Gene-Based Approaches to Inherited Neurometabolic Diseases.

In the last decade, the gene therapy (GT) field experienced a renaissance, thanks to crucial understandings and innovations in vector design, stem cell manipulation, conditioning protocols, and cell/vector delivery. These efforts were successfully coupled with unprecedented clinical results of the trials employing the newly developed technology and with the novel establishment of academic-industrial partnerships. A renewed and strengthened interest is rising in the development of gene-based approaches for inherited neurometabolic disorders with severe neurological involvement. Inherited metabolic disorders are monogenetic diseases caused by enzymatic or structural deficiencies affecting the lysosomal or peroxisomal metabolic activity. The metabolic defect can primarily affect the central nervous system, leading to neuronal death, microglial activation, inflammatory demyelination, and axonal degeneration. This review provides an overview of the GT strategies currently under clinical investigation for neurometabolic lysosomal and peroxisomal storage diseases, such as adrenoleukodystrophy and metachromatic leukodystrophy, as well as novel emerging indications such as mucopolysaccharidoses, gangliosidoses, and neuronal ceroid lipofuscinoses, with a comprehensive elucidation of the main features and mechanisms at the basis of a successful GT approach for these devastating diseases.

Infantile gangliosidoses: Mapping a timeline of clinical changes.

BACKGROUND: Infantile gangliosidoses include GM1 gangliosidosis and GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease). To date, natural history studies in infantile GM2 (iGM2) have been retrospective and conducted through surveys. Compared to iGM2, there is even less natural history information available on infantile GM1 disease (iGM1). There are no approved treatments for infantile gangliosidoses. Substrate reduction therapy using miglustat has been tried, but is limited by gastrointestinal side effects. Development of effective treatments will require identification of meaningful outcomes in the setting of rapidly progressive and fatal diseases. OBJECTIVES: This study aimed to establish a timeline of clinical changes occurring in infantile gangliosidoses, prospectively, to: 1) characterize the natural history of these diseases; 2) improve planning of clinical care; and 3) identify meaningful future treatment outcome measures. METHODS: Patients were evaluated prospectively through ongoing clinical care. RESULTS: Twenty-three patients were evaluated: 8 infantile GM1, 9 infantile Tay-Sachs disease, 6 infantile Sandhoff disease. Common patterns of clinical change included: hypotonia before 6months of age; severe motor skill impairment within first year of life; seizures; dysphagia and feeding-tube placement before 18months of age. Neurodevelopmental testing scores reached the floor of the testing scale by 20 to 28months of age. Vertebral beaking, kyphosis, and scoliosis were unique to patients with infantile GM1. Chest physiotherapy was associated with increased survival in iGM1 (p=0.0056). Miglustat combined with a low-carbohydrate ketogenic diet (the Syner-G regimen) in patients who received a feeding-tube was associated with increased survival in infantile GM1 (p=0.025). CONCLUSIONS: This is the first prospective study of the natural history of infantile gangliosidoses and the very first natural history of infantile GM1. The homogeneity of the infantile gangliosidoses phenotype as demonstrated by the clinical events timeline in this study provides promising secondary outcome measure candidates. This study indicates that overall survival is a meaningful primary outcome measure for future clinical trials due to reliable timing and early occurrence of this event. Combination therapy approaches, instead of monotherapy approaches, will likely be the best way to optimize clinical outcomes. Combination therapy approaches include palliative therapies (e.g., chest physiotherapy) along with treatments that address the underlying disease pathology (e.g. miglustat or future gene therapies).

Gangliosides and gangliosidoses: principles of molecular and metabolic pathogenesis.

Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes, involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular trafficking. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusively in mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoses have long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes, they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomal catabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respective hydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects of ganglioside hydrolases, e.g., of ß-galactosidase and ß-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis, dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with small residual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology. Apart from patients' differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificities and functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism by membrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mouse models and patients with juvenile forms of gangliosidoses are discussed.

The beta-hexosaminidase deficiency disorders: development of a clinical paradigm in the mouse.

Tay-Sachs disease and Sandhoff disease are severe neurodegenerative disorders caused by a deficiency of beta-hexosaminidase A and resultant accumulation of its substrate, GM2 ganglioside, in neuronal lysosomes. The three clinical forms of the disorders (infantile, juvenile and adult) are of varying severity and onset, and have been correlated with the amount of residual GM2 ganglioside-degrading activity present in patients' cells. Through targeted disruption of the murine beta-hexosaminidase genes in embryonic stem cells, we have developed a set of mice that vary in their GM2 ganglioside-degrading capacity and exhibit many of the clinical features of the human diseases. These mice are valuable for the study of pathogenic mechanisms and for devising novel therapeutic strategies in these disorders.

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.

Modified beta-D-N-acetylhexosaminidase isozymes for enzyme replacement in GM2 gangliosidosis.

The therapeutic potential of enzyme replacement in lysosomal storage disorders has remained largely unfulfilled, perhaps because of negative reactions to the initial disappointing results. Despite the existence of several animal models that can be utilized to explore solutions to the problems of exogenous enzyme targeting, the interest in ERT prevalent during the 1970's seems to have subsided to be replaced by active interest in bone marrow transplantation (BMT, Krivit and Paul [1986]). This is a logical approach to enzyme replacement in storage disorders of the RE system, and indeed some encouraging results have been obtained. However, in addition to having high morbidity and mortality, in the ultimate analysis BMT presents the same targeting problems as conventional ERT. In our opinion, these problems can be solved more easily in the case of ERT by exploiting the existing cellular uptake mechanisms and infusing enzymes whose structure has been suitably modified by simple biochemical manipulations. Accordingly, we have explored a methodology that takes advantage of negative charges on the cell surface to obtain nonspecific but effective membrane binding of beta-hex coupled to the highly positively charged PLL, followed by internalization and routing to the lysosomes. This system increases uptake of exogenous enzyme by some neurons in vitro and possibly in vivo, but its efficiency depends on the cells' endocytic activity that, in the case of neuronal soma, apparently is low. Thus, we have chosen as recognition marker for specific neuronal uptake a nontoxic fragment of TTx that is efficiently taken up by these cells. The initial results are encouraging; they support our contention that effective enzyme replacement methodologies can be devised, and encourage us to continue our work in this direction. Finally, recombinant DNA techniques are now being applied to a number of LSD, and the genes for several of the pertinent enzymes have been or are being isolated. In addition to representing a first step towards gene replacement therapy, the results of this work will permit the generation of large amounts of human enzymes from bacteria by recombinant DNA methods, thus obviating the problem of enzyme supply for ERT. Since human lysosomal enzymes obtained from bacteria will be nonglycosylated, to obtain cell uptake it will be necessary to resort to the type of modifications that we are trying to develop at this time, i.e., covalent linkage to moieties that allow non-glycosyl-mediated cellular uptake. Thus, our work on beta-hex may provide a model for biochemical manipulations of bacterially produced enzymes applicable to several LSD.

A therapeutic trial of amniotic epithelial cell implantation in patients with lysosomal storage diseases.

To determine whether allografts of normal amniotic epithelium might provide a nonimmunogenic cellular source of exogenous lysosomal enzymes, subcutaneous implants of amniotic epithelium were performed in six children with clinically advanced storage diseases. The clinical and the biochemical status of each patient was observed for several weeks after amniotic epithelial cell implantation (AECI). Serial studies of blood samples from each patient in the post-AECI period did not demonstrate any increase in levels of deficient lysosomal hydrolase. In two patients, quantitative urinary excretion of substrate was also studied and did not show consistent alterations after AECI. No patient had objective improvement in clinical or neurodevelopmental status following AECI. Two patients died with progressive disease at 2 1/2 and 3 1/2 mo after AECI; no residual amniotic epithelium was found at postmortem examination. Four patients are alive with progressive disease at 6-14 mo after AECI. We conclude that allografts of normal human amnion do not provide sufficient replacement hydrolases for clinical or biochemical improvement in lysosomal storage diseases.

Amniotic tissue transplantation as a trial of treatment in some lysosomal storage diseases.

This communication reports the clinical and biochemical results in six patients: four with mucopolysaccharidosis, one with GM1 gangliosidosis (Morquio B) and one with I-cell disease, who were treated by amniotic tissue transplantation. The sole evident clinical result was the diminishing of corneal clouding in three cases. A slight increase of beta-galactosidase activity in one patient's plasma was observed. The time of improvement was about 2 months after the transplantation and was transitory.