1st Year Research Reviews – 2009

Dr. Sara Cathey One year Partnership Grant with ISMRD
Greenwood Genetic Center, North Charleston, SC
Natural history study for mucolipidosis

In 2008 and 2009 Partnership Grants with ISMRD supported natural history studies of Mucolipidosis (ML) II and ML III. The ML Project of the Greenwood Genetic Center (GGC) is an ongoing effort to understand the disease course in people affected by these rare conditions. Fourteen individuals with ML II or III and their families participated in GGCs Second ML Clinic, held July 27-29, 2009 at the South Carolina Center for the Treatment of Genetic Disorders in Greenwood, South Carolina. During this special clinic, participants were evaluated by clinical geneticists, psychologists, an orthopedic surgeon, and an ophthalmologist. All patients had skeletal x-rays to evaluate the progression of bone disease. Many patients (and their parents!) provided samples for laboratory studies. Most of these same patients participated in the first ML Clinic held in 2006 at GGC. ML affects people from around the world, and each patient can contribute to better understanding. The natural history study went international in November 2009 when ML patients from Australia and New Zealand participated in special clinics held in Sydney, Australia and Wellington, New Zealand.

The ML Project has had a positive impact in many ways, including important contributions to the medical literature about ML. Large collections of clinical and laboratory data have been established. Samples collected are used by scientists at GGC and researchers around the world. Patients and their doctors have another source of information and support. Following a group of patients with this rare disease over time lets us learn the natural history of the illness, evaluate the usefulness of potential therapies, and set sights on effective treatments. To conquer the illness we have to learn about the people affected by ML.

Lysosomal Disease Network, one year grant
University of Minnesota, Minneapolis, MN
Neuroimaging Core activities of the projects of the LDN grant during the first year of work

Progress Report for MPS Society Award
This award is being used to support the Neuroimaging Core of the Lysosomal Disease Network, specifically the work of Dr. Alia Ahmed in volumetric neuroimaging (about 50% of her salary). The award thus supports the following LDN studies, all of whom employ neuroimaging:

  • Longitudinal Study Project 1 (Shapiro, PI Longitudinal Studies of Brain Structure and Function in MPS Disorders (MPS I, II, and VI),
  • Longitudinal Study Project 2 ( Polgreen PI, Longitudinal study of bone disease and the impact of growth hormone treatment in MPS I, II, and VI).
  • Pilot Study Project 16, (Potegal PI, Characterizing the Neurobehavioral Phenotype in MPS III.
  • Pilot Study Project 12 (Dickson PI, Intrathecal ERT for Cognitive Decline in MPS I.

The neuroimaging core has four functions, 1) to collect imaging data in a comparable manner suitable for natural history determination, 2) Develop and validate disease specific standardized imaging protocols, semi-quantitative MRI clinical ratings scales, and formal quantitative measures to be used as biomarkers for natural history and clinical trials, 3) demonstrate that standardized protocols will identify optimal imaging sequences, appropriate intervals for follow-up, and best clinical practices, 4) archives of scans for educational purposes. Dr. Ahmed’s work supports items 2 and 3.

With regard to optimal imaging seqences, protocols have been established by our neuroimaging core for volumetric analysis. For quantitative volumetric analysis of scans, we have established methods for reliable determination of hippocampus and amygdala using a three dimensional manual tracing program called BRAINS2. Dr. Alia Ahmed is expert at this demanding task.

Prior to funding, to develop her skills, she became proficient in tracing on 10 hippocampi of tremor patients and 5 caudates of normal subjects, using ImageJ, a two dimensional program. We decided to change to BRAINS2, which is a three dimensional program allowing tracing on coronal, saggital, and axial images which can be then combined to produce a structural image. To trace the hippocampus for each patient, she traced one image in a saggittal section and two in a coronal section, one with and one without white matter. Thus for each brain scan she traces each (left and right) hippocampus 3 times, making 6 tracings. For the amygdala, which also requires manual tracing, for each brain scan she does one sagital and one coronal tracing of the left and of the right amygdala.

So far to trace the hippocampus, she has traced 42 MPS I brain scans, 9 MPS II, 10 MPS III, 7 MPS VI, 2 MPS VII , and 9 control brain scans ( Total 79 brains scans X 6 tracings = 474). For the amygdala, she has traced 16 brains (we are only doing it for MPS II and III) totaling 64 tracings. She has also traced the caudate in 13 patients totaling 26 tracings.
Thus, in summary, Dr. Ahmed has successfully traced hippocampus on 70 brains of MPS children and 9 controls and has successfully traced the amygdala in 16 patients. These tracings can be seen in three dimensional space and we are anticipating that we can evaluate changes in configuration as well as volume over time. We are comparing some of Dr. Ahmed’s tracings to an automated program called FreeSurfer which is implemented at the Minnesota Supercomputer Center (they have provided a grant to cover our costs). She also did intra rater reliability studies on ten brain scans (rating each one twice) and now is working on inter rater reliability studies (comparing her ratings with those of Dr. Nestrasil, our expert in image acquisition and other methods of image analysis. Thus far, excellent manual tracing reliabilities on MPS I patients and preliminary data on brain volumes in 18 MPS I patients and 9 MPS II patients have been obtained. The results of the reliability study were presented at the WORLD symposium in February; this presentation is now being written for publication. It is attached to this progress report as well as the volumetric comparisons of attenuated MPS I and MPS II.

In addition, clinical ratings scales (such as the Matheus scale for MPS disorders) are now being refined to increase their reliability. Dr. Ahmed is also carrying out this analysis together with a member of our radiology department to determine its reliability and to determine if we can improve its sensitivity.

With respect to her involvement in the four studies: she has analyzed brains for 40 children in the Shapiro study, 25 for the Polgreen study (overlaps with Shapiro study), 10 for the Potegal study, and 3 for the Dickson study.
Funds have been used for 1) Dr. Ahmed’s salary, 2) a computer for her image analysis, and 3) travel for meetings and training.

Ahmed, A., Nestrasil, I., Rudser, K,, Shapiro, E. Reliability of manual and automated tracing of hippocampal volumes in MPS patients and normal controls: A report of the neuroimaging core of the lysosomal disease network. Molecular Genetics and Metabolism, 99 (2); 2010, S9.

Dr. Maria Pia Cosma
TIGEM, Naples, Italy
AAV2/5CMV-IDS therapy in MPSII mice: correction of CNS defects through IDS
delivery across the blood-brain barrier.

Results funded by the MPS society have been published in the following paper:

Polito V and Cosma MP (2009). IDS crossing of the blood-brain barrier corrects CNS defects in MPSII mice. AJHG 85(2):296-301.

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, arises from a deficiency in iduronate 2-sulfatase (IDS), and it is characterized by progressive somatic and neurological involvement. The MPSII mouse model reproduces the features of MPSII patients. Systemic administration of the AAV2/5CMV-hIDS vector in MPSII mouse pups results in the full correction of glycosaminoglycan (GAG) accumulation in visceral organs and in the rescue of the defects and GAG accumulation in the central nervous system (CNS). Remarkably, in treated MPSII animals, this CNS correction arises from the crossing of the blood-brain barrier by the IDS enzyme itself, not from the brain transduction. Thus, we show here that early treatment of MPSII mice with one systemic injection of AAV2/5CMV-hIDS results in prolonged and high levels of circulating IDS that can efficiently and simultaneously rescue both visceral and CNS defects for up to 18 months after therapy.

We are now testing if the treatment of the CNS defects can be also achieved in juvenile and adult MPSII mice. For that we injected groups of ids-/y animals with AAV2/5CMV-IDS viral particles. We are evaluating the IDS activities in the plasma and GAG contents in the urines to monitor the efficiency of the therapy. In addition the mice were tested with rotarod tests which evaluate the sensorimotor coordination. Indeed, we have truly characterized groups of ids-/y mice at different ages and found that Purkynje cells in the cerebellum strongly degenerate through the progression of the disease. Thus we are testing the rescue of the cerebellum defects after the therapy.

In addition to these gene therapy experiments we are also setting up enzyme replacement protocols to treat simultaneously the visceral defects and the CNS features of the MPSII mice. We are developing efficient protocols that allow the IDS to cross the blood brain barrier, to correct CNS phenotype and to reverse neurobehavioural features.

Dr. Jeffrey Esko
University of California, San Diego, CA
Substrate reduction strategy for MPS IIIA

The original focus of this grant was to demonstrate that substrate reduction would prove effective as a treatment for Sanfiippo syndromes. We found that reducing the level of reduced heparan sulfate biosynthesis in MPSIIIa fibroblasts by ~40% diminished lysosomal storage by ~50%. These studies are limited to short-term experiments because of the way that we silenced the expression of certain genes in the system. To get at this question in vivo, we have crossed the MPSIIIa mouse with mice carrying mutations in genes involved in heparan sulfate biosynthesis. The objective is to test the impact of substrate reduction on lysosomal storage and pathology in specific cell types in vivo, specifically hepatocytes, astrocytes, neurons and macrophages. We should have our first results in the next 6 months.

We also found that MPSIIIa cells (as well as MPSIIIb, MPSIIIc, and MPSIIId cells) store a significant amount of chondroitin/dermatan sulfate in addition to heparan sulfate. This finding was unexpected since the primary genetic defects in Sanfilippo syndrome are in enzymes specifically involved in heparan sulfate biosynthesis. Enzyme replacement therapy using recombinant sulfamidase, the enzyme missing in MPSIIIa, resolved both the primary and secondary storage (Fig. 1), suggesting that the accumulation of heparan sulfate caused the secondary storage of chondroitin/dermatan sulfate.

To understand the cause of secondary storage, we assessed whether heparan sulfate inhibited enzymes involved in chondroitin/dermatan sulfate degradation. We found that heparin and heparan sulfate strongly inhibited the enzyme iduronate-2-sulfatase (IC50 values of ~3 ?g/mL and ~14 ?g/mL, respectively). We are currently testing whether supplementing iduronate-2-sulfatase can reduce secondary storage and whether strategies that reduce both primary and secondary substrate accumulation are more effective at treating lysosomal storage.


Dr. Alessandro Fraldi
TIGEM, Naples, Italy
Developing a systemic AAV-mediated gene therapy to cross the blood-brain barrier and treat the brain pathology in MPS IIIA

The aim of this project is to develop a low-invasive systemic gene therapy strategy based on the intravenous injection of AAV serotype 8. This serotype displays high tropism to the liver and will be used to delivery of an engineered gene encoding a chimeric modified sulfamidase optimized to be: (i) highly secreted from the liver thus reaching high levels of circulatingenzyme in the blood stream; (ii) to efficiently cross the BBB.

Construction and validation of the engineered sulfamidase
In order to increase sulfamidase secretion from the liver and thus the amount of the enzyme in the blood stream available to specifically target the brain, we engineered the sulfamidase by replacing its own signal peptide (SP) with an alternative one. Two signal peptides have been tested, the Iduronate-2-sulfatase (IDS) signal peptide and the human antitrypsin (AAT) signal peptide. The rationale behind the use of these two signal peptides is that IDS is a lysosomal enzyme that has been demonstrated to be secreted at high levels from the liver while the AAT is a highly secreted enzyme. To test the functionality of chimeric sulfamidase coding sequences containing either the IDS-SP or the AAT-SP we transfected the constructs in Hela and MEF cells derived from MPS-IIIA mice; as control, the cells were also transfected with not-engineered SGSH enzyme. Two days after transfection we measured the SGSH activity in the pellet and in conditioned medium of transfected cells. In the cells transfected with either hAAT(SP)-SGSH-Flag or IDS(SP)-SGSH-Flag, the level of sulfamidase protein detected in the pellet and the medium was increased compared to the level detected in control cells (transfected with the not-engineered sulfamidase) (Fig.1). Such an increase in sulfamidase protein levels was due to both increased efficiency in secretion (medium/total activity; see graph on the right of figure 2) and increased stability of engineered sulfamidase (Fig.1). The analysis of SGSH activity in MEF cells derived from MPSIIIA mice confirmed the results obtained in the Hela cells (not shown).

Engineering the final chimeric sulfamidase. The final goal of our project is to product a modified sulfamidase capable to cross the BBB and target the CNS via receptor-mediated transcytosis. For this reason before starting the experiments aimed at evaluating the therapeutic efficacy of the substituting SP signal in SGSH, we further engineered the modified SGSH with a specific brain-targeting protein domain, the Low Density Lipoprotein receptor (LDLR)-binding domain of the Apolipoprotein B (ApoB LDLR-BD) (Fig.3). The Binding Domain of ApoB will allow the sulfamidase to reach the brain cells by binding LDL receptors, which are abundant on the endothelial cells of BBB. The two final engineered sulfamidase constructs comprise at C-terminal the ApoB LDLR-BD and at N-terminal either an IDS or an hAAT signal peptide (IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag-ApoB) (Fig. 3). To evaluate the functionality of IDSsp-SGSHflag-ApoB and hAATsp-SGSHflag- ApoB we transfected MPSIIIA MEF cells with these final constructs and compared the outcomes with those resulting from the transfections with partial modified (hAATsp-SGSHFlag, IDSsp-SGSH-Flag) and not modified SGSH constructs. Surprisingly, we observed that SGSH activity in the pellet and in the conditioned medium was higher in the cells transfected with the final chimeric constructs compared with the activity measured in the cells transfected with the other constructs, indicating that final engineered sulfamidase were efficiently secreted and even more stable compared to partial engineered sulfamidase (Fig.4). These results were associated with a higher secretion efficiency of the final engineered sulfamidase enzymes with respect to not-engineered sulfamidase. The secretion efficiency of the final chimeric constructs was similar to that measured after transfection of partial chimeric sulfamidase (containing only the alternative signal peptide).

In conclusion these results demonstrate that: (i) the chimeric sulfamidase enzymes containing the alternative signal peptide are functional and active; (ii) they are more stable with respect to not-modified sulfamidase; (iii) they are secreted with increased efficiency compared to not engineered sulfamidase enzyme; (iv) the introduction of the ApoB LDLR-BD to produce the final engineered sulfamidase did not affect neither the functionality nor the increased secretion efficiency observed in the cells transfected with the partial engineered sulfamidase.
In addition, the final engineered constructs appear to be more stable compared to partial engineered constructs.



Preliminary in vivo results in MPS IIIA mice injected with final engineered sulfamidase

We obtained very preliminary but extremely encouraging results in MPS-IIIA injected with the final constructs hAATsp-SGSHflag-ApoB. Adult MPS-IIIA mice were systemically injected with AAV2/8-TBG- hAATsp-SGSHflag-ApoB (the TBG is a liver specific promoter). A group of MPS-IIIA were also injected with AAV2/8-TBG-SGSH (containing the not modified sulfamidase) as control. The mice were sacrificed one month after injection. In the mice injected with the chimeric sulfamidase we observed higher liver sulfamidase activity and a very strong increase in the sulfamidase secretion respect to control mice. Moreover, we detected a significant increase in SGSH activity into the brain of mice injected with the chimeric sulfamidase. We are now evaluating the effective cross of BBB by the chimeric sulfamidase. As planned in the proposal, we have also designed an in vivo large study aimed to evaluate the rescue of pathological phenotype in MPS-IIIA. We plan to complete this study within one year.


Dr. Katherine Ponder
Washington University, St. Louis, MO
The role of cathepsin K in cardiac valve disease in MPS

Mucopolysaccharidosis (MPS) is due to a genetic deficiency in the activity of an enzyme that degrades glycosaminoglycans. One of the serious manifestations of MPS is the development of heart disease, which can result in reduced delivery of oxygenated blood to the body. This can involve thickened heart valves that block the flow of blood, and/or heart valves that are leaky and allow blood to flow in the wrong direction. The goal of this project is to understand what causes heart valve problems, which may ultimately allow us to identify a therapy to prevent these heart valve abnormalities from developing.

I. Evaluate collagen and elastin in the extracellular matrix (ECM) of mitral valves, chordae tendineae, and aortic valves in MPS I and MPS VII dogs by light and electron microscopy

The goal of this aim is to determine if collagen and elastin structure are abnormal in heart valve tissues using histochemical techniques. Collagen is the major extracellular matrix protein of the heart valves. We have demonstrated that the amount of structurally-intact collagen in the mitral valve is approximately 2% of normal at 2 years of age in MPS VII dogs, and that neonatal gene therapy with a retroviral vector can improve the collagen structure. This is consistent with the hypothesis that abnormal collagen structure is responsible for the abnormal valve function. The aortic valves and chordae tendinae have some reduction in structurally intact collagen, but this is less severe. We are in the process of quantifying these results at various ages in different treatment groups.

II. Determine if RNA for cathepsin K or other genes involved in ECM assembly or degradation are upregulated in the mitral valves, chordae tendineae, and aortic valves in MPS VII dogs

Abnormal collagen structure could reflect a failure to synthesize or assemble collagen or elastin properly, or an increase in expression of enzymes with collagenase activity. Mitral valves, chordae tendineae, and aortic valves have been isolated from normal, MPS VII, and retroviral vector-treated MPS VII dogs at 6 months of age, when collagen fragmentation is apparent in untreated MPS VII dogs, and expression of several genes from the mitral valve have been evaluated. Cathepsins and matrix metalloproteinases (MMP) are enzymes that can degrade collagen. RNA analysis demonstrated that cathepsin B, S, and W, and MMP 8, 9, and 12 were markedly upregulated (10- to 100-fold) in the mitral valves of untreated MPS VII dogs. Although cathepsin K RNA was not significantly elevated, several of the other cathepsins and the MMPs have collagen-degrading activity. We are in the process of using microarray analysis to look for changes in expression of other genes that are involved in collagen assembly or degradation, and evaluating the signal transduction pathways that may be involved. This may identify targets that can be inhibited in order to reduce collagen abnormalities.

III. Evaluate enzyme activities and GAG levels in the mitral valves, chordae tendineae, and aortic valves in MPS VII dogs

The final aim of this project will evaluate extracts from the mitral valves, chordae tendineae, and aortic valves of normal, MPS VII, and RV-treated MPS VII dogs for several biochemical parameters. Homogenized samples have been tested and found to have markedly elevated cathepsin activities, although the specific cathepsins that are involved are still being evaluated, and MMP activities have not yet been tested. These data further substantiate our hypothesis that increases in cathepsin activities contribute to abnormal collagen structure,
although additional assays still need to be performed.

Dr. Calogera Simonaro
Mount Sinai School of Medicine, New York, NY
Novel anti-inflammatory therapies for the mucopolysaccharidoses

Current therapeutic strategies for the MPS disorders, including enzyme replacement therapy (ERT), have limited effects on the bones and joints. The premise of our research is that new and improved treatment approaches are necessary for these disorders, and that this is best achieved by an improved understanding of the underlying pathogenic mechanisms. Our recent research has focused on the involvement of the toll-like receptor 4 (TLR4) signaling (inflammatory) pathway in MPS bone and joint disease, and the use of anti-inflammatory agents for the treatment of these diseases. The most recent studies were published in the Proceedings from the National Academy of Sciences (Simonaro et al., Proc Natl Acad Sci USA. 2010 Jan 5;107(1):222-7), and will be summarized below.

TLR4 knockout (KO) mice were bred to MPS type VII mice. Inactivation of this proinflammatory pathway in double KO mice corrected many biochemical and clinical features of the MPS disease, suggesting that drugs targeting this pathway could be effective in the treatment of these disorders. Double KO animals grew substantially better than MPS VII mice alone, and had longer and thinner bones. The levels of several inflammatory cytokines, including TNF-?, also were substantially reduced in the double KO mice, leading us to evaluate the effects of the FDA-approved anti-TNF-? drug, RemicadeTM, in MPS VI rats. When initiated pre-symptomatically, intravenous RemicadeTM treatment prevented the elevation of TNF-? and other inflammatory molecules in the blood. Importantly, the levels of these markers also were markedly reduced in cartilage (chondrocytes) and synovial membranes of the treated animals. Although the overall growth of these animals was not improved by RemicadeTM treatment, the number of apoptotic or dead chondrocytes were reduced by ~50%, as was the infiltration of synovial tissue into the underlying bone. These results indicated positive effects of RemicadeTM treatment at the sites of pathology. RemicadeTM treatment also reversed the established inflammatory disease in older animals. Thus, these studies revealed the important role of TLR4 signaling in the pathogenesis of MPS bone and joint disease, and suggested that targeting a downstream mediator of this pathway, TNF-?, might have a positive effect in attenuating the inflammatory response in MPS. This could lead to improved joint/bone pathology and also increase the accessibility of synovial tissues to recombinant proteins, thus improving the efficacy of ERT.

Towards this end, we have recently finished our combination studies in the MPS VI rats with RemicadeTM and ERT (NaglazymeTM), and are in the process of performing a comprehensive evaluation of the effects on the bone and joint disease. We hope that these studies will provide essential data that might fast-track this therapy into the clinic to be evaluated in MPS patients.