Evolutionary redesign of the lysosomal enzyme arylsulfatase A increases efficacy of enzyme replacement therapy for metachromatic leukodystrophy.

Protein engineering is a means to optimize protein therapeutics developed for the treatment of so far incurable diseases including cancers and genetic disorders. Here we report on an engineering approach in which we successfully increased the catalytic rate constant of an enzyme that is presently evaluated in enzyme replacement therapies (ERT) of a lysosomal storage disease (LSD). Although ERT is a treatment option for many LSDs, outcomes are lagging far behind expectations for most of them. This has been ascribed to insufficient enzyme activities accumulating in tissues difficult to target such as brain and peripheral nerves. We show for human arylsulfatase A (hARSA) that the activity of a therapeutic enzyme can be substantially increased by reversing activity-diminishing and by inserting activity-promoting amino acid substitutions that had occurred in the evolution of hominids and non-human mammals, respectively. The potential of this approach, here designated as evolutionary redesign, was highlighted by the observation that murinization of only 1 or 3 amino acid positions increased the hARSA activity 3- and 5-fold, with little impact on stability, respectively. The two kinetically optimized hARSA variants showed no immunogenic potential in ERT of a humanized ARSA knockout mouse model of metachromatic leukodystrophy (MLD) and reduced lysosomal storage of kidney, peripheral and central nervous system up to 3-fold more efficiently than wild-type hARSA. Due to their safety profile and higher therapeutic potential the engineered hARSA variants might represent major advances for future enzyme-based therapies of MLD and stimulate analogous approaches for other enzyme therapeutics.

Arylsulfatase A bound to poly(butyl cyanoacrylate) nanoparticles for enzyme replacement therapy--physicochemical evaluation.

Arylsulfatase A (ASA) deficiency is the cause of metachromatic leucodystrophy (MLD), a lysosomal storage disease associated with severe neurological disorders. Poly(butyl cyanoacrylate) (PBCA) nanoparticles overcoated with polysorbate 80 enabled the delivery of several drugs across the blood-brain barrier to the brain suggesting that these nanoparticles also may transport ASA across this barrier. The objective of this research, therefore, was to evaluate the feasibility of loading ASA onto PBCA nanoparticles. A stable ASA-loaded PBCA nanoparticle formulation was developed that could be easily freeze-dried and stored over a period of more than 8 weeks. The maximum loading capacity for this enzyme was -59 microg per 1 mg of PBCA. In the presence of 3% sucrose as a lyoprotector the activity of freeze-dried ASA was found to be 100% recoverable.

Long-term correction of biochemical and neurological abnormalities in MLD mice model by neonatal systemic injection of an AAV serotype 9 vector.

As both the immune system and the blood-brain barrier (BBB) are likely to be developmentally immature in the perinatal period, neonatal gene transfer may be useful for the treatment of lysosomal storage disease (LSD) with neurological involvements such as metachromatic leukodystrophy (MLD). In this experiment, we examined the feasibility of single-strand adeno-associated viral serotype-9 (ssAAV9)-mediated systemic neonatal gene therapy of MLD mice. ssAAV9 vector expressing human arylsulfatase A (ASA) and green fluorescent protein (GFP) (ssAAV9/ASA) was injected into the jugular vein of newborn MLD mice. High levels of ASA expression were observed in the muscle and heart for at least 15 months. ASA was continuously secreted into plasma without development of antibodies against ASA. Global gene transfer into the brain and spinal cord (SC), across the BBB, and long-term ASA expression in the central nervous system were detected in treated mice. Significant inhibition of the accumulation of sulfatide (Sulf) in the brain and cervical SC was confirmed by Alcian blue staining and biochemical analysis of the Sulf content. In a behavior test, treated mice showed a greater ability to traverse narrow balance beams than untreated mice. These data clearly demonstrate that MLD mice model can be effectively treated through neonatal systemic injection of ssAAV9/ASA.

Transport of arylsulfatase A across the blood-brain barrier in vitro.

Enzyme replacement therapy is an option to treat lysosomal storage diseases caused by functional deficiencies of lysosomal hydrolases as intravenous injection of therapeutic enzymes can correct the catabolic defect within many organ systems. However, beneficial effects on central nervous system manifestations are very limited because the blood-brain barrier (BBB) prevents the transfer of enzyme from the circulation to the brain parenchyma. Preclinical studies in mouse models of metachromatic leukodystrophy, however, showed that arylsulfatase A (ASA) is able to cross the BBB to some extent, thus reducing lysosomal storage in brain microglial cells. The present study aims to investigate the routing of ASA across the BBB and to improve the transfer in vitro using a well established cell culture model consisting of primary porcine brain capillary endothelial cells cultured on Transwell filter inserts. Passive apical-to-basolateral ASA transfer was observed, which was not saturable up to high ASA concentrations. No active transport could be determined. The passive transendothelial transfer was, however, charge-dependent as reduced concentrations of negatively charged monosaccharides in the N-glycans of ASA or the addition of polycations increased basolateral ASA levels. Adsorptive transcytosis is therefore considered to be the major transport pathway. Partial inhibition of the transcellular ASA transfer by mannose 6-phosphate indicated a second route depending on the insulin-like growth factor II/mannose 6-phosphate receptor, MPR300. We conclude that cationization of ASA and an increase of the mannose 6-phosphate content of the enzyme may promote blood-to-brain transfer of ASA, thus leading to an improved therapeutic efficacy of enzyme replacement therapy behind the BBB.

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.

Enzyme replacement improves ataxic gait and central nervous system histopathology in a mouse model of metachromatic leukodystrophy.

Inherited deficiencies of lysosomal hydrolases cause lysosomal storage diseases (LSDs) that are characterized by a progressive multisystemic pathology and premature death. Repeated intravenous injection of the active counterpart of the deficient enzyme, a treatment strategy called enzyme replacement therapy (ERT), evolved as a clinical option for several LSDs without central nervous system (CNS) involvement. To assess the efficacy of long-term ERT in metachromatic leukodystrophy (MLD), an LSD with prevailing nervous system disease, we treated immunotolerant arylsulfatase A (ASA) knockout mice with 52 doses of either 4 or 50 mg/kg recombinant human ASA (rhASA). ERT was tolerated without side effects and improved disease manifestations in a dose-dependent manner. Dosing of 4 mg/kg diminished sulfatide storage in kidney and peripheral nervous system (PNS) but not the CNS, whereas treatment with 50 mg/kg was also effective in the CNS in reducing storage in brain and spinal cord by 34 and 45%, respectively. Histological analyses revealed regional differences in sulfatide clearance. While 70% less storage profiles were detectable, for example, in the hippocampal fimbria, the histopathology of the brain stem was unchanged. Both enzyme doses normalized the ataxic gait of ASA knockout mice, demonstrating prevention of nervous system dysfunctions that dominate early stages of MLD.

Metachromatic leukodystrophy: an overview of current and prospective treatments.

Nowadays, different treatment options are available for an extending list of lysosomal storage diseases (LSDs). Hematopoietic stem cell transplantation (HSCT) can benefit selected subsets of patients with some LSDs, but results have been poor in several other disorders, including metachromatic leukodystrophy (MLD), outlining the need for innovative therapeutic approaches in this field. Enzyme replacement therapy has been developed recently for MLD, and a Phase I/II trial is ongoing. However, the blood-brain barrier limits the access of the recombinant product to the nervous tissues. Autologous hematopoietic stem/progenitor cells can be genetically modified to constitutively express supra-physiological levels of arylsulfatase-A and may become a quantitatively more effective source of functional enzyme than normal donor cells when transplanted in patients with MLD, thus possibly overcoming the limits of HSCT. Moreover, autologous transplantation might be associated with a significantly reduced transplant-related morbidity and TRM avoiding the risk of GVHD. Therefore, such a gene therapy strategy could represent a significant advance in comparison to conventional allogeneic HSCT.

Induction of tolerance to human arylsulfatase A in a mouse model of metachromatic leukodystrophy.

A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.

Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.

The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.

A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy, which is characterized by accumulation of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide). Sphingolipid storage results in progressive demyelination and severe neurologic symptoms. The disease is lethal, and curative therapy is not available. To assess the therapeutic potential of enzyme replacement therapy (ERT), ASA knockout mice were treated by intravenous injection of recombinant human ASA. Plasma levels of ASA declined with a half-time of approximately 40 min, and enzyme was detectable in tissues within minutes after injection. The uptake of injected enzyme was high into liver, moderate into peripheral nervous system (PNS) and kidney and very low into brain. The apparent half-life of endocytosed enzyme was approximately 4 days. A single injection led to a time- and dose-dependent decline of the excess sulfatide in PNS and kidney by up to 70%, but no reduction was seen in brain. Four weekly injections with 20 mg/kg body weight not only reduced storage in peripheral tissues progressively, but also were surprisingly effective in reducing sulfatide storage in brain and spinal cord. The histopathology of kidney and central nervous system was ameliorated. Improved neuromotor coordination capabilities and normalized peripheral compound motor action potential demonstrate the benefits of ERT on the nervous system function. Enzyme replacement may therefore be a promising therapeutic option in this devastating disease.

Biochemical aspects of globoid and metachromatic leukodystrophies.

Galactosylceramides and sulfogalactosylceramides are characteristic lipids of the myelin sheath. Two genetically determined leukodystrophies are caused by an inability to enzymically hydrolyze these glycolipids. Thus, a deficiency of galactocerebroside beta-galactosidase results in globoid cell leukodystrophy, whereas a reduced activity of arylsulfatase A is responsible for metachromatic leukodystrophy. Besides these disorders, deficiencies of arylsulfatases A, B, C, and other sulfatases have been shown in a distinct condition called "multiple sulfatase deficiency." All of these disorders are fatal and are characterized by marked demyelination and severe mental retardation. The cause of this demyelination is not known. However, cytotoxic galactosylsphingosine and sulfogalactosylsphingosine have been suggested as the agents responsible for this demyelination. Recent immunological studies have also shown that patients with globoid and metachromatic leukodystrophies contain a mutant galactocerebroside beta-galactosidase and arylsulfatase A, respectively. The mutant enzymes have different kinetic properties compared to the enzymes from normal subjects. However, they can cross-react with antibodies to these enzymes. Since partially purified preparations of galactocerebroside beta-galactosidase and homogeneous arylsulfatase A are now available, the possibility of enzyme replacement therapy in globoid and metachromatic leukodystrophies is discussed.

Recent developments in the biochemistry of globoid and metachromatic leucodystrophies.

Galactosylceramides and their sulphates are the main constituents of myelin sheath of the nerve cell. Two genetically determined disorders are the results of an inability to enzymatically hydrolyse these glycolipids. Thus the deficiency of galactosylceramide beta-galactosidase results in globoid cell leucodystrophy and the reduced activity of enzyme, arylsulphatase A is responsible for the disease Metachromatic leucodystrophy. Both these disorders are fatal and are characterized by marked demyelination and severe mental retardation. Since homognenous enzyme preparations of galactosylceramide beta-galactosidase and arylsulphatase A are now available, a possibility of enzyme replacement therapy in globoid and metachromatic leucodystrophies has been discussed.

Fetal metachromatic leukodystrophy: pathology, biochemistry and a study of in vitro enzyme replacement in CNS tissue.

Brain tissue from a fetus with the diagnosis of metachromatic leukodystrophy (MLD) became available at autopsy. Pathologic studies of the CNS showed inclusion bodies within oligodendroglia. The morphology of myelin was normal. Cells and myelin were isolated from the cerebrum; there was an increased level of sulfatide present in both fractions. In vitro studies of enzyme replacement in cultured MLD brain cells indicated that it may be possible to correct the abnormal sulfatide accumulation.