Development Of Enzyme Replacement Therapy Research Articles

Mechanistic Insights into S-Depalmitolyse Activity of Cln5 Protein Linked to Neurodegeneration and Batten Disease: A QM/MM Study.

Ceroid lipofuscinosis neuronal protein 5 (Cln5) is encoded by the CLN5 gene. The genetic variants of this gene are associated with the CLN5 form of Batten disease. Recently, the first crystal structure of Cln5 was reported. Cln5 shows cysteine palmitoyl thioesterase S-depalmitoylation activity, which was explored via fluorescent emission spectroscopy utilizing the fluorescent probe DDP-5. In this work, the mechanism of the reaction between Cln5 and DDP-5 was studied computationally by applying a QM/MM methodology at the ωB97X-D/6-31G(d,p):AMBER level. The results of our study clearly demonstrate the critical role of the catalytic triad Cys280-His166-Glu183 in S-depalmitoylation activity. This is evidenced through a comparison of the pathways catalyzed by the Cys280-His166-Glu183 triad and those with only Cys280 involved. The computed reaction barriers are in agreement with the catalytic efficiency. The calculated Gibb's free-energy profile suggests that S-depalmitoylation is a rate-limiting step compared to the preceding S-palmitoylation, with barriers of 26.1 and 25.3 kcal/mol, respectively. The energetics were complemented by monitoring the fluctuations in the electron density distribution through NBO charges and bond strength alterations via local mode stretching force constants during the catalytic pathways. This comprehensive protocol led to a more holistic picture of the reaction mechanism at the atomic level. It forms the foundation for future studies on the effects of gene mutations on both the S-palmitoylation and S-depalmitoylation steps, providing valuable data for the further development of enzyme replacement therapy, which is currently the only FDA-approved therapy for childhood neurodegenerative diseases, including Batten disease.

Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal−/− mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

Advancing the Research and Development of Enzyme Replacement Therapies for Lysosomal Storage Diseases

With the increasing interest in developing gene therapies for rare diseases, it is easy to overlook that there are numerous rare lysosomal storage diseases (LSD) with treatments that have been approved by regulatory agencies in the United States and Europe. These primarily consist of enzyme replacement therapies (ERT), which are recombinant human proteins that are delivered for the life of the patient via different routes and may have distinct safety and distribution advantages over gene therapies. The research and development of ERT is a lengthy and expensive process, which is usually performed in academic laboratories before transfer to pharmaceutical companies and is hence a process ripe for disruption. There may still be considerable scientific and investment potential for ERT, however we need to develop a pipeline of proteins analogous to what has been created in some open science efforts as well as apply technologies to decrease manufacturing costs. In this Perspective, we illustrate the opportunity to fill the rare LSD treatment gap with ERTs while gene therapies are in development for these life-shortening diseases.

Enzyme Replacement Therapy for Succinic Semialdehyde Dehydrogenase Deficiency: Relevance in γ-Aminobutyric Acid Plasticity.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare inborn metabolic disorder caused by the functional impairment of SSADH (encoded by the ALDH5A1 gene), an enzyme essential for metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). In SSADHD, pathologic accumulation of GABA and its metabolite γ-hydroxybutyrate (GHB) results in broad spectrum encephalopathy including developmental delay, ataxia, seizures, and a heightened risk of sudden unexpected death in epilepsy (SUDEP). Proof-of-concept systemic SSADH restoration via enzyme replacement therapy increased survival of SSADH knockout mice, suggesting that SSADH restoration might be a viable intervention for SSADHD. However, before testing enzyme replacement therapy or gene therapy in patients, we must consider its safety and feasibility in the context of early brain development and unique SSADHD pathophysiology. Specifically, a profound use-dependent downregulation of GABAA receptors in SSADHD indicates a risk that any sudden SSADH restoration might diminish GABAergic tone and provoke seizures. In addition, the tight developmental regulation of GABA circuit plasticity might limit the age window when SSADH restoration is accomplished safely. Moreover, given SSADH expressions are cell type-specific, targeted instead of global restoration might be necessary. We therefore describe 3 key parameters for the clinical readiness of SSADH restoration: (1) rate, (2) timing, and (3) cell type specificity. Our work focuses on the construction of a novel SSADHD mouse model that allows "on-demand" SSADH restoration for the systematic investigation of these key parameters. We aim to understand the impacts of specific SSADH restoration protocols on brain physiology, accelerating bench-to-bedside development of enzyme replacement therapy or gene therapy for SSADHD patients.

Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept

Galactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and β-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and β-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms. Here, we describe a proof-of-concept, preclinical study toward the development of enzyme replacement therapy for galactosialidosis, using a recombinant human PPCA. We show that the recombinant enzyme, taken up by patient-derived fibroblasts, restored cathepsin A, neuraminidase-1, and β-galactosidase activities. Long-term, bi-weekly injection of the recombinant enzyme in a cohort of mice with null mutation at the PPCA (CTSA) locus (PPCA–/–), a faithful model of the disease, demonstrated a dose-dependent, systemic internalization of the enzyme by cells of various organs, including the brain. This resulted in restoration/normalization of the three enzyme activities, resolution of histopathology, and reduction of sialyloligosacchariduria. These positive results underscore the benefits of a PPCA-mediated enzyme replacement therapy for the treatment of galactosialidosis.

Development of enzyme replacement therapy using liver organoids derived from human iPS cells

Development of enzyme replacement therapy using liver organoids derived from human iPS cells

A lipoprotein lipase–GPI-anchored high-density lipoprotein–binding protein 1 fusion lowers triglycerides in mice: Implications for managing familial chylomicronemia syndrome

Lipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol-anchored high-density lipoprotein–binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex and its derivative, the LPL–GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3 (ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL–GPIHBP1 fusion protein exhibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL–GPIHBP1 fusion protein has potential for use as a therapeutic for managing FCS.

Skeletal mineralization: mechanisms and diseases.

Skeletal mineralization is initiated in matrix vesicles (MVs), the small extracellular vesicles derived from osteoblasts and chondrocytes. Calcium and inorganic phosphate (Pi) taken up by MVs form hydroxyapatite crystals, which propagate on collagen fibrils to mineralize the extracellular matrix. Insufficient calcium or phosphate impairs skeletal mineralization. Because active vitamin D is necessary for intestinal calcium absorption, vitamin D deficiency is a significant cause of rickets/osteomalacia. Chronic hypophosphatemia also results in rickets/osteomalacia. Excessive action of fibroblast growth factor 23 (FGF23), a key regulator of Pi metabolism, leads to renal Pi wasting and impairs vitamin D activation. X-linked hypophosphatemic rickets (XLH) is the most common form of hereditary FGF23-related hypophosphatemia, and enhanced FGF receptor (FGFR) signaling in osteocytes may be involved in the pathogenesis of this disease. Increased extracellular Pi triggers signal transduction via FGFR to regulate gene expression, implying a close relationship between Pi metabolism and FGFR. An anti-FGF23 antibody, burosumab, has recently been developed as a new treatment for XLH. In addition to various forms of rickets/osteomalacia, hypophosphatasia (HPP) is characterized by impaired skeletal mineralization. HPP is caused by inactivating mutations in tissue-nonspecific alkaline phosphatase, an enzyme rich in MVs. The recent development of enzyme replacement therapy using bone-targeting recombinant alkaline phosphatase has improved the prognosis, motor function, and quality of life in patients with HPP. This links impaired skeletal mineralization with various conditions, and unraveling its pathogenesis will lead to more precise diagnoses and effective treatments.

The role of patient advocacy organizations in shaping medical research: the Pompe model.

The Pompe model is the term used by the Pompe community to describe the relationship that exists between the patient community, the medical/scientific community, and industry. The development of the Pompe model represented a new paradigm for the involvement of patients in new treatments-and also for scientists and pharmaceutical companies. It saw patients developing a sense of agency, of involvement in the process of treatment development rather than powerless recipients or (if lucky) occasional spectators. At the same time, as described below, it benefited the other partners in the process with the result that the different components of the model added up to more than the sum of their parts. However, in order for this to happen, each part had to undergo a transformation in mindset. The development of enzyme replacement therapy (ERT) for Pompe disease represented a unique set of circumstances and individuals that helped to bring about this change and, in doing so, created a model that has had far wider applications.

Quantitative natural history characterization in a cohort of 142 published cases of patients with galactosialidosis-A cross-sectional study.

Galactosialidosis (GS; OMIM #256540) is a rare multisystemic inborn glycoprotein storage disease caused by biallelic mutations in the cathepsin A gene resulting in combined deficiency of the lysosomal enzymes β-galactosidase and α-neuraminidase. The precise understanding of the natural course of the disease is limited. Development of enzyme replacement therapy is at the preclinical stage. The purpose of this research project was to quantitatively characterize the natural history of the condition. Quantitative analysis of all published cases in the literature with sufficient data (N = 142 patients) was carried out. Main outcome variables were survival, diagnostic delay, description of symptoms, biomarker-phenotype associations, and radiological findings. STROBE criteria were respected. Median survival age of the cohort was 48 years. Median age of onset was 4.25 years with interquartile range (IQR) 1 to 16 years. Median age at diagnosis was 19 (IQR: 8.92-29) years, with median diagnostic delay of 8 (IQR: 4-12) years. Patients with residual β-galactosidase activity of more than 8.6% (leukocytes) survived significantly longer than patients with lower enzyme activities.

N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-L-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I.

Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-L-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man5 (cgl-IDUA) or Man6-8 (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K m) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases.

Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches

Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σs was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proUmod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.

A transgenic silkworm overexpressing human lysosomal enzyme as a novel resource for producing recombinant glycobiologics and its application to development of enzyme replacement therapy for lysosomal diseases

A transgenic silkworm overexpressing human lysosomal enzyme as a novel resource for producing recombinant glycobiologics and its application to development of enzyme replacement therapy for lysosomal diseases

Pathophysiology of hypophosphatasia and the potential role of asfotase alfa.

Hypophosphatasia (HPP) is an inherited systemic bone disease that is characterized by bone hypomineralization. HPP is classified into six forms according to the age of onset and severity as perinatal (lethal), perinatal benign, infantile, childhood, adult, and odontohypophosphatasia. The causative gene of the disease is the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed ubiquitously, and its physiological role is apparent in bone mineralization. A defect in bone mineralization can manifest in several ways, including rickets or osteomalacia in HPP patients. Patients with severe forms suffer from respiratory failure because of hypoplastic chest, which is the main cause of death. They sometimes present with seizures due to a defect in vitamin B6 metabolism resulting from the lack of alkaline phosphatase activity in neuronal cells, which is also lethal. Patients with a mild form of the disease exhibit rickets or osteomalacia and a functional defect of exercise. Odontohypophosphatasia shows only dental manifestations. To date, 302 mutations in the ALPL gene have been reported, mainly single-nucleotide substitutions, and the relationships between phenotype and genotype have been partially elucidated. An established treatment for HPP was not available until the recent development of enzyme replacement therapy. The first successful enzyme replacement therapy in model mice using a modified human TNAP protein (asfotase alfa) was reported in 2008, and subsequently success in patients with severe form of the disease was reported in 2012. In 2015, asfotase alfa was approved in Japan in July, followed by in the EU and Canada in August, and then by the US Food and Drug Administration in the USA in October. It is expected that therapy with asfotase alfa will drastically change treatments and prognosis of HPP.

Development of enzyme replacement therapy for Fabry disease with a modified α-N-acetylgalactosaminidase

Development of enzyme replacement therapy for Fabry disease with a modified α-N-acetylgalactosaminidase

Development of enzyme replacement therapy for Farber disease and other disorders with ceramide storage

while white matter and cerebellar volumes decreased. Mean diffusivity (MD) was increased and fractional anisotropy (FA) values were below normal in all analyzed brain regions. There was a positive correlation between motor scores of the Vineland scale and the FA values in the corticospinal tract (corr coef 0.39) and a negative correlation with the MD values (corr coef. -0.50) in the same brain region. We conclude from these initial findings that deficiency in mucolipin-1 affects the entire brain but that there might be a selective regional cerebral neurodegenerative process in MLIV. In addition, these data suggest that diffusion-weighted imaging might be a good biomarker for following patients with MLIV. Therefore, our findings may be helpful for designing of future clinical trials.

Imaging of Gaucher disease.

Gaucher disease is the prototypical lysosomal storage disease. It results from the accumulation of undegraded glucosylceramide in the reticuloendothelial system of the bone marrow, spleen and liver due to deficiency of the enzyme glucocerebrosidase. This leads to hematologic, visceral and skeletal maifestions. Build up of glucosylceramide in the liver and spleen results in hepatosplenomegaly. The normal bone marrow is replaced by the accumulating substrate leading to many of the hematologic signs including anemia. The visceral and skeletal manifestations can be visualized with various imaging modalities including radiography, computed tomography, magnetic resonance imaging (MRI) and radionuclide scanning. Prior to the development of enzyme replacement therapy, treatment was only supportive. However, once intravenous enzyme replacement therapy became available in the 1990s it quickly became the standard of care. Enzyme replacement therapy leads to improvement in all manifestations. The visceral and hematologic manifestations respond more quickly usually within a few months or years. The skeletal manifestations take much longer, usually several years, to show improvement. In recent years newer treatment strategies, such as substrate reduction therapy, have been under investigation. Imaging plays a key role in both initial diagnosis and routine monitoring of patient on treatment particularly volumetric MRI of the liver and spleen and MRI of the femora for evaluating bone marrow disease burden.

Toward the successful delivery of lysosomal enzymes across the blood–brain barrier

AbstractThe development of enzyme replacement therapy (ERT) has been one of the most remarkable successes of translational studies, bridging laboratory discoveries to clinical settings. Although ERT is very effective in clearing peripheral tissue storage in many lysosomal storage diseases (LSD), negotiating the blood‐brain barrier (BBB) remains a key challenge in delivering corrective lysosomal enzymes into the brain. In recent years, brain delivery of lysosomal enzymes has been vigorously pursued for treating neurodegenerative LSD. Delivery strategies include high‐dose and long‐term treatment regimens, manipulation of receptor‐mediated transport, biochemical engineering of enzymes, use of transport vectors, and enzyme administration by alternative routes. Most studies have investigated biochemical or histological improvements in central nervous system (CNS) storage after treatment. Few studies, however, have directly focused on the mechanisms of lysosomal enzyme transport across the BBB. In previous studies, we discovered that the luminally expressed mannose 6‐phosphate/insulin‐like growth factor‐II receptor is a universal transporter of lysosomal enzymes. We and others have also successfully manipulated receptor‐mediated transport by using endogenous characteristics of the BBB. Interestingly, accumulating evidence indicates that some lysosomal enzymes employ multiple mechanisms for their transport across the BBB. This review provides a detailed discussion of the kinetics of lysosomal enzyme transport across the BBB, and the manipulation of transport characteristics based on studies of the transport of β‐glucuronidase, sulfamidase, arylsulfatase A and acid α‐glucosidase. Furthermore, recent achievements in treating abnormalities in CNS storage and their contribution to developing new therapeutic approaches are also discussed.

Mucopolysaccharidoses (MPS)

This special edition of the Journal of Inherited Metabolic Disease brings together several publications focused on the mucopolysaccharidoses (MPS). The development of enzyme replacement therapy (ERT) for many of these disorders has led to a burgeoning interest in this field, as reflected in this group of articles. The general theme of phenotypic presentation is highlighted in the report by Hendriksz and colleagues (10.1007/s10545-011-9410-9), who present the natural history and treatment outcomes from a clinical surveillance program for MPS 6 (Maroteaux-Lamy disease). The focus then shifts to organ systems in the MPS disorders, centered primarily cardiovascular manifestations. Three reports firstly by van der Ploeg and co-workers (10.1007/s10545-011-9444-z) summarize the cardiac features of MPS I (Hurler syndrome), MPS II (Hunter syndrome) and MPS VI, and the associated outcomes with ERT. The report by Harmatz and co-workers (10.1007/s10545-012-9481-2) summarizes the long term cardiac outcomes in a cohort of MPS VI patients, while the report of Wynn and colleagues (10.1007/s10545-012-9500-3) highlights cardiac outcomes in a subgroup of MPS I patients who manifest cardiomyopathy. The latter report provides management guidelines and represents an important adjuvant for clinicians dealing with MPS patients. The critical association between the timeline of ERT prior to BMT, and its relationship to ablation therapy geared to optimize outcomes, is highlighted in this paper. Rounding out the discussion of cardiac findings is the report by Braunlin and co-workers (10.1007/s10545-011-9438-x) who overview the endothelial dysfunction of cardiac tissue in MPS patients, which may help to explain the increased risk for cardiovascular complications in these disorders. The overall theme of systems and organ involvement is continued with reports on the respiratory and central nervous system (CNS) in MPS patients. The respiratory system, poorly understood in most MPS disorders, is closely linked to airway patency in many of these disorders. Berger and colleagues (10.1007/s10545-012-9555-1) examines this topic in conjunction with a supporting article on anaesthesia management by Walker and colleagues (10.1007/s10545-012-9563-1). The latter should become a pivotal “must read” for anaesthetists involved in the management of MPS patients. Moving to the theme of CNS disorders, Cross and co-workers (10.1007/s10545-012-9572-0) evaluate the behavioural phenotypes of several MPS disorders, followed by reports from the Hendriksz (10.1007/s10545-011-9430-5 and 10.1007/s10545-012-9459-0) and Guigliani (10.1007/s10545-012-9559-x) groups that begin to describe the CNS involvement in MPS IVa and MPS VI, which is at variance with the classical concept that these disorders have no impact on the brain. For both disorders, the pathology of the cervical spine has been very topical, and Solanki and colleagues (10.1007/s10545-013-9585-3) address this issue via morphometric evaluation of the spine in MPSV IA patients followed by an overview of management guidelines for spinal manifestations (10.1007/s10545-013-9586-2). The latter paper overviews anatomy, neuroimaging, and the approach to follow-up, and may well become an important future treatment reference for MPS disorders. Moving from phenotype to genotype, Pollard and co-workers (10.1007/s10545-012-9533-7) present 104 novel mutations in a cohort of 355 MPS patients, representing a significant contribution to the molecular diagnostic armamentarium. Progressing to a discussion of diagnosis, Wood and colleagues (10.1007/s10545-013-9587-1) present an excellent overview on the diagnostic approach to MPS IVA (Morquio A syndrome) that further highlights the diagnostic pitfalls and conundrums with this disorder. Wijburg and colleagues continue the theme of diagnosis with two papers evaluating the effects of therapy on urinary glycosaminoglycans and frequently employed biomarkers for disease severity in the MPS disorders (10.1007/s10545-012-9535-5 and 10.1007/s10545-012-9538-2). Readers who seek a “taste” of basic science and animal models are not forgotten in this issue. Tomatso and colleagues (10.1007/s10545-012-9522-x) report on the micro CT evaluation of the bone dysplasia in different MPS mouse models, while Fildes and co-workers (10.1007/s10545-012-9508-8) characterize the immune system of the murine model of MPS1, with a focus on both T and dendritic cells. Auricchio and co workers (10.1007/s10545-012-9521-y) explore the potential of some MPSVI mutations as potential targets for read through therapies. The effects of disease on the family are not forgotten as well, and Hare and colleagues (10.1007/s10545-012-9558-y) assess the coping strategies of parents of Sanfilippo syndrome in comparison to parental strategies for handling other intellectual disabilities. The authors and Editorial Board are confident that this special edition will evolve into an invaluable resource for clinicians and laboratory personnel dealing with MPS patients. None.

143 Porous three-dimensional DNA crystals as biomolecular containers for catalysis

The major goal of DNA nanotechnology has been the design and manufacture of artificial DNA structures for technological uses. The porous three-dimensional DNA crystals have been proposed as macromolecular scaffolds for host–guest structure determination, as molecular sieves and as molecular containers for catalysis. By using fluorescence dequenching technique, we have demonstrated that a protein enzyme adsorbed in a designed three-dimensional DNA crystal is capable of performing catalysis. The axially distinct aperture sizes in the crystal design allowed us to improve the enclosing of the enzyme with a protective protein-based “coating” cross-linked over the crystal surface. This coating allows entry and exit of small molecules through the crystal while restricting enzymes inside the crystal. This enzyme-enclosed DNA crystal is capable of performing multiple cycles of catalysis and it retained its enzymatic activity over numerous days after being protein-coated. The concepts of the enzyme-enclosed DNA crystal and the unique protein coating technique provide possibilities to the development of enzyme replacement therapies and biodegradable solid-state catalysts and biosensors.