1st Year Research Reviews – 2013

1st year Research Reviews – 2013

General Grant

Lachlan Smith, PhD
University of Pennsylvania
Philadelphia, PA

“Pathogenesis of Bone Disease in Mucopolysaccharidosis Disorders”

The broad objectives of this proposal are to establish the molecular mechanisms of failed bone formation in MPS disorders, focusing primarily on MPS types VII and I. Our overall hypothesis is that accumulation of partially degraded, functionally abnormal GAGs in developing MPS vertebrae impairs chondrocyte proliferation and differentiation by disrupting key regulatory signaling pathways. Highlights of our research over the past 12 months are as follows:

We have pinpointed the critical developmental window and chondrocyte differentiation stage when failed bone formation first manifests in MPS VII dogs:

We collected thoracic vertebrae from 9 day and 14 day old litter-matched normal and MPS VII affected dogs for quantitative PCR and micro-computed tomography (microCT) analyses. This age range was selected based on radiographic, longitudinal studies of vertebral bone formation in MPS VII dogs. Comparison of mRNA expression of chondrocyte differentiation (SOX9, RUNX2, COL10A1) and osteoblast (ALPL, BGLAP) markers, and microCT visualization of vertebral bodies of normal and MPS VII affected dogs showed striking differences in bone formation (Figure 1) at 14 days whereas at 9 days, no significant differences besides a trend towards lower RUNX2 expression were detected. Interestingly, SOX9 expression was downregulated at 14 days for both normal and affected dogs suggesting that both chondrocyte populations receive regulatory signals for proliferation but that MPS VII chondrocytes fail to progress to hypertrophy. Furthermore, bone volume fraction and bone mineral density quantification of the vertebral primary ossification centers showed no differences between normal and affected dogs suggesting that at this stage, the most affected developmental pathways involve activation of secondary ossification centers. This work lays the foundation for future mechanistic investigations into bone disease in MPS VII.







Figure 1. MicroCT and mRNA analysis of vertebral epiphyses of developing MPS VII dogs. A. 9-days Normal. B. 9-days MPS VII. C. 14-days Normal, epiphyseal bone formation (*). D. 14-days MPS VII, absence of epiphyseal bone formation. E. mRNA expression of chondrocyte differentiation (SOX9, RUNX2, COL10A1) and osteoblast (ALPL, BGLAP) markers. ***p<0.001; **p<0.01; *p<0.05; +p<0.1.

We have identified the key pathways required for bone formation that are dysregulated in MPS VII dogs using whole transcriptome next generation sequencing (RNA-Seq):

 We have used RNA-Seq to map the transcriptome of chondrocytes isolated from the vertebral epiphyses of normal and MPS VII dogs, initially at 14-days. RNA-Seq revealed ~4000 genes that were significantly differentially expressed between MPS VII and normal. Key bone formation pathways that were implicated included: Wnt/beta-catenin (179 genes); TGF-beta (87 genes); BMP (77 genes); and FGF (89 genes) (Figure 2). We are currently confirming these results using additional assays, and in the coming months will conduct cell culture assays to elucidate differential responses of normal and MPS VII chondrocytes to regulators of hypertrophic differentiation.






Figure 2. Highly dysregulated pathways (MPS VII vs Normal) implicated in endochondral ossification identified through RNA-Seq.

Key goals for year 2 of this grant include: 1) Undertaking RNA-Seq for tissue from 9-day old MPS VII animals (tissue already collected) and performing confirmatory assays for both developmental stages. 2) Examining dysregulation of additional pathways from RNA-Seq data. 3) Performing functional cell culture assays as described above. 4) Comparing these results for MPS VII to those for MPS I (tissue already collected), to identify potential common disease mechanisms. Our long-term goal is to identify new therapeutic targets for treating bone disease in MPS.

 General Grant

Richard Steet, PhD               Dr. Dwight Koeberl
University of Georgia           Duke University
Athens, GA                               Durham, NC

“Adjunctive therapy for Hurler syndrome.”

Background: Enzyme replacement therapy (ERT) remains an important therapeutic option for many lysosomal storage disorders. ERT relies on the ability of recombinant enzyme to be taken up into tissues by cell surface receptors and trafficked to the lysosome, where it would replace the missing or defective enzyme and be able to clear intralysosomal storage. Although several receptor systems have been proposed to mediate cell surface uptake, mannose 6-phosphate (M6P)-dependent mechanisms are the best studied. Recombinant enzymes that are modified with M6P residues on their sugar chains can bind to the cation-independent M6P receptor (CI-MPR) on the cell surface and target back to the lysosome. This cell surface localization is critical for ERT since it provides both a means of entry into the cell and a route to the lysosome.

Several factors have limited the efficacy of ERT in lysosomal storage disorders. Some tissues such as muscle have low levels of CI-MPR expression, limiting enzyme uptake. The abundance of carbohydrate-dependent receptors such as the mannose receptor in the liver can result in rapid clearance of recombinant enzymes in this tissue, thus lowering the amount that can be taken up by affected tissues. Furthermore, the presence of intracellular storage can alter receptor trafficking and the fusion of endosomes and lysosomes, limiting the delivery of internalized enzymes to the lysosomes. The specific aims shown below will test the hypothesis that ß2-agonist adjunctive therapy can be used to improve ERT in the context of MPS I by altering the expression and/or trafficking of the CI-MPR. We will 1) test the ability of ß2-agonists such as clenbuterol or albuterol to increase receptor-mediated uptake of AldurazymeTM into MPS I fibroblasts, 2) determine the mechanisms whereby ß2-agonist treatment improves enzyme uptake and/or intracellular trafficking, and 3) investigate whether ß2-agonist treatment increases AldurazymeTM uptake into MPS I mouse tissues such as the brain.

Progress: We have demonstrated that ß2-agonist (e.g. albuterol) treatment increases the uptake and activity of therapeutic enzyme in both MPS I and Pompe fibroblasts by 2-3 fold. This effect is rapid, dose-dependent and CI-MPR-dependent since the presence of free mannose 6-phosphate completely blocks uptake. We further showed that addition of the ß2-antagonist, propranolol, along with albuterol, suppresses the increased uptake of enzyme, supporting an essential role for ß2-adrenergic receptor activation in the enhancement of enzyme uptake. The increase in enzyme uptake into MPS I fibroblasts upon albuterol treatment also corresponded with a modest decrease in GAG storage when compared to enzyme alone. Western blot analysis of albuterol-treated MPS I and Pompe fibroblasts showed no differences in the total level of the CI-MPR upon ß2-agonist exposure, suggesting that the increased uptake of therapeutic enzyme may be regulated instead by receptor recycling or internalization.

Collectively, these findings: 1) point to a novel mechanism of action for ß2-agonists in the CIMPR-dependent internalization of lysosomal enzymes and 2) have important clinical implications for the use of ß2-agonists and antagonists in the context of LSDs. Over the next year we plan to further explore the mechanisms of action using a combination of microscopy-based studies and biochemical analysis to determine the relevant intracellular signaling pathways that mediate the positive effect of albuterol. The ability of ß2-agonist treatment to stimulate enzyme uptake into MPS I mouse tissues will also be further tested. Our preliminary studies show that the combination of albuterol treatment with ERT further reduced GAGs in liver, in comparison with ERT alone in mice with MPS I.


Vito Ferro, PhD
University of Queensland
Brisbane, Queensland

“Development of pharmacological chaperone therapy for MPS II.

First Year Report: National MPS Society Research Initiative 2013

“Development of Pharmacological Chaperone Therapy for MPS II”
Investigators: A/Prof Vito Ferro, School of Chemistry & Molecular Biosciences, the University of Queensland; and Prof John Hopwood, LDRU, Adelaide.

Summary (in lay terms):
The aim of this project is the development of small molecule drugs for MPS II. Conventional small molecule drugs can be taken orally as a pill and have the potential to reach the brain in order to treat the more severe forms of MPS II, unlike enzyme replacement therapy (Elaprase) which can’t cross the “blood-brain barrier”. Our approach is to develop compounds for so-called Pharmacological Chaperone Therapy (PCT; aka Enzyme Enhancement Therapy or EET). This is an approach to treatment that has shown great promise in other lysosomal storage disorders, e.g., Gaucher’s and Fabry
disease, but has not been fully exploited yet for the mucopolysaccharidoses such as MPS II. This is thus the first time that this promising approach has been attempted for MPS
II. PCT works by having a small molecule drug (a “chaperone”) attach itself to the defective
enzyme, in this case iduronate sulfatase, and stabilizing it so it can do its intended job: to
degrade the mucopolysaccharides in the cell. In order to prepare compounds for PCT that are suitable for testing we need to synthesize small molecules that resemble the sugar iduronic acid, the component of the mucopolysaccharides that is degraded by the enzyme iduronate sulfatase. Two approaches have been explored for this purpose: (i) the synthesis of modified iduronic acid derivatives, and (ii) the synthesis of a compound known to bind to iduronate sulfatase, and derivatives of this compound. The initial stages of this process involved developing chemistries to prepare the required compounds. The first set of test compounds are now in hand and we are currently waiting for their testing using purified enzyme and cell preparations from MPS II patients at the Lysosomal Diseases
Research Unit in Adelaide.

Description of Experiments
There are two distinct series of compounds we have been working on:

(i) Compounds derived from iduronic acid. After settling on a preferred route to iduronic acid derivatives via epimerization at C5 of diacetone glucose, we prepared the advanced intermediate 1 (Fig. 1). This compound has the required protecting groups at positions C3, C4 and C6 and has the OH group at C2 free for subsequent transformations. We have successfully transformed this into two compounds for testing (2 and 3). However, a number of problems have been encountered with elimination side reactions as well as the final oxidation step and this has hampered efforts to obtain additional compounds in this series to date. Significant time was spent to try to overcome these problems. The PhD student who has been working on this series, Shifaza Mohamed, is currently on maternity leave.(ii) Compounds derived from anhydromannitol-1-sulfate. Following discussions with Prof John Hopwood, anhydromannitol-1-sulfate (compound 9) was identified as a potential chaperone. 9 had previously been prepared in 1990 in small quantities via an enzymatic route and found to be a potent inhibitor of iduronate sulfatase (IDS). As such, it is a potential chaperone and thus we have synthesized it on a multi-milligram scale from glucosamine. We have also prepared a number of derivatives  using  different,  non-hydrolysable  groups  as  sulfate  mimics  as  well  as  some  more lipophilic “prodrugs”, ie, compounds with greater lipophilicity which should be better taken up by cells.

Compound 1 was previously converted into ido-configured methyl ester 2 via a series of reactions involving oxidation and Emmons-Wadsworth reaction followed by hydrogenation. Both compound 2 and its peracetate 3 will be submitted for testing. However, attempts to oxidize C6 to the carboxylate to provide a second series of compounds with structure closer to iduronic acid have been problematic. We have also sought to prepare analogues of 2 and 3 with alternative sulfate group mimics following similar sequences. Initial efforts were promising, however, the use of different
Emmons-Wadsworth reagents have been plagued by elimination side reactions across C4 and C5 due to the strongly basic conditions required. Considerable time has thus been expended in finding alternative protecting group strategies that avoid the use of locked 4,6-benzylidene acetal which is prone to elimination.


Jeffrey Esko, PhD
University of California, San Diego
La Jolla, CA

“Delivery of sulfamidase to the brain.”

2013-2014 Progress Report

Over the last year we successfully produced recombinant sulfamidase (SGSH) by transient transfection of HEK293 cells in large-scale suspension culture under serum free conditions. Purity was estimated to >98% and the enzyme was found to be free of several other lysosomal enzyme activities (-iduronidase and acid -glucosidase). Enzyme activity was greater than commercially available material. We also optimize conditions for conjugating the enzyme with our carrier system (guanidinylated neomycin, GNeo), which had no impact on the activity of the enzyme.

We had shown previously that SGSH and GNeo-SGSH were cleared very rapidly after intravenous injection. Attempts to increase the half-life in the plasma by adding heparin, by repetitive dosing, and by continuous infusion the enzyme had no effect. To circumvent this problem, we tested if we might be able to deliver enzyme intranasally, with particular interest in delivery of the enzyme to the brain. In preliminary studies we showed that GNeo-conjugation allowed efficient delivery to the brain after intranasal delivery. GNeo does not appear to have adverse side effects based on the lack of obvious changes in gross behavior, feeding behavior, and growth of the mice after administering the conjugate every other day for four weeks.

MPSIIIA is characterized by accumulation of heparan sulfate and the pathological carbohydrate biomarker N-sulfoglucosamine on the non-reducing end (NRE) of the chain. Analysis of the NRE in the glycosaminoglycans extracted from whole brain after treatment of mice every other day for 1 month with GNeo-SGSH showed a ~25% reduction in the biomarker. GNeo-SGSH was ~5-fold more effective than SGSH. These studies will be extended to two months to determine if further reduction of the biomarker occurs.

It remains unclear how HS storage leads to neurological behavioral phenotypes and whether damage resulting from HS storage in the brain is reversible. Therefore, in the most ideal circumstances enzyme replacement should begin prior to or at the onset of pathological changes in the brain. To study this problem, we conducted a natural history study using a hypomorphic MPSIIIA mouse model (Sgshh/h) that exhibits 3-4% residual enzyme activity. Our analysis showed that accumulation was detectable at birth in the Sgshh/h mice and increased in proportion to brain growth during the first several postnatal weeks. Our data show that heparan sulfate accumulation is present in the early postnatal brain and therefore enzyme replacement therapy should begin shortly following birth. We have also begun to examine the impact of early storage on neurodevelopment in the mice in order to better understand the relationship of storage to early behavioral changes that occur prior to overt neuropathological changes.



Adriana Montano, PhD                       Raymond Wang, MD

St. Louis University                            CHOC Children’s Hospital
St. Louis, MO                                        Orange, CA

“Manifestations of Cardiovascular Disease in Morquio A: Evaluation, Assessment, and Therapy”

Cardiovascular disease (CVD) is an increasingly recognized disease manifestation of mucopolysaccharidoses (MPS). While glycosaminoglycans (GAGs), including chondroitin, heparan, and dermatan sulfates, are normally present in the proteoglycans of major arteries such as the aorta, the enzymatic deficiencies leading to MPS result in vascular GAG accumulation and subsequent CVD. The broad goal of this study is to characterize the extent of the Morquio A disease (MPS IVA) in the cardiovascular system of human patients and mouse model, and to assess and potentially enhance the effect of intravenous enzyme replacement therapy (ERT) upon the pathophysiology of cardiovascular disease.

1.   Comparison of carotid intima-media thickness and carotid stiffness in individuals with MPS IVA (treated and untreated) vs. healthy age- and gender-matched controls.

 Carotid intima-media thickness (cIMT) is a non-invasive ultrasonographic measurement that correlates well with carotid histology and has been repeatedly validated as a marker and predictive risk factor for cardiovascular events. During the first year of support, we received IRB approval from three institutions: Saint Louis University, CHOC Children’s and the University of Minnesota to conduct the human studies in MPS IVA population. After biochemical and/or molecular confirmation of MPS IVA diagnosis, we have consented four patients and have performed bilateral carotid artery scanning in three of them (Fig. 1). The cIMT is compared between treated and untreated MPS IVA patients, and healthy age- and gender-matched controls. Additional patients are being recruited for this study.

Figure 1.   Carotid  intima-media thickness (cIMT) of an MPS IVA patient. 






2. Evaluation of heart failure in the MPS IVA knock-out mouse model and the  effect  of Enzyme replacement therapy on cardiac  dysfunction.

 We have maintained our mouse colony, determined  genotype  and  selected  mice  for the treatment. The mouse protocol was amended to  perform  echocardiograms.  We are continuously producing and purifying GALNS enzyme for enzyme  replacement therapy (ERT).  Two  different groups  of  mice are currently under  treatment  with  ERT  and one group is our untreated control. We have obtained urine every week  and  blood  every other week from the treated and untreated mice.

Figure 2. A. B-mode and B. M-mode images of the left ventricle of an untreated C57BL mouse.





We also obtained hearts from mice treated for 2 months and fixed them in 10% formalin and 4% paraformaldehyde. For the evaluation of cardiac systolic and diastolic performance in mice we have used transthoracic echocardiogram which is a non-invasive procedure. Figure 2 shows an example of a two- dimensional imaging (A) of the parasternal short axis of the left ventricle, and the assessment  of  left ventricle chamber dimensions by M-mode echocardiography (B).


ML II/II (Partnership Grant with ISMRD)

Heather Flanagan-Steet, PhD
Complex Carbohydrate Research Center
University of Georgia

“Investigating the role of cathepsin proteases in NL II cardiac pathology”

Progress Report Year 1 MPS/ISMRD Grant to Heather Flanagan-Steet – Investigating the Role of Cathepsin Proteases in MLII Cardiac Pathology

In the last year we have made progress (detailed below) on several aspects of the aims outlined in this grant. In addition to progress on the proposed studies, we have also recently made two additional advances that will have a major impact on our ongoing pursuit of molecular mediators of MLII cardiac pathology.  These include 1) the generation of a TALEN-mediated MLII mutant line and 2) the isolation of a separate MLII mutant line from a sperm TILLING screen. These two unique zebrafish lines are essential to confirm all of our morpholino-based findings, for analyses of later stage aspects of disease, and for small molecule screens. When published we will credit MPS/ISMRD for contributing to the establishment of these vital tools.

Aim 1: Assess individual contribution of cathepsin K and L toward cardiac defects in ML zebrafish.

1) Cathepsin expression and generation of transgenic animals.  Immunohistochemical analyses of cathepsin K and L demonstrate that both proteases are expressed in multiple heart tissues, including the myocardium and epicardium. These analyses have guided the choice of tissue specific promoters for transgenic constructs that will be used to generate zebrafish expressing fluorophor tagged WT and mutant versions of Cts K and L. Toward this goal, we have now successfully shown that following mRNA injection, the CtsK-mCherry fusion protein is both expressed and active in developing embryos. Establishing these parameters was critical before proceeding with transgenesis. Therefore the final DNA constructs for transgenesis are currently in production at Gene Art. Initial injections for the generation of transgenic lines should be completed by early next year.

2) Inhibition of cathespin K in MLII. To assess ctsK’s contribution toward ML cardiac pathology, its expression or activity was inhibited in the MLII background.  CtsK expression was genetically inhibited using one of two gene specific morpholinos and its activity reduced pharmacologically using the FDA-approved cathepsin K inhibitor Odanacatib.  Preliminary analyses of multiple aspects of cardiac morphology and function, including formation of the AV valve and unidirectional blood flow, suggest both treatments substantially improve MLII cardiac development.  Additional experiments to confirm and extend these findings are currently underway.

Aim 2Determine whether increased cathepsin activity impacts TGFß signaling in MLII hearts.

1) Are there differences in either TGFß or BMP signaling in WT and MLII hearts? In combination with TGFß and BMP-reporter transgenic animals, immunohistochemicalanalyses of pSmad levels is being used to assess differences in signaling between WT and MLII hearts. In parallel studies pharmacological inhibition of TGFß/BMP receptor activation is being used to address the pathogenic significance of noted differences in pathway activation. These studies are currently underway. Once these analyses are completed we will address whether genetic or pharmacological inhibition of cathepsin K also affects pSmad levels and reporter transgene activation in MLII hearts.

Aim 3 – Determine which patient mutations are pathogenic for cardiac dysfunction.

To identify which patient mutations are associated with altered cardiac morphology and function, we have introduced mRNA bearing specific lesions in the morphant background and compared their ability to rescue heart phenotypes with ML animals co-injected with WT mRNA.  We have analyzed three different sets of mutations including several within the DMAP domain (including K732N), three mutations within Notch domain 1 (C442Y, C461G, and C468S), and one mutation in Notch domain 2. Thus far analyses have been limited to gross morphology. Unlike WT gnptab mRNA, which reduces cardiac edema, restores normal cardiac morphology, and increases blood flow in ³85% of the animals analyzed, mRNA bearing the K732N mutation did not significantly restore any of the tested parameters.  Therefore, as previously noted in the cartilage, the DMAP domain is essential for normal phosphotransferase function – and mutations within it are highly likely to be pathogenic in both the cartilage/bone and the heart.  In contrast, preliminary analyses of the three Notch domain 1 mutations suggest that the C442Y lesion, which has been associated with MLIII, is less pathogenic in the heart than the ML-intermediate lesion C468S.  This is supported by the fact that mRNA“rescue” experiments show improved cardiac morphology and reduced edema in 85% of the MLII animals injected with the C442Y-containing mRNA versus only 40% recovery with the mRNA carrying the C468S lesion. mRNAs bearing either the C461G (Notch 1/MLIII) or C505Y (Notch2/MLIII) rescued gross cardiac pathology ~60% of the time, suggesting that in addition to differences in pathogenic severity between MLII and MLIII, different mutations within a single ML class may have different degrees of pathogenicity. More in depth experiments are currently underway. Collectively these data support the idea that genotype-phenotype correlations can be established in this system, and as such may eventually combine with patient data to predict the severity and course of mutation specific tissue pathologies.

Summary of progress to date:

Although all studies are still in progress and much work remains, we are highly encouraged by the findings to date.  First, the fact that both cathepsin K knockdown and treatment with Odanacatib improved MLII heart development is very promising.  These data not only support a central role for cathepsin(s) in primary pathogenesis but point to FDA-approved drugs like Odanacatib for future consideration. Although these findings are preliminary and need to be independently confirmed, they are certainly encouraging.  Second, the fact that both cathepsin K and TGFß signaling may be involved in cardiac pathology suggests common mechanisms may underlie ML skeletal and heart dysfunction.  If true, this would not only be an important advance for future scientific investigation, but also mean that an individual modality may therapeutically improve both systems. Third, the tools generated thus far and those underway will serve as novel platforms to both further investigate these and other emerging mechanisms and to rapidly screen the efficacy of potential drugs.

Comment on year 1 budget residual funds:

As clear from the financial report of year 1’s expenditures there was a $2300 carry-over.  I wanted to qualify that this money has essentially been spent, but because the “reagents” are in production they will not post on this year’s financial report.  $800 of the remaining balance will be payed to the Zebrafish TILLING Project Consortium at The Fred Hutchinson Cancer Research Center.  This money covers part of the cost for them to screen for the gnptab mutant, which is now identified and being raised for shipment to us.  The remaining $1500 is also already allocated to pay Gene Art for synthesizing one of the large DNA constructs necessary to generate the cathespin transgenic animals.