Shih-hsin Kan

Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I

Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB

Significance Mucopolysaccharidosis type IIIB (MPS IIIB) is a devastating and currently untreatable disease affecting mainly the brain. The cause is lack of the lysosomal enzyme, α–N-acetylglucosaminidase (NAGLU), and storage of heparan sulfate. Using a mouse model of MPS IIIB, we administered a modified NAGLU by injection into the left ventricle of the brain, bypassing the blood–brain barrier. The modification consisted of a fragment of IGFII, which allows receptor-mediated uptake and delivery to lysosomes. The modified enzyme was taken up avidly by cells in both brain and liver, where it reduced pathological accumulation of heparan sulfate and other metabolites to normal or near-normal levels. The results suggest the possibility of treatment for MPS IIIB. Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α–N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood–brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1–28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Immune response to intrathecal enzyme replacement therapy in mucopolysaccharidosis I patients

Background:Intrathecal (IT) enzyme replacement therapy with recombinant human α-L-iduronidase (rhIDU) has been studied to treat glycosaminoglycan storage in the central nervous system of mucopolysaccharidosis (MPS) I dogs and is currently being studied in MPS I patients.Methods:We studied the immune response to IT rhIDU in MPS I subjects with spinal cord compression who had been previously treated with intravenous rhIDU. We measured the concentrations of specific antibodies and cytokines in serum and cerebrospinal fluid (CSF) collected before monthly IT rhIDU infusions and compared the serologic findings with clinical adverse event (AE) reports to establish temporal correlations with clinical symptoms.Results:Five MPS I subjects participating in IT rhIDU trials were studied. One subject with symptomatic spinal cord compression had evidence of an inflammatory response with CSF leukocytosis, elevated interleukin-5, and elevated immunoglobulin G. This subject also complained of lower back pain and buttock paresthesias temporally correlated with serologic abnormalities. Clinical symptoms were managed with oral medication, and serologic abnormalities were resolved, although this subject withdrew from the trial to have spinal decompressive surgery.Conclusion:IT rhIDU was generally well tolerated in the subjects studied, although one subject had moderate to severe clinical symptoms and serologic abnormalities consistent with an immune response.

Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I.

BACKGROUND Treatment with intravenous enzyme replacement therapy and hematopoietic stem cell transplantation for mucopolysaccharidosis (MPS) type I does not address joint disease, resulting in persistent orthopedic complications and impaired quality of life. A proof-of-concept study was conducted to determine the safety, tolerability, and efficacy of intra-articular recombinant human iduronidase (IA-rhIDUA) enzyme replacement therapy in the canine MPS I model. METHODS Four MPS I dogs underwent monthly rhIDUA injections (0.58 mg/joint) into the right elbow and knee for 6 months. Contralateral elbows and knees concurrently received normal saline. No intravenous rhIDUA therapy was administered. Monthly blood counts, chemistries, anti-rhIDUA antibody titers, and synovial fluid cell counts were measured. Lysosomal storage of synoviocytes and chondrocytes, synovial macrophages and plasma cells were scored at baseline and 1 month following the final injection. RESULTS All injections were well-tolerated without adverse reactions. One animal required prednisone for spinal cord compression. There were no clinically significant abnormalities in blood counts or chemistries. Circulating anti-rhIDUA antibody titers gradually increased in all dogs except the prednisone-treated dog; plasma cells, which were absent in all baseline synovial specimens, were predominantly found in synovium of rhIDUA-treated joints at study-end. Lysosomal storage in synoviocytes and chondrocytes following 6 months of IA-rhIDUA demonstrated significant reduction compared to tissues at baseline, and saline-treated tissues at study-end. Mean joint synovial GAG levels in IA-rhIDUA joints were 8.62 ± 5.86 μg/mg dry weight and 21.6 ± 10.4 μg/mg dry weight in control joints (60% reduction). Cartilage heparan sulfate was also reduced in the IA-rhIDUA joints (113 ± 39.5 ng/g wet weight) compared to saline-treated joints (142 ± 56.4 ng/g wet weight). Synovial macrophage infiltration, which was present in all joints at baseline, was abolished in rhIDUA-treated joints only. CONCLUSIONS Intra-articular rhIDUA is well-tolerated and safe in the canine MPS I animal model. Qualitative and quantitative assessments indicate that IA-rhIDUA successfully reduces tissue and cellular GAG storage in synovium and articular cartilage, including cartilage deep to the articular surface, and eliminates inflammatory macrophages from synovial tissue. CLINICAL RELEVANCE The MPS I canine IA-rhIDUA results suggest that clinical studies should be performed to determine if IA-rhIDUA is a viable approach to ameliorating refractory orthopedic disease in human MPS I.

Biochemical characterization of fluorescent‐labeled recombinant human alpha‐l‐iduronidase in vitro

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low‐molecular‐weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the proteins characteristics. We tested the in vitro properties of fluorescent‐labeled recombinant human alpha‐l‐iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6‐phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

Behavioral deficits and cholinergic pathway abnormalities in male Sanfilippo B mice.

Sanfilippo B syndrome is a progressive neurological disorder caused by inability to catabolize heparan sulfate glycosaminoglycans. We studied neurobehavior in male Sanfilippo B mice and heterozygous littermate controls from 16 to 20 weeks of age. Affected mice showed reduced anxiety, with a decrease in the number of stretch-attend postures during the elevated plus maze (p=0.001) and an increased tendency to linger in the center of an open field (p=0.032). Water maze testing showed impaired spatial learning, with reduced preference for the target quadrant (p=0.01). In radial arm maze testing, affected mice failed to achieve above-chance performance in a win-shift working memory task (t-test relative to 50% chance: p=0.289), relative to controls (p=0.037). We found a 12.4% reduction in mean acetylcholinesterase activity (p<0.001) and no difference in choline acetyltransferase activity or acetylcholine in whole brain of affected male animals compared to controls. Cholinergic pathways are affected in adult-onset dementias, including Alzheimer disease. Our results suggest that male Sanfilippo B mice display neurobehavioral deficits at a relatively early age, and that as in adult dementias, they may display deficits in cholinergic pathways.

Genetically Corrected iPSC-Derived Neural Stem Cell Grafts Deliver Enzyme Replacement to Affect CNS Disease in Sanfilippo B Mice

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB [MPS IIIB]) is a lysosomal storage disorder primarily affecting the brain that is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. Here we report that, ex vivo, lentiviral correction of Naglu−/− neural stem cells derived from Naglu−/− mice (iNSCs) corrected their lysosomal pathology and allowed them to secrete a functional NAGLU enzyme that could be taken up by deficient cells. Following long-term transplantation of these corrected iNSCs into Naglu−/− mice, we detected NAGLU activity in the majority of engrafted animals. Successfully transplanted Naglu−/− mice showed a significant decrease in storage material, a reduction in astrocyte activation, and complete prevention of microglial activation within the area of engrafted cells and neighboring regions, with beneficial effects extending partway along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu−/− mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver enzymes and treat MPS IIIB.

A Humoral Immune Response Alters the Distribution of Enzyme Replacement Therapy in Murine Mucopolysaccharidosis Type I

Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase (rhIDU). Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by partial inhibition compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naive mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than in hepatocytes in antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.

364. Neurological Correction of Mucopolysaccharidosis IIIB Mice by Haematopoietic Stem Cell Gene Therapy

Mucopolysaccharidosis Type IIIB (MPSIIIB) is a paediatric, autosomal recessive Lysosomal Storage Disease (LSD) caused by deficiency of α-N-acetylglucosaminidase (NAGLU), an enzyme in the heparan sulfate (HS) degradation pathway. Absence of NAGLU leads to the accumulation of partially degraded HS glycosaminoglycan in cell lysosomes, giving rise to cellular dysfunction with devastating clinical consequences. Individuals affected by this fatal disease exhibit severe central nervous system degeneration with progressive cognitive impairment and behavioural problems, alongside more attenuated somatic symptoms. There are currently no effective treatments available. Enzyme replacement therapy with recombinant NAGLU enzyme is ineffective for MPSIIIB since enzyme cannot cross the blood brain barrier (BBB) to where it is needed. Modified recombinant NAGLU enzymes that utilise the insulin growth factor II (IGFII) peptide to facilitate improved uptake across the BBB are currently in development. Haematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy that can circumvent the BBB via monocyte trafficking and engraftment in the brain, allowing delivery of enzyme by cross correction. We have developed a novel stem cell gene therapy approach to investigate the therapeutic potential of HSCGT for MPSIIIB. We designed two lentiviral vectors expressing therapeutic enzyme; the first vector expressing codon optimised NAGLU, and the second expressing a NAGLU.IGFII fusion to aid cellular uptake, both driven by the myeloid specific promoter CD11b and compared these in autologous MPSIIIB transplants against a normal WT bone marrow transplant. Here we present for the first time neurological correction of MPSIIIB mice by HSCGT. We observed correction of the MPSIIIB behavioural phenotype in treated mice to wild-type levels with normalisation of path length, average speed, frequency entering the centre and duration of speed >100mm/s in open field tests. In addition, we observed a significant correction of astrogliosis and lysosomal compartment size in the brains of CD11b. NAGLU LV treated mice with an accompanied normalisation of inflammatory cytokines TNFα, IL1B and IL1RN. Furthermore, NAGLU enzyme activity was substantially increased in the brain. Interestingly, WT transplant alone was able to mediate a partial brain correction, although levels of inflammation and lysosomal storage remain high.

430. Antibodies to Recombinant Human α-L-Iduronidase Enhance Uptake into Macrophages in Murine Mucopolysaccharidosis Type I

Mucopolysaccharidosis I (MPS I) is an inherited, and progressive lysosomal storage disease caused by lack or low level of α-L-iduronidase (IDU), altering the catabolism of glycosaminoglycans (GAG) in lysosomes. Some clinical improvement in patients has been observed by enzyme replacement therapy with recombinant human IDU (rhIDU), but studies suggested that anti-rhIDU antibodies reduce the effectiveness of treatment. Most MPS I treated patients develop a humoral immune response against rhIDU. To determine how anti-rhIDU antibodies alter the tissue and cellular distribution of rhIDU in vivo, we sensitized MPS I mice to rhIDU by administering rhIDU via tail-vein injection from 4 to 16 weeks of age. Mice that developed anti-rhIDU antibodies showed a shift in rhIDU uptake preferentially towards tissues with high reticuloendothelial (RE) content (liver, spleen, thymus) vs. low RE content (lung, kidney, heart, brain). Furthermore, in mice with anti-rhIDU antibodies, rhIDU was mainly localized in Kupffer cells, with relatively less available for hepatocytes. In order to further understand how the humoral immune response against rhIDU alters its distribution, we comparedin vitro uptake of rhIDU in the absence or presence of antibodies against rhIDU in MPS I fibroblasts and macrophages. Bone marrow monocytes were harvested from MPS I mice and derived into macrophages. MPS I macrophages were incubated with rhIDU with or without immunized rabbit serum, pre-immune serum, murine Fc receptor blocking agent and mannose 6-phosphate. We evaluated two types of immunized rabbit serum: one that completely abolished rhIDU uptake into human MPS I fibroblasts (inhibiting serum) and one that only partially (30%) inhibited rhIDU uptake into human fibroblasts (partially-inhibiting serum). The uptake of rhIDU in the presence of serum containing rhIDU antibodies was enhanced into MPS I mouse macrophages when both inhibiting and partially-inhibiting serum was applied. Uptake of rhIDU per cell was higher in macrophages when immune serum than was uptake of rhIDU into human fibroblasts normalized per cell. Treatment with mannose 6-phosphate or Fc receptor block partially inhibited the uptake in macrophages. Our study demonstrated that even a low-level humoral immune response against rhIDU, which only partially inhibits rhIDU uptake into fibroblasts in vitro, nevertheless might alter its tissue distribution in vivo. At the cellular level, antibody-positive mice show reduced rhIDU distribution in hepatocytes, while distribution to tissue macrophages is maintained. In vitro, immunized serum reduced rhIDU uptake into human fibroblasts but increased uptake into murine macrophages. Our findings imply that the altered tissue distribution of rhIDU caused by anti-rhIDU antibodies is partly due to reduced uptake into fibroblasts and partly due to enhanced uptake into tissue macrophages. These results imply that functional immune assays of rhIDU uptake in vitro into fibroblasts may not completely predict the impact of the humoral immune response against enzyme replacement therapy.