Enzyme replacement and enhancement therapies: lessons from lysosomal disorders

The past decade has witnessed remarkable advances in our ability to treat inherited metabolic disorders, especially the lysosomal storage diseases, a group of more than 40 disorders, each of which is caused by the deficiency of a lysosomal enzyme or protein. During the past few years, both enzyme replacement and enhancement therapies have been developed to treat these disorders. This review discusses the successes and shortcomings of these therapeutic strategies, and the contributions that they have made to treating lysosomal storage diseases.     

Storage solutions: treating lysosomal disorders of the brain

Many neurodegenerative diseases are characterized by the accumulation of undegradable molecules in cells or at extracellular sites in the brain. One such family of diseases is the lysosomal storage disorders, which result from defects in various aspects of lysosomal function. Until recently, there was little prospect of treating storage diseases involving the CNS. However, recent progress has been made in understanding these conditions and in translating the findings into experimental therapies. We review the developments in this field and discuss the similarities in pathological features between these diseases and some more common neurodegenerative disorders.     

Treatments for lysosomal storage disorders

There are over 70 human diseases that are caused by defects in various aspects of lysosomal function. Until 20 years ago, the only specific therapy available for lysosomal storage disorders was allogeneic haemopoietic stem cell transplantation. Over the last two decades, there has been remarkable progress and there are now licensed treatments for seven of these diseases. In some cases, a choice of agents is available. For selected enzyme-deficiency disordes, ERT (enzyme-replacement therapy) has proved to be highly effective. In other cases, ERT has been less impressive, and it seems that it is not possible to efficiently deliver recombinant enzyme to all tissues. These difficulties have led to the development of other small-molecule-based therapies, and a drug for SRT (substrate-reduction therapy) is now licensed and potential chaperone molecules for ERT are in the late stages of clinical development. Nonetheless, there is still significant unmet clinical need, particularly when it comes to treating LSDs which affect the brain. LSDs have led the way in the development of treatment for genetic disorders, and it seems likely that there will be further therapeutic innovations in the future.     

Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders

Glycosphingolipid (GSL) lysosomal storage disorders are a small but challenging group of human diseases to treat. Although these disorders appear to be monogenic in origin, where the catalytic activity of enzymes in GSL catabolism is impaired, the clinical presentation and severity of disease are heterogeneous. Present attitudes to treatment demand individual therapeutics designed to match the specific disease-related gene defect; this is an acceptable approach for those diseases with high frequency, but it lacks viability for extremely rare conditions. An alternative therapeutic approach termed 'substrate deprivation' or 'substrate reduction therapy' (SRT) aims to balance cellular GSL biosynthesis with the impairment in catalytic activity seen in lysosomal storage disorders. The development of N-alkylated iminosugars that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase, offers a generic therapeutic for the treatment of all glucosphingolipidoses. The successful use of N-alkylated iminosugars to establish SRT as an alternative therapeutic strategy has been demonstrated in in vitro, in vivo and in clinical trials for type 1 Gaucher disease. The implications of these studies and the prospects of improvement to the design of iminosugar compounds for treating Gaucher and other GSL lysosomal storage disorders will be discussed.     

Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2)

Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.     

Mass spectrometry in the study of lysosomal storage disorders.

Lysosomal storage disorders represent a group of over 45 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins that are involved in lysosomal biogenesis. A common biochemical feature of this group of disorders is the accumulation within lysosomes of undegraded or partially degraded substrates that are normally degraded within, and transported out of the lysosome. The particular substrates stored and the site(s) of storage vary with disease type and enzyme/protein deficiency. The nature of the substrate can be used to group the disorders into broad categories including the mucopolysaccharidoses, lipidoses, glycogenoses and oligosaccharidoses. These categories show many clinical similarities within groups as well as significant similarities between groups. For most lysosomal storage disorders the relationship between the stored substrates (type, amount and location) and the disease pathology is not well understood. The use of mass spectrometry and in particular tandem mass spectrometry provides a powerful tool for the investigation of stored substrates in this group of disorders. In this review we will describe the use of mass spectrometry for the analysis of stored substrates. We will discuss progress in the field, limitations of current methods, and summarise issues relating to the diagnosis and treatment of some of the more prevalent lysosomal storage disorders.     

Glycoprotein lysosomal storage disorders: alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency

Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan catabolism. Most of the lysosomal enzymes involved in the hydrolysis of glycoprotein carbohydrate chains are exo-glycosidases, which stepwise remove terminal monosaccharides. Thus, the deficiency of a single enzyme causes the blockage of the entire pathway and induces a storage of incompletely degraded substances inside the lysosome. Different mutations may be observed in a single disease and in all cases account for the nonexpression of lysosomal glycosidase activity. Different clinical phenotypes generally characterize a specific disorder, which rather must be described as a continuum in severity, suggesting that other biochemical or environmental factors influence the course of the disease. This review provides details on clinical features, genotype-phenotype correlations, enzymology and biochemical storage of four human glycoprotein lysosomal storage disorders, respectively alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency. Moreover, several animal disorders of glycoprotein metabolism have been found and constitute valuable models for the understanding of their human counterparts.     

Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses

The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties.     

Lysosomes and lysosomal storage diseases

Lysosomal storage disorders (LSDs) are monogenic inborn errors of metabolism. Various groups have been delineated according to the affected pathway and the accumulated substrate, and new entities are still being identified. They are severe disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement, and high morbidity and mortality. Most of the genes encoding the lysosomal enzymes have been cloned, and animal models have been obtained for almost each disease. In the last decades, LSDs have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have allowed the successful development of bone marrow transplantation, substrate deprivation, enzyme replacement therapy and gene transfer methods as therapeutic options for several LSDs. The aim of this paper is to review the biology of acid hydrolases and lysosomal membrane proteins, to describe the systematic classification of LSDs and the most recently identified entities, and to briefly review novel therapeutic approaches for two lipidoses: Gaucher disease and Fabry disease.     

Stop-codon read-through for patients affected by a lysosomal storage disorder

Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.     

Significance of immune response to enzyme-replacement therapy for patients with a lysosomal storage disorder

Lysosomal storage disorders are collectively important because they cause significant morbidity and mortality. Patients can present with severe symptoms that include somatic tissue and bone pathology, developmental delay and neurological impairment. Enzyme-replacement therapy has been developed as a treatment strategy for patients with a lysosomal storage disorder, and for many of these disorders this treatment is either in clinical trial or clinical practice. One major complication arising from enzyme infusion into patients with a lysosomal storage disorder is an immune response to the replacement protein. From clinical trials, it is clear that there is considerable variability in the level of immune response to enzyme-replacement therapy, dependent upon the replacement protein being infused and the individual patient. Hypersensitivity reactions, neutralizing antibodies to the replacement protein and altered enzyme targeting or turnover are potential concerns for patients exhibiting an immune response to enzyme-replacement therapy. The relative occurrence and significance of these issues have been appraised.     

Gene Transfer Approaches to the Lysosomal Storage Disorders

The work summarized in this paper used animal and cell culture models systems to develop gene therapy approaches for the lysosomal storage disorders. The results have provided the scientific basis for a clinical trial of gene transfer to hematopoietic stem cells (HSC) in Gaucher disease which is now in progress. The clinical experiment is providing evidence of HSC transduction, competitive engraftment of genetically corrected HSC, expression of the GC transgene, and the suggestion of a clinical response. In this paper we will review the progress made in Gaucher disease and include how gene transfer might be studied in other lysosomal storage disorders.     

New therapeutic options for lysosomal storage disorders: enzyme replacement,small molecules and gene therapy

During the last few years, much progress has been made in the treatment of lysosomal storage disorders. In the past, no specific therapy was available for the affected patients, and management consisted solely of supportive care and treatment of complications. Since enzyme replacement therapy has been successfully introduced for patients with Gaucher disease, this principle of treatment has been taken into consideration for other lysosomal storage disorders as well. Clinical trials could demonstrate the clinical benefit of this therapeutic principle in Fabry disease, mucopolysaccharidoses type I, II and VI and in Pompe disease. However, the usefulness of enzyme replacement therapy is limited due to the fact that a given enzyme preparation does not have beneficial effects on all aspects of a disorder in the same degree. Additionally, clinical studies have shown that many symptoms of a lysosomal storage disorder even after long-term treatment are no more reversible. A further novel therapeutic option for lysosomal storage disorders consists of the application of small molecules that either inhibit a key enzyme which is responsible for substrate synthesis (substrate deprivation) or act as a chaperone to increase the residual activity of the lysosomal enzyme (enzyme enhancing therapy). Various gene therapeutic techniques (in vivo and ex vivo technique) have been developed in order to administer the gene that is defective in a patient to the bloodstream or directly to the brain in order to overcome the blood–brain barrier. This review will give an insight into these newly developed therapeutic strategies and will discuss their advantages and limitations.     

Selection in favor of lysosomal storage disorders?

Four examples of Israeli communities or large families in which high consanguinity is common are presented, with two different lysosomal storage disorders within each community. In each of the four cases the stored substances share common chemical structure, despite the different lysosomal hydrolases involved in each disease. A similar phenomenon is known among the Ashkenazi Jews, in whom four of the most frequent hereditary disorders are lysosomal storage disorders, which are characterized by storage of sphingolipid derivatives. Similar findings are reported in the literature in other communities. We suggest that this phenomenon indicates a selection in favor of lysosomal storage disorders of similar nature in certain populations. The selection forces leading to this phenomenon have not been identified yet, and it has not yet been determined whether these forces are the same in the different communities presented here.     

Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment

Lysosomal storage diseases are characterized by deficiencies in lysosomal enzymes, allowing accumulation of target substrate in cells and eventually causing cell death. Enzyme replacement therapy is the principal treatment for most of these diseases. However, these therapies are often complicated by immune responses to the enzymes, blocking efficacy and causing severe adverse outcomes by neutralizing product activity. It is thus crucial to understand the relationships between genetic mutations, endogenous residual enzyme proteins (cross-reactive immunologic material), development of neutralizing antibodies and their impact on clinical outcomes of lysosomal storage diseases. For patients in whom neutralizing antibodies may cause severe adverse clinical outcomes, it is paramount to develop tolerance inducing protocols to preclude, where predictable, or treat such life-threatening responses.     

Disorders of lysosomal membrane transport--cystinosis and Salla disease

Two lysosomal storage diseases are now known to result from impaired transport of small molecules across the lysosomal membrane. In cystinosis, the disulfide amino acid, cystine, accumulates and in free sialic acid storage disorders, N-acetylneuraminic acid is stored. The lysosomal cystine carrier exhibits saturability, counter-transport, temperature dependence, and stereospecificity; it is highly specific for molecules resembling cystine. Less is known about sialic acid transport, but its temperature dependence and deficiency in certain autosomal-recessive human mutations strongly suggests that it is a carrier-mediated process. Cystine and sialic acid serve as prototypes for amino acids and sugars transported by specific lysosomal membrane carriers, whose impairment results in lysosomal storage disorders.     

Future perspectives for glycolipid research in medicine

Medical interest in glycolipids has been mainly directed to the rare and complex glycosphingolipid storage disorders that are principally caused by unitary deficiencies of lysosomal acid hydrolases. However, glycolipids are critical components of cell membranes and occur within newly described membrane domains known as lipid rafts. Glycolipids are components of important antigen systems and membrane receptors; they participate in intracellular signalling mechanisms and may be presented to the immune system in the context of the novel CD1 molecules present on T lymphocytes. A knowledge of their mechanism of action in the control of cell growth and survival as well as developmental pathways is likely to shed light on the pathogenesis of the glycosphingolipid storage disorders as well as the role of lipid second messengers in controlling cell mobility and in the mobilization of intracellular calcium stores (a biological role widely postulated particularly for the lysosphingolipid metabolite sphingosine 1-phosphate). Other sphingolipid metabolites such as ceramide 1-phosphate may be involved in apoptotic responses and in phagocytosis and synaptic vesicle formation. The extraordinary pharmaceutical success of enzymatic complementation for Gaucher's disease using macrophage-targeted human glucocerebrosidase has focused further commercial interest in other glycolipid storage diseases: the cost of targeted enzyme therapy and its failure to restore lysosomal enzymatic deficiencies in the brain has also stimulated interest in the concept of substrate reduction therapy using diffusible inhibitory molecules. Successful clinical trials of the iminosugar N-butyldeoxynojirimycin in type 1 Gaucher's disease prove the principle of substrate reduction therapy and have attracted attention to this therapeutic method. They will also foster important further experiments into the use of glycolipid synthesis inhibitors for the severe neuronopathic glycosphingolipidoses, for which no definitive treatment is otherwise available. Future glycolipid research in medicine will be directed to experiments that shed light on the role of sphingolipids in signalling pathways, and in the comprehensive characterization and their secretory products in relation to the molecular pathogenesis of the storage disorders; experiments of use to improve the efficiency of complementing enzymatic delivery to the lysosomal compartment of storage cells are also needed. Further systematic screening for inhibitory compounds with specific actions in the pathways of glycosphingolipid biosynthesis will undoubtedly lead to clinical trials in the neuronopathic storage disorders and to wider applications in the fields of immunity and cancer biology.     

Characterization of lipid-linked oligosaccharide accumulation in mouse models of Batten disease

The neuronal ceroid lipofuscinoses (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain and other tissues. A number of genes underlying various forms of NCL have been cloned, but the basis for the neurodegeneration in any of these is unknown. High levels of dolichol pyrophosphoryl oligosaccharides have previously been demonstrated in brain tissue from several NCL patients, but the specificity of the effect for the NCLs has been unclear. In the present study, we examine eight mouse models of lysosomal storage disorders by modern FACE and found striking lipid-linked oligosaccharide (LLO) accumulation in NCL mouse models (especially CLN1, CLN6, and CLN8 knockout or mutant mice) but not in several other lysosomal storage disorders affecting the brain. Using a mouse model of the most severe form of NCL (the PPT1 knockout mouse), we show that accumulated LLOs are not the result of a defect in LLO synthesis, extension, or transfer but rather are catabolic intermediates derived from LLO degradation. LLOs are enriched about 60-fold in the autofluorescent storage material purified from PPT1 knockoutmouse brain but comprise only 0.3% of the autofluorescent storage material by mass. The accumulation of LLOs is postulated to result from inhibition of late stages of lysosomal degradation of autophagosomes, which may be enriched in these metabolic precursors.     

Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases

As a novel neurotherapeutic strategy, stem cell transplantation has received considerable attention. However, little focus of this attention has been devoted to the probabilities of success of stem cell therapies for specific neurological disorders. Given the complexities of the cellular organization of the nervous system and the manner in which it is assembled during development, it seems unlikely that a cellular replacement strategy will succeed for any but the simplest of neurological disorders in the near future. A general strategy for stem cell transplantation to prevent or minimize neurological disorders is much more likely to succeed. The lysosomal storage diseases represent the quintessential neurodegenerative diseases for which preventative stem cell transplantation will both likely succeed and set the stage for therapeutic approaches to other neurodegenerative diseases.