Efficacy of enzyme replacement therapy in alpha-mannosidosis mice: a preclinical animal study

Alpha-mannosidosis is a lysosomal storage disorder which manifests itself in the excessive storage of mannose-containing oligosaccharides in the lysosomes of multiple peripheral tissues and in the brain. Here we report on the correction of storage in a mouse model of alpha-mannosidosis after intravenous administration of lysosomal acid alpha-mannosidase (LAMAN) from bovine kidney, and human and mouse recombinant LAMAN. The bovine and the human enzyme were barely phosphorylated, whereas the bulk of the mouse LAMAN contained mannose 6-phosphate recognition markers. The clearance decreased from bovine to human to mouse LAMAN with plasma half-times of 4, 8 and 12 min, respectively. The apparent half-life of the internalized enzyme was dependent on the enzyme source as well as tissue type and varied between 3 and 16 h. The corrective effect on the storage of neutral oligosaccharides was time-, tissue- and dose-dependent, and the effects were observed to be transient. After a single dose of LAMAN the maximum corrective effect was observed between 2 and 6 days after injection. In general the corrective effect of the human LAMAN was higher than that of the mouse LAMAN and lowest for the bovine LAMAN. Injection of 250 mU human LAMAN/g body weight followed by a subsequent injection 3.5 days later was sufficient to clear liver, kidney and heart from neutral oligosaccharides. Surprisingly a decrease in mannose containing oligosaccharides was also observed in the brain, with storage levels reported at <30% than that found in controls. These data clearly underline the efficacy of enzyme replacement therapy for the correction of storage in alpha-mannosidosis and suggest that this treatment can substantially decrease storage in the brain.     

Enzyme replacement therapy in feline mucopolysaccharidosis I

Enzyme replacement therapy (ERT) has long been considered an approach to treating lysosomal storage disorders caused by deficiency of lysosomal enzymes. ERT is currently used to treat Gaucher disease and is being developed for several lysosomal storage disorders now that recombinant sources of the enzymes have become available. We have continued development of ERT for mucopolysaccharidosis I (MPS I) using the feline model. Recombinant alpha-L-iduronidase was administered intravenously at low dose (approximately 0.1 mg/kg or 25,000 units/kg) to four cats and high dose (0.5 mg/kg or 125,000 units/kg) to two cats on a weekly basis for 3- or 6-month terms. Clinical examinations showed distinct clearing of corneal clouding in one cat although clinical effects in the others were not evident. Biochemical studies of the cats showed that the enzyme was distributed to a variety of tissues although the liver and spleen contained the highest enzyme activities. Glycosaminoglycan storage was decreased in liver and spleen, and the histologic appearance improved in liver, spleen, and renal cortex. Enzyme was not consistently detected in cerebral cortex, brainstem, or cerebellum and the histologic appearance and ganglioside profiles did not improve. A variety of other tissues showed low variable uptake of enzyme and no distinct improvement. IgG antibodies to alpha-L-iduronidase were observed in five cats with higher titers noted when higher doses were administered. Mild complement activation occurred in three cats. Enzyme replacement therapy was effective in reversing storage in some tissues at the biochemical and histologic level in MPS I cats but an improved tissue distribution and prevention of a significant immune response could make the therapy more effective.     

First experience of combined enzyme replacement therapy and hematopoietic stem cell transplantation in alpha-mannosidosis

We describe the first case of bridge therapy in alpha-mannosidosis (AM) in an infant diagnosed at only 5 months of life who underwent enzyme replacement therapy (ERT) in the pre- and peri-transplant phases. Eight ERT infusions were administered before hematopoietic stem cell transplantation (HSCT) and continued for additional 90 days until complete engraftment. The clinical and laboratory data after 3 years post-HSCT show that the early combined intervention may reduce the disease progression and the urine and plasma content of mannosyl-oligosaccharides (OS) monitored by liquid chromatography tandem mass spectrometry (LC–MS/MS). This report highlights that early diagnosis and prompt initiation of such treatments in AM are the best chance to minimize the progression of symptoms.     

Reversal of peripheral and central neural storage and ataxia after recombinant enzyme replacement therapy in alpha-mannosidosis mice

Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including alpha-mannosidosis. Here we show that the neuropathology of a mouse model for alpha-mannosidosis can be efficiently treated using recombinant human alpha-mannosidase (rhLAMAN). After intravenous administration of different doses (25-500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (<70%) in visceral tissues, higher doses were needed for a clear effect in central and peripheral nervous tissues. A distinct reduction (<50%) of brain storage required repeated high-dose injections (500 U/kg), whereas lower doses (250 U/kg) were sufficient for clearance of stored substrates in peripheral neurons of the trigeminal ganglion. Successful transfer across the blood-brain barrier was evident as the injected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storage vacuoles. Importantly, the decrease in neuronal storage in the brain correlated with an improvement of the neuromotor disabilities found in untreated alpha-mannosidosis mice. Uptake of rhLAMAN seems to be independent of mannose-6-phosphate receptors, which is consistent with the low phosphorylation profile of the enzyme. These data suggest that high-dose injections of low phosphorylated enzymes might be an interesting option to efficiently treat LSDs with CNS involvement.     

Enzyme replacement therapy in the mouse model of Pompe disease

Deficiency of acid alpha-glucosidase (GAA) results in widespread cellular deposition of lysosomal glycogen manifesting as myopathy and cardiomyopathy. When GAA-/- mice were treated with rhGAA (20 mg/kg/week for up to 5 months), skeletal muscle cells took up little enzyme compared to liver and heart. Glycogen reduction was less than 50%, and some fibers showed little or no glycogen clearance. A dose of 100 mg/kg/week resulted in approximately 75% glycogen clearance in skeletal muscle. The enzyme reduced cardiac glycogen to undetectable levels at either dose. Skeletal muscle fibers with residual glycogen showed immunoreactivity for LAMP-1/LAMP-2, indicating that undigested glycogen remained in proliferating lysosomes. Glycogen clearance was more pronounced in type 1 fibers, and histochemical analysis suggested an increased mannose-6-phosphate receptor immunoreactivity in these fibers. Differential transport of enzyme into lysosomes may explain the strikingly uneven pattern of glycogen removal. Autophagic vacuoles, a feature of both the mouse model and the human disease, persisted despite glycogen clearance. In some groups a modest glycogen reduction was accompanied by improved muscle strength. These studies suggest that enzyme replacement therapy, although at much higher doses than in other lysosomal diseases, has the potential to reverse cardiac pathology and to reduce the glycogen level in skeletal muscle.     

Enzyme replacement therapy in a murine model of Morquio A syndrome

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to accumulation of keratan sulfate (KS) and chrondroitin-6-sulfate. The pharmacokinetics and biodistributions were determined for two recombinant human GALNSs produced in CHO cell lines: native GALNS and sulfatase-modifier-factor 1 (SUMF1) modified GALNS. Preclinical studies of enzyme replacement therapy (ERT) by using two GALNS enzymes were performed on MPS IVA mice. The half-lives in blood circulation of two phosphorylated GALNS enzymes were similar (native, 2.4 min; SUMF1, 3.3 min). After intravenous doses of 250 units/g body weight were administered, each enzyme was primarily recovered in liver and spleen, with detectable activity in other tissues including bone and bone marrow. At 4 h post-injection, enzyme activity was retained in the liver, spleen, bone and bone marrow at levels that were 20-850% of enzyme activity in the wild-type mice. After intravenous doses of 250 units/g of native GALNS, and 250, 600 or 1000 units/g of SUMF1-GALNS were administered weekly for 12 weeks, MPS IVA mice showed marked reduction of storage in visceral organs, sinus lining cells in bone marrow, heart valves, ligaments and connective tissues. A dose-dependent clearance of storage material was observed in brain. The blood KS level assayed by tandem mass spectrometry was reduced nearly to normal level. These preclinical studies demonstrate the clearance of tissue and blood KS by administered GALNS, providing the in vivo rationale for the design of ERT trials in MPS IVA.     

JCL Roundtable: Enzyme replacement therapy for lipid storage disorders

There are several inherited disorders that involve abnormal storage of lipids in tissues leading to severe compromise of organs. Sadly, these are often accompanied by lifelong morbidity and early mortality. Disorders such as Gaucher, Fabry, and lysosomal acid lipase deficiencies (Wolman and cholesteryl ester storage diseases) have been known for many years, and provide a difficult and frustrating set of problems for patients, their families, and their physicians. With recombinant methods of protein synthesis, it is now possible to literally replace the defective enzymes that underlie the basic pathophysiology of many such disorders. The delivery of these enzymes into the affected cells is possible because of their location in the lysosomes where the natural degradation of their lipid substrates occurs. I have asked 2 well-known investigators to join us for this Roundtable. These are professors who have been involved with the research that has made this type of therapy possible and who have participated in the clinical trials that demonstrated the value of enzyme replacement therapy. They are Dr. Robert Desnick, dean of Genetic and Genomic Medicine and professor and chairman emeritus of the Department of Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai in New York City, and Dr. Gregory Grabowski, professor of Microbiology, Biochemistry, and Pediatrics, at the University of Cincinnati College of Medicine. Dr. Grabowski recently retired from that school to become the chief science officer of Synageva, a company involved in producing enzymes for this type of therapy.     

Enzyme replacement therapy with agalsidase beta improves cardiac involvement in Fabry's disease

Fabry's disease is an X-linked lysosomal storage disease caused by a deficiency of alpha-galactosidase that results in an accumulation of neutral glycosphingolipids throughout the body, including the cardiovascular system. Fabry cardiomyopathy, characterized by progressive severe concentric left ventricular (LV) hypertrophy, is very frequent and is the most important cause of death in affected patients. Enzyme replacement therapy (ERT) allows a specific treatment for this disease, however, there are very few data on the effectiveness of therapy on cardiac involvement. Nine patients with Fabry cardiac disease were studied on basal condition and after 6 and 12 months of treatment with algasidase beta (Fabrazyme). A complete clinical, electrocardiographic and echocardiographic evaluation was performed in all patients. Interpretable Doppler recordings of transmitral flow and pulmonary flow velocity curves were also acquired. At baseline, the patients with Fabry's disease had increased LV septum and posterior wall thickness, normal LV fractional shortening, LV ejection fraction, normal Doppler parameters of mitral inflow but a duration of pulmonary vein flow velocity wave exceeding that of the mitral wave at atrial systole. ERT did not affect heart rate and arterial pressure. LV internal diameters did not change, there was a slight but not significant decrease in the LV posterior wall thickening and a progressive decrease in the interventricular septum thickening (p < 0.025) and in LV mass (p < 0.001) The difference in duration between pulmonary vein flow velocity wave and mitral wave at atrial systole significantly decreased (p < 0.001). These results suggest that ERT in patients with Fabry cardiomyopathy is able to reduce the LV mass and ameliorate the LV stiffness.     

Enzyme replacement therapy prevents loss of bone and fat mass in murine homocystinuria

Skeletal and connective tissue defects are the most striking symptoms in patients suffering from classical homocystinuria (HCU). Here, we determined body composition and bone mass in three mouse models of HCU and assessed whether a long‐term administration of enzyme replacement therapy (ERT) corrected the phenotype. The mouse models of HCU were analyzed using dual‐energy X‐ray absorptiometry and the data were complemented by plasma biochemical profiles. Both the mouse model lacking CBS (KO) and the one expressing human CBS mutant transgene on a mouse CBS null background (I278T) showed marked bone loss and decreased weight mostly due to a lower fat content compared with negative controls. In contrast, the HO mouse expressing the human CBS WT transgene on a mouse CBS null background showed no such phenotype despite similar plasma biochemical profile to the KO and I278T mice. More importantly, administration of ERT rescued bone mass and changes in body composition in the KO mice treated since birth and reversed bone loss and improved fat content in the I278T mice injected after the development of clinical symptoms. Our study suggests that ERT for HCU may represent an effective way of preventing the skeletal problems in patients without a restricted dietary regime.     

Enzyme replacement therapy attenuates disease progression in two Japanese siblings with mucopolysaccharidosis type VI

Mucopolysaccharidosis type VI (MPS VI) is a progressive, multisystem autosomal recessive lysosomal disorder resulting from deficient N-acetylgalactosamine-4-sulphatase (ASB) and the consequent accumulation of glycosaminoglycan (GAG). Preclinical and clinical studies had demonstrated clinical benefits of early initiation of systemic therapies in patients with MPS. In this case report, two siblings with MPS VI started enzyme replacement therapy (ERT) with weekly infusions of recombinant human ASB (Galsulfase) at 1 mg/kg. Sibling 1 started ERT 5.6 years of age and Sibling 2 was 6 weeks old. The disease status in these two siblings prior to and for no less than 36 months of ERT was followed up and compared. The treatment was well tolerated by both siblings. During 36 months of ERT, symptoms typical of MPS VI including short stature, progressive dysmorphic facial features, hepatosplenomegaly, hearing impairment, corneal clouding, and dysostosis multiplex were largely absent in the younger sibling. Her cardiac functions and joint mobility were well preserved. On the other hand, her affected brother had typical MPS VI phenotypic features described above before commencing ERT at the equivalent age, of 3 years. There was significant improvement in the shoulder range of motion and hearing loss after 36 months of treatment and cardiac function was largely preserved. His skeletal deformity and short stature remained unchanged. The results showed that early ERT initiated at newborn is safe and effective in preventing or slowing down disease progression of MPS VI including bone deformities. These observations indicate that early diagnosis and treatment of MPS VI before development of an irreversible disease is critical for optimal clinical outcome.     

Enzyme replacement therapy in mucopolysaccharidosis I: altered distribution and targeting of alpha-L-iduronidase in immunized rats

Enzyme replacement therapy (ERT) has been developed and trialed for the treatment of human lysosomal storage disorder patients. The viability of ERT for the treatment of these severe multiple pathology disorders has subsequently been established. However, in both animal model studies and human clinical trials, some individuals have been shown to develop an immune response to the replacement protein. This potential complication for treatment has been investigated by the infusion of recombinant human alpha-L-iduronidase (rh-alpha-L-iduronidase) into nonimmune and immunized rats to simulate mucopolysaccharidosis type I ERT in the presence of different levels of antibody. In rats with high antibody titers to rh-alpha-L-iduronidase (titer 1,024,000) there was evidence of altered organ distribution and subcellular targeting when compared to either lower titer immunized rats (titers less than 64,000) or nonimmune rats (titers 512-1024). In addition, hypersensitivity reactions were observed for high titer rats (titer 1,024,000) during rh-alpha-L-iduronidase infusion, but not for the other two treatment groups. A rat with an antibody titer of 64,000 had only minor changes in subcellular targeting and organ distribution when infused with rh-alpha-L-iduronidase. This implied that a high level of antibody was required to effect changes in alpha-L-iduronidase enzyme targeting and distribution. Notably, in the high titer rats, the antibody produced appeared to increase the tissue and subcellular level of rh-alpha-L-iduronidase specific activity. This suggested that antibody production may not always result in an adverse effect on ERT.     

Enzyme replacement therapy partially prevents invariant Natural Killer T cell deficiency in the Fabry disease mouse model

Fabry disease is a lysosomal storage disease caused by deficient activity of the α-Galactosidase A (α-Gal A) enzyme, which leads to abnormal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in the lysosome. Glycosphingolipids are known to be invariant Natural Killer T (iNKT) cell antigens. Several animal models of lysosomal storage diseases, including Fabry disease, present a defect in iNKT cell selection by the thymus. We have studied the effect of age and the impact of enzyme replacement therapy on Gb3 accumulation and iNKT cells of Fabry knockout mice. At 4 weeks of age, Fabry knockout mice already showed Gb3 accumulation and a reduction in the percentage of iNKT cells. In older mice (12-week old), we observed an accentuated peripheral iNKT deficiency. 12-week old animals also showed a reduced splenic CD4+/CD4- iNKT cell ratio due to greater loss in the iNKT CD4+ subset. Treatment of Fabry knockout mice with α-Gal A replacement therapy efficiently reduced Gb3 deposition in the liver and spleen. Moreover, enzyme replacement therapy had a positive effect on the number of iNKT cells in an organ-dependent fashion. Indeed, treatment of Fabry knockout mice with α-Gal A did not alter iNKT cell percentage in the thymus and liver but increased splenic iNKT cell percentage when compared to untreated mice. Study of animals prior to treatment indicates that enzyme replacement therapy stabilized iNKT cell percentage in the spleen. This stabilization is due to a specific effect on the iNKT CD4+ subset, preventing the decrease on the number of these cells that occurs with age in Fabry knockout mice. This study reveals that enzyme replacement therapy has a positive organ and subset-dependent effect in iNKT cells of Fabry knockout mice.