Successful AAV8 readministration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

BACKGROUND: A major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice. METHODS: An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood. RESULTS: In näive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration. CONCLUSIONS: Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naïve mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.

Identification of a potent regulatory T cell epitope in factor V that modulates CD4+ and CD8+ memory T cell responses.

Identification of T cell epitopes that are recognized by Tregs may elucidate the relative contributions of thymic Tregs and induced Tregs to control of autoimmune diseases and allergy. One such T regulatory cell epitope or 'Tregitope', derived from blood Factor V, is described here. Tregs responding to Tregitope FV621 are potent suppressors of CD4+ T effector responses to Tetanus Toxoid in an in vitro bystander suppression assay, strongly inhibit proliferation of effector CD8+ T cells, down-modulate CD86 and HLA DR on antigen-presenting cells, and enhance expression of granzyme B in Tregs. Tregitope FV621 also suppresses anti-OVA immune responses in vivo. The immunomodulatory effect of Tregitope FV621 is enhanced when conjugated to albumin, suggesting that the short half-life of Tregitope peptides can be prolonged. The in silico tools used to prospectively identify the FV Tregitope described here, when combined with in vitro /in vivo validating assays, may facilitate future Tregitope discoveries.

Transforming the clinical outcome in CRIM-negative infantile Pompe disease identified via newborn screening: the benefits of early treatment with enzyme replacement therapy and immune tolerance induction.

PURPOSE: To assess the magnitude of benefit to early treatment initiation, enabled by newborn screening or prenatal diagnosis, in patients with cross-reactive immunological material (CRIM)-negative infantile Pompe disease (IPD), treated with enzyme replacement therapy (ERT) and prophylactic immune tolerance induction (ITI) with rituximab, methotrexate, and intravenous immunoglobulin (IVIG). METHODS: A total of 41 CRIM-negative IPD patients were evaluated. Among patients who were treated with ERT + ITI (n = 30), those who were invasive ventilator-free at baseline and had ≥6 months of follow-up were stratified based on age at treatment initiation: (1) early (≤4 weeks), (2) intermediate (>4 and ≤15 weeks), and (3) late (>15 weeks). A historical cohort of 11 CRIM-negative patients with IPD treated with ERT monotherapy served as an additional comparator group. RESULTS: Twenty patients were included; five, seven, and eight in early, intermediate, and late treatment groups, respectively. Genotypes were similar across the three groups. Early-treated patients showed significant improvements in left ventricular mass index, motor and pulmonary outcomes, as well as biomarkers creatine kinase and urinary glucose tetrasaccharide, compared with those treated later. CONCLUSION: Our preliminary data suggest that early treatment with ERT + ITI can transform the long-term CRIM-negative IPD phenotype, which represents the most severe end of the Pompe disease spectrum.

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.

Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues.

BACKGROUND: Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls METHODS: Because Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 "classical" reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs. RESULTS: We affirmed that 17 out of 21 classical reference genes had upregulated expression in tumors compared to normal colonic epithelial tissue and dramatically so in some cases. Indeed, tumors were distinguished from normal controls in both unsupervised hierarchical clustering analyses (HCA) and principal component analyses (PCA). We then identified 42 novel potential reference genes with minimal coefficients of variation (CV) across 79 CRC tumor pairs. Though largely consistently expressed across tumors and normal control tissues, a subset of high stage tumors (HSTs) as well as some normal tissue samples (HSNs) located adjacent to these HSTs demonstrated dysregulated expression, thus identifying a subset of tumors with a potentially distinct and aggressive biological profile. CONCLUSION: While classical CRC reference genes were found to be differentially expressed between tumors and normal controls, novel reference genes, identified via NGS, were more consistently expressed across malignant and normal colonic tissues. Nonetheless, a subset of HST had profound dysregulation of such genes as did many of the histologically normal tissues adjacent to such HSTs, indicating that the HSTs so distinguished may have unique biological properties and that their histologically normal tissues likely harbor a small population of microscopically undetected but metabolically active tumors.

Capnocytophaga spp. infection causing chorioamnionitis: an unusual suspect.

Capnocytophaga spp. are not often considered as a cause of perinatal infection. However, Capnocytophaga spp. are a commensal of the human oral cavity and can be opportunistic pathogens. The present case illustrates the ability of a species from the human oral cavity, C. sputigena, to cause an ascending infection of the genital tract in a healthy pregnant woman.

Hepatotoxicity of immune checkpoint inhibitors: An evolving picture of risk associated with a vital class of immunotherapy agents.

Immune checkpoint inhibitors (ICIs) block CTLA-4, PD-1 and PD-L1, or other molecules that control antitumour activities of lymphocytes. These products are associated with a broad array of immune-related toxicities affecting a variety of organs, including the liver. ICI-associated immune-mediated hepatitis (IMH) ranges in severity between mild and life-threatening and is marked by findings that bear both similarities as well as differences with idiopathic autoimmune hepatitis. Hepatotoxic events are often detected in clinical trials of ICIs that are powered for efficacy. Risk levels for ICI-induced liver injury may be impacted by the specific checkpoint molecule targeted for treatment, the ICI dose levels, and the presence of a pre-existing autoimmune diathesis, chronic infection or tumour cells which infiltrate the liver parenchyma. When patients develop liver injury during ICI treatment, a prompt assessment of the cause of injury, in conjunction with the application of measures to optimally manage the adverse event, should be made. Strategies to manage the risk of IMH include the performance of pretreatment liver tests with regular monitoring during and after ICI treatment and patient education. Using Common Terminology Criteria for Adverse Events developed at the National Cancer Institute to measure the severity level of liver injury, recommended actions may include continued ICI treatment with close patient monitoring, ICI treatment suspension or discontinuation and/or administration of corticosteroids or, when necessary, a non-steroidal immunosuppressive agent. The elucidation of reliable predictors of tumour-specific ICI treatment responses, as well as an increased susceptibility for clinically serious immune-related adverse events, would help optimize treatment decisions for individual 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.

Immune response to enzyme replacement therapies in lysosomal storage diseases and the role of immune tolerance induction.

The US Food and Drug Administration (FDA) and National Organization for Rare Disease (NORD) convened a public workshop titled "Immune Responses to Enzyme Replacement Therapies: Role of Immune Tolerance Induction" to discuss the impact of anti-drug antibodies (ADAs) on efficacy and safety of enzyme replacement therapies (ERTs) intended to treat patients with lysosomal storage diseases (LSDs). Participants in the workshop included FDA staff, clinicians, scientists, patients, industry, and advocacy group representatives. The risks and benefits of implementing prophylactic immune tolerance induction (ITI) to reduce the potential clinical impact of antibody development were considered. Complications due to immune responses to ERT are being recognized with increasing experience and lengths of exposure to ERTs to treat several LSDs. Strategies to mitigate immune responses and to optimize therapies are needed. Discussions during the workshop resulted in the identification of knowledge gaps and future areas of research, as well as the following proposals from the participants: (1) systematic collection of longitudinal data on immunogenicity to better understand the impact of ADAs on long-term clinical outcomes; (2) development of disease-specific biomarkers and outcome measures to assess the effect of ADAs and ITI on efficacy and safety; (3) development of consistent approaches to ADA assays to allow comparisons of immunogenicity data across different products and disease groups, and to expedite reporting of results; (4) establishment of a system to widely share data on antibody titers following treatment with ERTs; (5) identification of components of the protein that are immunogenic so that triggers and components of the immune responses can be targeted in ITI; and (6) consideration of early ITI in patients who are at risk of developing clinically relevant ADA that have been demonstrated to worsen treatment outcomes.

CRIM-negative infantile Pompe disease: characterization of immune responses in patients treated with ERT monotherapy.

PURPOSE: Enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) prolongs survival in infantile Pompe disease (IPD). However, the majority of cross-reactive immunologic material (CRIM)-negative (CN) patients have immune responses with significant clinical decline despite continued ERT. We aimed to characterize immune responses in CN patients with IPD receiving ERT monotherapy. METHODS: A chart review identified 20 CN patients with IPD treated with ERT monotherapy for ≥6 months. Patients were stratified by anti-rhGAA antibody titers: high sustained antibody titers (HSAT; ≥51,200) at least twice; low titers (LT; <6,400) throughout treatment; or sustained intermediate titers (SIT; 6,400-25,600). RESULTS: Despite early initiation of treatment, the majority (85%) of CN patients developed significant antibody titers, most with HSAT associated with invasive ventilation and death. Nearly all patients with HSAT had at least one nonsense GAA mutation, whereas the LT group exclusively carried splice-site or frameshift mutations. Only one patient in the HSAT group is currently alive after successful immune modulation in the entrenched setting. CONCLUSION: Immunological responses are a significant risk in CN IPD; thus induction of immune tolerance in the naive setting should strongly be considered. Further exploration of factors influencing immune responses is required, particularly with the advent of newborn screening for Pompe disease.

A novel function of RNAs arising from the long terminal repeat of human endogenous retrovirus 9 in cell cycle arrest.

The human genome contains approximately 50 copies of the replication-defective human endogenous retrovirus 9 (ERV-9) and thousands of copies of its solitary long term repeat (sLTR) element. While some sLTRs are located upstream of critical genes and have enhancer activity, other sLTRs are located within introns and may be transcribed as RNAs. We found that intronic RNAs arising from U3 sLTRs of ERV-9 were expressed as both sense (S) and antisense (AS) transcripts in all human cells tested but that expression levels differed in malignant versus nonmalignant cells. In nonmalignant cells, AS was expressed at higher levels than S and at higher levels than in malignant cells; in malignant cells, AS was expressed at amounts equivalent to those of S RNA. Critically, U3 AS RNA was found to physically bind to key transcription factors for cellular proliferation, including NF-Y, p53, and sp1, indicating that such RNA transcripts may function as decoy targets or traps for NF-Y and thus inhibit the growth of human cancer cells. Indeed, short U3 oligodeoxynucleotides (ODNs) based on these RNA sequences ably inhibited proliferation of cancer cell lines driven by cyclins B1/B2, the gene targets of NF-Y.

In silico methods for immunogenicity risk assessment and human homology screening for therapeutic antibodies.

In silico immunogenicity risk assessment has been an important step in the development path for many biologic therapeutics, including monoclonal antibodies. Even if the source of a given biologic is 'fully human', T cell epitopes that are contained in the sequences of the biologic may activate the immune system, enabling the development of anti-drug antibodies that can reduce drug efficacy and may contribute to adverse events. Computational tools that identify T cell epitopes from primary amino acid sequences have been used to assess the immunogenic potential of therapeutic candidates for several decades. To facilitate larger scale analyses and accelerate preclinical immunogenicity risk assessment, our group developed an integrated web-based platform called ISPRI, (Immunogenicity Screening and Protein Re-engineering Interface) that provides hands-on access through a secure web-based interface for scientists working in large and mid-sized biotech companies in the US, Europe, and Japan. This toolkit has evolved and now contains an array of algorithms that can be used individually and/or consecutively for immunogenicity assessment and protein engineering. Most analyses start with the advanced epitope mapping tool (EpiMatrix), then proceed to identify epitope clusters using ClustiMer, and then use a tool called JanusMatrix to define whether any of the T cell epitope clusters may generate a regulatory T cell response which may diminish or eliminate anti-drug antibody formation. Candidates can be compared to similar products on a normalized immunogenicity scale. Should modifications to the biologic sequence be an option, a tool for moderating putative immunogenicity by editing T cell epitopes out of the sequence is available (OptiMatrix). Although this perspective discusses the in-silico immunogenicity risk assessment for monoclonal antibodies, bi-specifics, multi-specifics, and antibody-drug conjugates, the analysis of additional therapeutic modalities such as enzyme replacement proteins, blood factor proteins, CAR-T, gene therapy products, and peptide drugs is also made available on the ISPRI platform.

ISPRI (Interactive Screening and Protein Reengineering Interface): Integrated, cloud-based, comprehensive toolkit for Immunogenicity Risk Assessment.EpiMatrix Immunogenicity Score: Combined T effector and Treg Epitope Content per unit protein.Tregitopes: Treg Epitopes found in IgG Framework that have been shown to modulate antigen-specific effector T cell responses.ClustiMer: Tool for identifying epitope rich polypeptides from within a given protein sequence.JanusMatrix: Tool for Predicting Tolerance, Putative Treg Epitopes, and Anti-self-immune responses.OptiMatrix: Tool for modifying T cell epitope sequences to reduce (or enhance) MHC binding.

Bortezomib in the rapid reduction of high sustained antibody titers in disorders treated with therapeutic protein: lessons learned from Pompe disease.

PURPOSE: High sustained antibody titers complicate many disorders treated with a therapeutic protein, including those treated with enzyme replacement therapy, such as Pompe disease. Although enzyme replacement therapy with alglucosidase alfa (Myozyme) in Pompe disease has improved the prognosis of this otherwise lethal disorder, patients who develop high sustained antibody titers to alglucosidase alfa enter a prolonged phase of clinical decline resulting in death despite continued enzyme replacement therapy. Clinically effective immune-tolerance induction strategies have yet to be described in the setting of an entrenched immune response characterized by high sustained antibody titers, wherein antibody-producing plasma cells play an especially prominent role. METHODS: We treated three patients with infantile Pompe disease experiencing marked clinical decline due to high sustained antibody titers. To target the plasma cell source of high sustained antibody titers, a regimen based on bortezomib (Velcade) was used in combination with rituximab, methotrexate, and intravenous immunoglobulin. RESULTS: The treatment regimen was well tolerated, with no obvious side effects. Patient 1 had a 2,048-fold, and patients 2 and 3 each had a 64-fold, reduction in anti-alglucosidase alfa antibody titer, with concomitant sustained clinical improvement. CONCLUSION: The addition of bortezomib to immunomodulatory regimens is an effective and safe treatment strategy in infantile Pompe disease, with potentially broader clinical implications.

Apolipoprotein B binding domains: evidence that they are cell-penetrating peptides that efficiently deliver antigenic peptide for cross-presentation of cytotoxic T cells.

Low-density lipoproteins (LDLs) are a good source of cholesterol, which is important in cellular homeostasis and production of steroids. Apolipoprotein B-100 (ApoB-100), the sole protein component of LDL, is known to bind to cell surface LDL receptor (LDLR) or cell surface-bound proteoglycans and to be internalized into cells. We found that APCs, consisting of macrophages and dendritic cells, upregulate LDLR on culture in vitro without obvious stimulation. In contrast, T cell populations only upregulate LDLR on activation. Thus, we strategized that tagging immunogens to ApoB-100 might be a useful means to target Ag to APCs. We generated fusion proteins consisting of receptor binding sites in ApoB-100, coupled to OVA peptide (ApoB-OVA), as Ag delivery vehicles and demonstrated that this novel delivery method successfully cross-presented OVA peptides in eliciting CTL responses. Surprisingly, internalization of ApoB-OVA peptide occurred via cell surface proteoglycans rather than LDLRs, consistent with evidence that structural elements of ApoB-100 indicate it to have cell-penetrating peptide properties. Finally, we used this strategy to assess therapeutic vaccination in a tumor setting. OVA-expressing EL-4 tumors grew progressively in mice immunized with ApoB-100 alone but regressed in mice immunized with ApoB-OVA fusion protein, coinciding with development of OVA-specific CTLs. Thus, to our knowledge, this is the first article to describe the cell-penetrating properties of a conserved human origin cell penetrating peptide that may be harnessed as a novel vaccination strategy as well as a therapeutics delivery device.

Immunoinformatic Risk Assessment of Host Cell Proteins During Process Development for Biologic Therapeutics.

The identification and removal of host cell proteins (HCPs) from biologic products is a critical step in drug development. Despite recent improvements to purification processes, biologics such as monoclonal antibodies, enzyme replacement therapies, and vaccines that are manufactured in a range of cell lines and purified using diverse processes may contain HCP impurities, making it necessary for developers to identify and quantify impurities during process development for each drug product. HCPs that contain sequences that are less conserved with human homologs may be more immunogenic than those that are more conserved. We have developed a computational tool, ISPRI-HCP, that estimates the immunogenic potential of HCP sequences by evaluating and quantifying T cell epitope density and relative conservation with similar T cell epitopes in the human proteome. Here we describe several case studies that support the use of this method for classifying candidate HCP impurities according to their immunogenicity risk.

Immunophenotype associated with high sustained antibody titers against enzyme replacement therapy in infantile-onset Pompe disease.

Introduction: The efficacy of enzyme replacement therapy (ERT) with alglucosidase alfa for infantile-onset Pompe disease (IOPD) is limited in some patients due to the development of high and sustained antibody titers (HSAT; ≥12,800). Methods: We carried out detailed immunophenotyping of IOPD patients (n=40), including analysis of circulating cell populations by flow cytometry and plasma cytokines by multiplex array, to determine whether patients with HSAT have unique immunological characteristics compared to those with low titers (LT; <12,800). Results: Compared to patients with LT, patients who develop HSAT were skewed toward a type 2 immune profile, with an increased frequency of Th2 cells that was positively correlated with levels of Th2 (IL-4, IL-5, IL-13) and pro-inflammatory (IL-6, TNF-α, MIP-1α, MIP-1ß) cytokines. B cells were increased in HSAT patients with a decreased fraction of unswitched memory B cells. Plasma GM-CSF concentrations were lower on average in HSAT patients, while CXCL11 was elevated. Finally, using principal components analysis, we derived an HSAT Signature Score that successfully stratified patients according to their antibody titers. Discussion: The immune profiles revealed in this study not only identify potential biomarkers of patients that developed HSAT but also provide insights into the pathophysiology of HSAT that will ultimately lead to improved immunotherapy strategies.

The role of immune tolerance induction in restoration of the efficacy of ERT in Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency in the enzyme acid α-glucosidase (GAA). Pompe disease is characterized by the accumulation of glycogen, predominantly in muscle tissue, leading to progressive muscle weakness, loss of motor, respiratory, and, in the infantile-onset form, cardiac function. Disease progression is highly variable depending on phenotype, but premature death due to respiratory complications occurs in most patients. Beginning in 2006, approved alglucosidase alfa enzyme replacement therapies [recombinant human (rh) GAA] have been available to treat Pompe patients. Treatment of classic infantile-onset patients, who manifest the severest form of the disease, with alglucosidase alfa (Myozyme®) has led to extended survival and an evolving understanding of the pathophysiology and course of the disease. Moreover, such treatment has brought to light the role of the immune response in abrogating the efficacy of rhGAA in classic infantile-onset patients with severe genetic mutations. Thus, optimization of treatment for such patients includes development and utilization of strategies to prevent or eliminate immune responses, including modulating the immune system (prophylactic and therapeutic immune tolerance induction regimens) and engineering the enzyme to be less immunogenic and more effective. Future research is also critical for evaluating and mitigating novel disease-associated pathologies uncovered by prolonged survival of infantile-onset patients including development of novel therapeutics, and for protein design strategies to increase delivery of enzyme replacement therapy to critical target tissues. Such efforts would be greatly bolstered by further development of predictive animal models and biomarkers to facilitate clinical trials and patient management. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.