha-glucosidase (rhGAA). rhGAA was found to complex with PS liposomes through hydrophobic interactions, and incubation PS-rhGAA with dendritic cells resulted in the increased secretion of transforming growth factor-ß. Immunization with PS-rhGAA or O-phospho-L-serine-rhGAA led to a reduction in anti-rhGAA antibody response which persisted despite rechallenge with free rhGAA. Importantly, the titer levels in a majority of these animals remained unchanged after rechallenge and can be considered nonresponders. These data provide evidence that PS liposomes can be used to induce tolerance toward therapeutic proteins, in general.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease.

Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa(-/-) mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders.

[Enzyme Replacement Therapy for Pompe Disease: The Long-Term Efficacy and Limitation].

The long-term efficacy of enzyme replacement therapy (ERT) in patients with Pompe disease has been unraveled. Cardiac muscle responds well to ERT, whereas motor and respiratory functions do not. The screening of newborns, which enables the early initiation of ERT, has improved outcomes of infantile Pompe patients. Myopathies still exist even in patients in whom ERT is initiated early, and have become recognized as an emerging new phenotype of IP on ERT. In addition, this shows the limitation of currently available ERT. It is expected that the development of therapy will improve the outcome of Pompe disease.

Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells.

Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

The role of antibodies in enzyme treatments and therapeutic strategies.

Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs.

BAFF blockade prevents anti-drug antibody formation in a mouse model of Pompe disease.

Antibodies formed against the therapeutic protein are a life-threatening complication that arises during enzyme replacement therapy for Pompe disease (acid α-glucosidase deficiency; GAA). To provide an effective alternative to current practices, we investigated the capacity of anti-B-cell activating factor (BAFF) as a novel drug candidate to prevent antibody formation in a Pompe disease mouse model. A BAFF-neutralizing antibody was administered prophylactically and with maintenance doses in association with enzyme replacement therapy using recombinant human GAA in Gaa(-/-) mice. BAFF blockade delayed antibody production and increased GAA activity within tissues with protection from anaphylaxis. Anti-BAFF also resolved antibody formation during an immune response and precluded the maturation of antibody secreting cells from entering the bone marrow compartment. This treatment modality may therefore be a viable alternative for the clinical management of antibody formation for Pompe disease and has potential use against antibody formation in other protein replacement therapies.