Treatment Of Lysosomal Storage Diseases Research Articles

Drug delivery to the CNS

Effective delivery of biological products to the central nervous system remains on the frontier of modern medicine. The blood–brain barrier (BBB) is an efficient and elegant system for regulating the influx and efflux of molecular entities. The selectivity of this endothelial layer is essential for maintaining a homeostatic CNS environment and protecting neuronal tissues from foreign or harmful blood born compounds. Therapeutic interventions for CNS diseases, to be effective, must contend with traversing this barrier and reaching their targets. Although there have been substantial advances in our understanding of the molecular biology of the BBB, we have yet to translate this knowledge into consistent and reliable modalities for delivering a diverse array of therapeutic biological agents. Circumventing rather than traversing the BBB has been successfully used to deliver small molecules to the CNS for pain management, treatment of spasticity, and cancer chemotherapy. Typically, this requires the use of mechanical strategies, such as ports and pumps, to deliver drugs directly into the CSF. This approach has an inherent risk of infection, and is also limited by the physical and physiological factors governing tissue penetration and distribution. A deeper understanding of receptor-mediated transport systems and CSF dynamics is needed in order to generalize this approach to treat a broader spectrum of CNS diseases. In July 2011, the Controlled Release Society held a workshop designed to bring together leading scientists in the fields of drug delivery, vascular biology, imaging, animal model development, and clinical research (1), with the goal of engaging in multidisciplinary discussions of the strategies needed to develop drug and biological products targeted to the CNS, from drug discovery through to clinical evaluation. The presenters were invited to participate in this special issue of Drug Delivery and Translational Research to capture the wealth of information and thought provoking material that was presented in this unique setting. Patricia Dickson, the workshop facilitator, describes the current state of CNS delivery and provides some insight into the challenges associated with conducting clinical trials, particularly in rare CNS diseases. William Banks provides a synopsis of the molecular interactions related to the influx/efflux modalities of the BBB and how they could be adapted to facilitate brain delivery from systemic administration. Recent work has shown that intranasal administration is a potential access point for the BBB. Leah Hanson et al. demonstrate that intranasal administration of an iron chelator reduces spatial memory loss in a mouse model of Alzheimer’s disease, suggesting that this approach may be a viable, noninvasive approach for CNS delivery. An area of intense research in CNS delivery of large molecule biologics is in the treatment of lysosomal storage diseases (LSD), a collection of ~50 different rare genetic disorders characterized by a deficiency in the activity of one of the lysosomal enzymes. In the majority of the LSDs, uncontrolled accumulation of substrate results in progressive CNS impairment and premature death. SilviaMuro provides a review of the underlying physiology of this class of diseases, and approaches to deliver protein therapeutics across the BBB. Another approach under investigation for the treatment of LSDs is the direct administration of enzyme to the CNS. Richard Pfeifer et al. provide evidence in nonhuman primates that lumbar administration is safe, well tolerated, and likely to deliver sufficient enzyme to the relevant anatomical sites within the brain. Another major challenge in developing delivery modalities for CNS therapeutics is accurately assessing where and how much product is delivered to the targeted tissue. Immunohistochemistry, the standard method for describing biodistribution in nonclinical studies, requires a large number of animals and is not applicable in the clinical setting. Mikhail Papisov et In addition to the contributing authors, I would like to thank Anne Pariser for her significant contributions to the CNS Workshop and her thoughtful review of this article.

The Two Faces of Iminoalditols: Powerful Inhibitors Trigger Glycosidase Activation

Understanding and controlling carbohydrate processing enzymes (CPE) have been major issues and challenges for chemists, biochemists and clinical practitioners alike. One of the most powerful families of substances for probing active sites as well as allosteric interactions with CPEs are basic sugar analogues, in particular iminoalditols. This compound class presents a basic trivalent nitrogen instead of oxygen in the sugar ring as the common feature. Depending on the task, such molecules may show two faces, acting as powerful competitive inhibitors or as folding templates for the same CPE protein. When applied at sub-inhibitory concentration iminoalditols and derivatives thereof have become attractive as pharmacological chaperones for the treatment of lysosomal storage diseases. As such these structures can restore protein activity by assisting correct folding of mutant enzymes thus facilitating transportation to the lysosome and consequently substrate hydrolysis. This review surveys iminoalditol structures which have recently been investigated as potential pharmacological chaperones for the treatment of lysosomal storage diseases.

A useful guide for your practice

I was very pleased to discover a very practical and comprehensive material, containing updated information on basic and clinical research in the field of lysosomal storage diseases, a field that has witnessed an extraordinary evolution in the last 15 years. The material is structured in four major parts: ‘general aspects', ‘clinical presentations in detail', ‘mucopolysaccharidoses from the specialists' point of view' and ‘specific treatments for lysosomal storage diseases', each of them rich in practical data, reflecting the author's experience in the field. A remarkable aspect is that all the presentations concentrate on early recognition and diagnosis, specific therapies being more efficient in incipient stages. The first part introduces lysosomal storage diseases, discussing common features that suggest the presence of a storage disorder, as well as specific features that make the difference between entities. The next section (epidemiology, biochemistry and genetics) illustrates how to read and interpret biochemical and molecular tests. Suddenly, the complex field of metabolic disorders becomes very understandable after reading this material. This part also presents organizational and ethical aspects of newborn screening, as well as pathophysiological aspects of lysosomal storage disorders in a very comprehensive, yet realistic way. Even if the text is clear, I think some schemes and graphs are necessary to illustrate biochemical pathways, pathophysiology and interpretation of lab results. The next section remarkably discusses two of the most complex lysosomal storage diseases – mucopolysaccharidoses and Anderson–Fabry disease. Illustrative photographs and imagistic investigations appropriately complete the text for both of them. In my opinion, the value of this material is given by the discussion in detail of early signs and symptoms, separately for different fields involved (dysmorphology, ENT, musculoskeletal, etc). Updated data are included and the most important mechanisms and theories are discussed. The third part refers to mucopolysaccharidoses from the specialists' point of view. Once you start reading this section, you cannot put it away – you become more and more eager to discover something new about the next specialist's opinion. Classical features are mentioned, but here, the accent is put on practical issues that practitioners have to solve in their daily activity – epilepsy, psychological assessment and support, radiological findings, anesthesia, as well as neurosurgical complications and their management. The rich selection of illustrations makes the material practical and instructive. Physicians should not miss the last part referring to specific treatments for lysosomal storage diseases. In addition to the presently used enzyme replacement therapy and hematopoetic stem cell transplantation, other therapeutic approaches are discussed (eg, allogeneic stem cell transplantation and hematopoetic stem cell gene therapy). Each of the chapters included in this section is a state-of-the-art description in the field. I am convinced that readers will appreciate the therapeutic goals and the follow-up and management indications added to the classical presentation of therapy. The bibliography is remarkable – it includes hundreds of very interesting articles, some historical, most of them recent ones. In conclusion, this is an excellent book that should be available in every medical service that deals with lysosomal storage disorders (pediatric and neurological wards, medical genetics centers, and medical school libraries). The material is dense, but provides you with comprehensive and updated data in the field▪

Fluorous iminoalditols act as effective pharmacological chaperones against gene products from GLB1 alleles causing GM1‐gangliosidosis and Morquio B disease

Unlike replacement therapy by infusion of exogenous recombinant lysosomal enzymes, pharmacological chaperones aim at a gain of function of endogenous gene products. Deficits resulting from missense mutations may become treatable by small, competitive inhibitors binding to the catalytical site and thus correcting the erroneous conformation of mutant enzymes. This may prevent their premature degradation and normalize intracellular trafficking as well as biological half-life. A major limitation currently arises from the huge number of individual missense mutations and the lack of knowledge on the structural requirements for specific interaction with mutant protein domains. Our previous work on mutations of the β-galactosidase (β-gal) gene, causing GM1 gangliosidosis (GM1) and Morquio B disease (MBD), respectively, characterized clinical phenotypes as well as biosynthesis, intracellular transport and subcellular localization of mutants. We recently identified an effective chaperone, DL-HexDGJ (Methyl 6-{[N(2)-(dansyl)-N(6)-(1,5-dideoxy-D-galactitol-1,5-diyl)- L-lysyl]amino} hexanoate), among a series of N-modified 1-deoxygalactonojirimycin derivatives carrying a dansyl group in its N-acyl moiety. Using novel and flexible synthetic routes, we now report on the effects of two oligofluoroalkyl-derivatives of 1-deoxygalactonojirimycin, Ph(TFM)(2)OHex-DGJ (N-(α,α-di-trifluoromethyl) benzyloxyhexyl-1,5-dideoxy-1,5-imino-D: -galactitol) and (TFM)(3)OHex-DGJ (N-(Nonafluoro-tert-butyloxy)hexyl-1,5-dideoxy-1,5-imino-D: -galactitol) on the β-gal activity of GM1 and MBD fibroblasts. Both compounds are competitive inhibitors and increase the residual enzyme activities up to tenfold over base line activity in GM1 fibroblasts with chaperone-sensitive mutations. Western blots showed that this was due to a normalization of protein transport and intralysosomal maturation. The fact that the novel compounds were effective at very low concentrations (0.5-10 μM) in the cell culture medium as well as their novel chemical character suggest future testing in animal models. This may contribute to new aspects for efficient and personalized small molecule treatment of lysosomal storage diseases.

PH-Responsive Polysaccharide-Based Polyelectrolyte Complexes As Nanocarriers for Lysosomal Delivery of Therapeutic Proteins

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ∼ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.

Intracerebroventricular enzyme infusion corrects central nervous system pathology and dysfunction in a mouse model of metachromatic leukodystrophy

Arylsulfatase A (ASA) catalyzes the desulfation of sulfatide, a major lipid component of myelin. Inherited functional deficiencies of ASA cause the lysosomal storage disease (LSD) metachromatic leukodystrophy (MLD), which is characterized by intralysosomal accumulation of sulfatide, progressive neurological symptoms and early death. Enzyme replacement therapy (ERT) using intravenous injection of active enzyme is a treatment option for many LSDs as exogenous lysosomal enzymes are delivered to lysosomes of patient's cells via receptor-mediated endocytosis. Efficient treatment of MLD and other LSDs with central nervous system (CNS) involvement is, however, hampered by the blood-brain barrier (BBB), which limits transfer of therapeutic enzymes from the circulation to the brain parenchyma. To bypass the BBB, we infused recombinant human ASA (rhASA) by implanted miniature pumps into the cerebrospinal fluid (CSF) of a conventional and a novel, genetically aggravated ASA knockout mouse model of MLD. rhASA continuously delivered to the lateral ventricle for 4 weeks penetrated the brain parenchyma and was targeted to the lysosomes of brain cells. Histological analysis revealed complete reversal of lysosomal storage in the infused hemisphere. rhASA concentrations and sulfatide clearance declined with increasing distance from the infusion site. Correction of the ataxic gait indicated reversal of central nervous system dysfunctions. The profound histopathological and functional improvements, the requirement of low enzyme doses and the absence of immunological side effects suggest intracerebroventricular ERT to be a promising treatment option for MLD and other LSDs with prevailing CNS disease.

Minicircle DNA-based Gene Therapy Coupled With Immune Modulation Permits Long-term Expression of α-L-Iduronidase in Mice With Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease characterized by mutations to the α-L-iduronidase (IDUA) gene resulting in inactivation of the IDUA enzyme. The loss of IDUA protein results in the progressive accumulation of glycosaminoglycans within the lysosomes resulting in severe, multi-organ system pathology. Gene replacement strategies have relied on the use of viral or nonviral gene delivery systems. Drawbacks to these include laborious production procedures, poor efficacy due to plasmid-borne gene silencing, and the risk of insertional mutagenesis. This report demonstrates the efficacy of a nonintegrating, minicircle (MC) DNA vector that is resistant to epigenetic gene silencing in vivo. To achieve sustained expression of the immunogenic IDUA protein we investigated the use of a tissue-specific promoter in conjunction with microRNA target sequences. The inclusion of microRNA target sequences resulted in a slight improvement in long-term expression compared to their absence. However, immune modulation by costimulatory blockade was required and permitted for IDUA expression in MPS I mice that resulted in the biochemical correction of pathology in all of the organs analyzed. MC gene delivery combined with costimulatory pathway blockade maximizes safety, efficacy, and sustained gene expression and is a new approach in the treatment of lysosomal storage disease.

Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders

Most lysosomal storage diseases (LSDs) are life-threatening genetic diseases. The pathogenesis of these diseases is poorly understood. Induced pluripotent stem (iPS) cell technology offers new opportunities for both mechanistic studies and development of stem cell- based therapies. Here we report the generation of disease-specific iPS cells from mouse models of Fabry disease, globoid cell leukodystrophy (GLD), and mucopolysaccharidosis VII (MPSVII). These mouse model-derived iPS cells showed defects in disease-specific enzyme activities and significant accumulation of substrates for these enzymes. In the lineage-directed differentiation studies, Fabry-iPS and GLD-iPS cells were efficiently differentiated into disease-relevant cell types, such as cardiomyocytes and neural stem cells, which might be useful in mechanistic and therapeutic studies. Notably, MPSVII-iPS cells demonstrated a markedly impaired ability to form embryoid bodies (EBs) in vitro. MPSVII-EBs exibited elevated levels of hyaluronan and its receptor CD44, and markedly reduced expression levels of E-cadherin and cell-proliferating marker. Partial correction of enzyme deficiency in MSPVII-iPS cells led to improved EB formation and reversal of aberrant protein expression. These data indicate a potential mechanism for the partial lethality of MPSVII mice in utero, and suggest a possible abnormality of embryonic development in MPSVII patients. Thus, our study demonstrates the unique promise of iPS cells for studying the pathogenesis and treatment of LSDs.

Stem cell transplantation for neurometabolic and neurodegenerative diseases

Over the last decade, the potential for therapeutic use of stem cell transplantation for cell replacement or as cellular vectors for gene delivery for neurometabolic and neurodegenerative diseases has received a great deal of interest. There has been substantial progress in our understanding of stem cell biology. Potential applications of cell-mediated therapy include direct cell replacement or protection and repair of the host nervous system. Given the complexities of the cellular organization of the nervous system, especially in diseased states, it seems that using stem cells as cellular vectors to prevent or ameliorate neurological disorders rather than cell replacement and the regrowth of damaged circuitry is more likely to succeed in the near term. Recent success in the treatment of lysosomal storage diseases with genetically modified stem cells support this notion. In Alzheimer's and Parkinson's diseases, stem cell therapy is at its early stages and data generated in animal models and clinical trials using other cell types suggest that a combination of gene and stem cell therapy may be an optimal therapeutic paradigm.

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Current Enzyme Replacement Therapy for the Treatment of Lysosomal Storage Diseases

&lt;P&gt;Lysosomal storage diseases (LSDs) are a group of more than 40 different inherited metabolic diseases, which arise from a gene defect or mutation that impairs proper functioning of a particular lysosomal enzyme or its transport protein. Clinical presentation is very variable among the diverse group of LSDs. A defect in one lysosomal enzyme can have a greater affect on one particular tissue, for example, skeletal muscle, whereas another may have its main effect on the central nervous system. At this time there is no cure for any LSD, and in the past only symptomatic treatment was available. However, throughout the past several decades, remarkable progress has been made in developing more effective treatments such as hematopoietic stem cell transplantation (HCT) and enzyme replacement therapy (ERT). These therapies have led to significant improvement in quality of life as well as reduction in clinical symptoms for some of the LSDs. A child’s general pediatrician, often following him/her from birth, may be the first to note clinical signs and symptoms of a LSD, leading to early diagnosis and treatment. What follows is a summary of several LSD, along with an update on available and upcoming treatment modalities.&lt;/P&gt; &lt;H4&gt;ABOUT THE AUTHORS&lt;/H4&gt; &lt;P&gt;Elizabeth R. Lim-Melia, MD, is Assistant Director, Regional Medical Genetics Center, and Instructor of Pediatrics, N.Y. Medical College, Valhalla, New York. David F. Kronn, MD, is Associate Professor of Pediatrics, N.Y. Medical College.&lt;/P&gt; &lt;P&gt;Address correspondence to: Elizabeth R. Lim-Melia, MD, 503 Grasslands Road, Valhalla, NY 10595; fax 914-345-1753; or e-mail &lt;A HREF="Elizabeth_LimMelia@nymc.edu" TARGET="_new"&gt;Elizabeth_LimMelia@nymc.edu&lt;/A&gt;.&lt;/P&gt; &lt;P&gt;Dr. Lim-Melia and Dr. Kronn have disclosed no relevant financial relationships.&lt;/P&gt; &lt;P&gt;doi: 10.32800904481-20090723-09 &lt;/P&gt;

Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics

Lysosomal storage diseases (LSDs) are a group of genetic disorders due to defects in any aspect of lysosomal biology. During the past two decades, different approaches have been introduced for the treatment of these conditions. Among them, enzyme replacement therapy (ERT) represented a major advance and is used successfully in the treatment of some of these disorders. However, ERT has limitations such as insufficient biodistribution of recombinant enzymes and high costs. An emerging strategy for the treatment of LSDs is pharmacological chaperone therapy (PCT), based on the use of chaperone molecules that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking. After proof-of-concept studies, PCT is now being translated into clinical applications for Fabry, Gaucher and Pompe disease. This approach, however, can only be applied to patients carrying chaperone-responsive mutations. The recent demonstration of a synergistic effect of chaperones and ERT expands the applications of PCT and prompts a re-evaluation of their therapeutic use and potential. This review discusses the strengths and drawbacks of the potential therapies available for LSDs and proposes that future research should be directed towards the development of treatment protocols based on the combination of different therapies to improve the clinical outcome of LSD patients.

The Pharmacological Chaperone N-butyldeoxynojirimycin Enhances Enzyme Replacement Therapy in Pompe Disease Fibroblasts

In spite of the progress in the treatment of lysosomal storage diseases (LSDs), in some of these disorders the available therapies show limited efficacy and a need exists to identify novel therapeutic strategies. We studied the combination of enzyme replacement and enzyme enhancement by pharmacological chaperones in Pompe disease (PD), a metabolic myopathy caused by the deficiency of the lysosomal acid α-glucosidase. We showed that coincubation of Pompe fibroblasts with recombinant human α-glucosidase and the chaperone N-butyldeoxynojirimycin (NB-DNJ) resulted in more efficient correction of enzyme activity. The chaperone improved α-glucosidase delivery to lysosomes, enhanced enzyme maturation, and increased enzyme stability. Improved enzyme correction was also found in vivo in a mouse model of PD treated with coadministration of single infusions of recombinant human α-glucosidase and oral NB-DNJ. The enhancing effect of chaperones on recombinant enzymes was also observed in fibroblasts from another lysosomal disease, Fabry disease, treated with recombinant α-galactosidase A and the specific chaperone 1-deoxygalactonojirimycin (DGJ). These results have important clinical implications, as they demonstrate synergy between pharmacological chaperones and enzyme replacement. A synergistic effect of these treatments may result particularly useful in patients responding poorly to therapy and in tissues in which sufficient enzyme levels are difficult to obtain.

A new surrogate marker for CNS pathology in Niemann–Pick disease type C?

Over the past 20 years there has been unprecedented progressin the development of treatments for lysosomal storage diseases(LSDs) [1]. Enzyme replacement therapy (ERT) continues to expandwith clinical programmes in Niemann–Pick type B, MPS IV andmetachromatic leucodystrophy. One small molecule drug (miglu-stat) is approved for type 1 Gaucher disease and other substratereduction therapy (SRT) and chaperone approaches are in the pipe-line [2]. Indeed, it is possible that in the future physicians treatingGaucher disease may be able to choose from three different en-zyme preparations, two inhibitors of glucosyl ceramide synthesisand a chaperone.However, these treatments are not all equally efficacious. Onemajor obstacle is that there is no means of delivering significantamounts of enzyme across the blood–brain barrier. This meansthat whilst ERT canbe highlyeffective against the visceralmanifes-tations of LSDs, patients in whom CNS involvement is the majorfeature do not benefit. For the majority of LSDs, which involve stor-age and pathology in the brain, ERT is not currently a realisticoption.Considerable research activity is therefore currently focused ondeveloping strategies to target brain disease in these disorders,either using small molecules, gene therapy or methods to deliverenzymes across the blood–brain barrier.One major obstacle in designingclinical trialsis the definitionofsuitable clinical endpoints. Regulatory authorities demand con-trolled trials, which demonstrate statistically significant changesin quantifiable variables. Pharmaceutical companies want trialsto be completed in months rather than years. Unfortunately, theseare rare, neurodegenerative diseases, which show variable andunpredictable progression over long periods. We do not knowwhich aspects of CNS pathology may be stabilised or improvedby treatment and it is very difficult to decide which clinical mani-festations should be measured or to know how long it may take tosee a significant change.The situation would be greatly improved if there were validatedsurrogate markers of brain disease that could be monitored as sec-ondaryclinical endpoints. These wouldpotentiallyprovidean earlyindicator of efficacy, prior to significant changes in clinicalsymptoms.The utility of surrogate markers in the storage disease field isbest exemplified by type 1 Gaucher disease [3]. This is a visceralstorage disorder with the Gaucher macrophage being the centralcell type in the pathogenic cascade [4]. These cells secrete a num-ber of factors that can be measured in peripheral blood and used assurrogate markers to estimate the total storage cell burden in un-treated patients and measure the degree of improvement followingthe initiation of ERT or SRT [5].However, currentlythere are no equivalent markersfor measur-ing storage in the brain. The Holy Grail in this field would be toidentify a non-invasive method that could be performed in mosthospitals where the parameter being measured was closely linkedto the primary pathological events occurring in the brain. It wouldneed to correlate with disease severity and be altered by diseasemodifying therapies.Miglustat, a small molecule inhibitor of glucosylceramide syn-thesis, does cross the blood–brain barrier and has shown benefitwhen used for SRT in a number of mouse models of CNS glyco-sphingolipid storage diseases [6], including Niemann–Pick type C

Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots: Application to Screening Newborns for Mucopolysaccharidosis I

Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, alpha-L-iduronidase. We synthesized a new alpha-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liquid-liquid extraction step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concentration of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision. When the assay was tested on dried blood spots, the alpha-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the alpha-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands. This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this laboratory and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.

Substrate reduction therapy

The therapeutic options for lysosomal storage diseases (LSDs) have expanded greatly over the past decade, although for many disorders there is still no effective treatment. Given that the majority of LSDs involve pathological changes in both the brain and peripheral tissues, effective treatment of central nervous system (CNS) and peripheral manifestations still remains a considerable technical challenge. Type 1 Gaucher disease has two approved treatment modalities - enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) - which have unique, independent and potentially complementary mechanisms of action. The availability of these two therapies has greatly increased the options for the effective clinical management of type 1 Gaucher disease. ERT involves the intravenous administration of fully functional enzyme that is taken up by cells and delivered to the lysosome, where it can compensate for the underlying enzyme deficiency. SRT uses an orally available, small molecule drug that inhibits the first committed step in glycosphingolipid biosynthesis. The aim is to reduce the rate of biosynthesis of glycosphingolipids to offset the catabolic defect, restoring the balance between the rate of biosynthesis and the rate of catabolism. SRT also has the potential to treat LSDs with CNS pathology, as the drug in clinical use (miglustat, Zavesca; Actelion Pharmaceuticals Ltd, Allschwil, Switzerland) crosses the blood-brain barrier. In this review, the current status of SRT for the treatment of Gaucher disease and other LSDs will be discussed, based upon preclinical and clinical studies. SRT is an oral alternative treatment option for patients with type 1 Gaucher disease unwilling or unable to receive ERT. With the recent reports of clinical improvement/stabilization of CNS manifestations following SRT in patients with Niemann-Pick disease type C, miglustat may also have a role to play in the management of patients with glycosphingolipid storage in the brain. Furthermore, as SRT synergises with other therapeutic modalities, it may also prove to be a key component of combination therapies in the future.

An Overview of Enzyme Replacement Therapy for Lysosomal Storage Diseases

The lysosomal storage diseases (LSDs) are a group of disorders heralding in a new era in the treatment of genetic diseases. Enzyme replacement therapy (ERT) moves the treatment of these disorders from symptomatic management to therapeutic interventions. ERT is not a cure for these disorders, but it can greatly modify or attenuate the phenotype. Treatment for LSDs is lifelong and the diseases affect multiple organ systems. It is possible that nurses in almost every specialty will encounter a patient with one of these conditions. Therefore knowledge of the conditions, the benefits and limitations of ERT, and its effective management are becoming more important for all nurses. This article will describe several LSDs, namely Gaucher disease, Fabry disease, Pompe disease, and the mucopolysaccharidoses. Each disease pathology, signs and symptoms, and effectiveness of ERT treatment will be discussed, as well as the administration of ERT, common side effects, management of the side effects, and nursing implications. Additionally drug costs and insurance concerns will be highlighted.

Enzyme reconstitution/replacement therapy for lysosomal storage diseases

Over the past 15 years, the lysosomal storage diseases have become paradigms for the specific treatment of monogenic disorders, particularly those affecting children. This review summarizes the phenotypes and recent literature regarding enzyme reconstitution (replacement) therapy and outcomes for such treatable lysosomal storage diseases: Gaucher disease, Fabry disease, Pompe disease and the mucopolysaccharidoses. Recent clinical trials have shown that enzyme reconstitution therapy effectively treats many of the manifestations of the lysosomal storage diseases. When initiated early in the disease course, enzyme reconstitution therapy can reverse some disease manifestations, but may not completely alleviate the disease progression. Enzyme reconstitution therapy is generally well tolerated. Many adverse events are antibody-related, but can be managed without requiring cessation of enzyme reconstitution therapy. Documented IgE reactions, i.e. anaphylactoid, are quite rare (fewer than 1%). Enzyme reconstitution therapy is a safe and effective treatment modality available for several of the lysosomal storage diseases. Owing to the short history of enzyme reconstitution therapy, the long-term outcomes of enzyme reconstitution therapy-treated individuals are unknown and require further investigation. Medical professionals must learn to identify patients likely to benefit from these life-changing therapies so as to prevent many of the devastating, irreversible complications of the lysosomal storage diseases.

Complete Correction of Enzymatic Deficiency and Neurochemistry in the GM1-gangliosidosis Mouse Brain by Neonatal Adeno-associated Virus–mediated Gene Delivery

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal), and characterized by accumulation of GM1-ganglioside and GA1 in the brain. Here we examined the effect of neonatal intracerebroventricular (i.c.v.) injection of an adeno-associated virus (AAV) vector encoding mouse betagal on enzyme activity and brain GSL content in GM1-gangliosidosis (betagal(-/-)) mice. Histological analysis of betagal distribution in 3-month-old AAV-treated betagal(-/-) mice showed that enzyme was present at high levels throughout the brain. Biochemical quantification showed that betagal activity in AAV-treated brains was 7- to 65-fold higher than in wild-type controls and that brain GSL levels were normalized. Cerebrosides and sulfatides, which were reduced in untreated betagal(-/-) mice, were restored to normal levels by AAV treatment. In untreated betagal(-/-) brains, cholesterol was present at normal levels but showed abnormal cellular distribution consistent with endosomal/lysosomal localization. This feature was also corrected in AAV-treated mice. The biochemical and histological parameters analyzed in this study showed that normal brain neurochemistry was achieved in AAV-treated betagal(-/-) mice. Therefore we show for the first time that neonatal AAV-mediated gene delivery of lysosomal betagal to the brain may be an effective approach for treatment of GM1-gangliosidosis.

Treatment of Lysosomal Storage Disorders

Enzyme replacement therapy (ERT) as treatment for lysosomal storage diseases (LSDs) was suggested as long ago as 1966 by De Duve and Wattiaux. However, it took >35 years to demonstrate the safety and effectiveness of ERT for type 1 Gaucher's disease. An important breakthrough was certainly the enactment of legislation in the US, designed to encourage commercialisation of products developed in academic institutions for pharmaceutical companies to invest in treatments for rare diseases. The principles elaborated in the development of the treatment of Gaucher's disease were subsequently applied to the development of ERT of other LSDs. The safety and effectiveness of ERT for Fabry's disease, mucopolysaccharidoses (MPS) I, MPS II and MPS VI, as well as for Pompe's disease have been demonstrated in well designed clinical trials, and the treatments are now commercially available throughout the world. Several questions remain to be answered. The long-term effectiveness of most of the treatments has not yet been established. What is reversible by ERT and what may not be reversible but is preventable, is not yet clear. The pathology in some tissues, such as the brain, is inaccessible to ERT, indicating that some manifestations of the LSD will not respond to the treatment. The extent of this problem is still unclear. The cost of ERT is very high, creating problems for third-party payers, which has strained reimbursement schemes based on the demonstration of acceptable cost effectiveness. ERT of LSDs represents the most important advance in the treatment of this class of diseases. The information that is currently being collected as part of large-scale observational studies will help to establish the full potential of the treatment.