2nd Year Research Reviews – 2011


Drs. Patricia Dickson and Stephen Kaler
UCLA Harbor, Los Angeles, CA

Choroid plexus-directed viral gene therapy as a source of cerebrospinal fluid NAGLU-IGF2 for Sanfilippo B syndrome

The goal of this project is to evaluate whether adeno-associated virus (AAV) gene therapy vectors can remodel choroid plexus epithelia to produce N-acetylglucosaminidase (NAGLU) fused to insulin-like growth factor 2 (IGF2).  The therapeutic purpose underlying our experiments is to enable secretion of the missing lysosomal enzyme into the ventricles of the brain in a mouse model of Sanfilippo B syndrome. Since choroid plexus epithelia turn over at an extremely slow rate, viral transduction of these cells with a correct version of NAGLU-IGF2 would potentially provide a permanent source of the enzyme in the cerebrospinal fluid for global brain distribution.

2011 2nd yr fig 1

Figure 1. Enhanced intracellular uptake of NAGLU-IGF2 (open circles) by Sanfilippo B fibroblasts compared to recombinant human NAGLU (rhNAGLU, black circles).

We produced a cDNA construct containing the gene that encodes NAGLU (the enzyme deficient in Sanfilippo B syndrome) fused to IGF2. Manufactured forms of NAGLU lack mannose 6-phosphorylation, which limits cellular uptake (Fig. 1). In contrast, NAGLU-IGF2 binds to the mannose 6-phosphate receptor so that delivery is greatly enhanced (Fig. 1). We documented robust expression and NAGLU enzyme activity (1.7 units/mg protein) in HEK293T cells transfected with the NAGLU-IGF2 construct. We next generated a recombinant AAV serotype 5 vector harboring NAGLU-IGF2 (rAAV5.NAGLU-IGF2) under the control of a chicken beta actin promoter and cytomegalovirus enhancer. The rAAV5 serotype is known from our previous work to target choroid plexus epithelia (Donsante et al., 2011), and we confirmed this in wild type neonatal mice (Fig. 2a). We then administered 5×1010 vector genomes of rAAV5.NAGLU-IGF2 to the left lateral brain ventricle of 12 week old Sanfilippo B mice (Naglu-/-). Correct ventricular localization technique was confirmed in a subset of mice by dye injection. NAGLU enzymatic activity in brain sections reached several fold-normal (Fig. 2b) and immunohistochemical evaluation detected NAGLU-IGF2 throughout the brain and into neurons (Fig. 2c).

Our ongoing testing is evaluating the safety and efficacy of this approach to prevent or correct brain pathology and neurological abnormalities in older Sanfilippo B mice. This unique therapeutic approach combines the benefits of M6P-independent endocytosis and viral gene therapy to enable efficient NAGLU-IGF2 distribution throughout central nervous system. Our exciting preliminary results were accepted for presentation at the American Society for Gene and Cell Therapy annual meeting (May 2013, Salt Lake City, UT). The potential impact on clinical practice in the field of LSD is high since, if the proposed aims are successfully achieved, the largest current barriers to health for patients with LSDs will be circumvented. In addition, the principles of gene transfer and CSF protein transport being investigated in this project will potentially be useful for other neurometabolic diseases with global effects on brain.

 2011 2nd yr fig 2

Figure 2. Transduction of choroid plexus epithelia by AAV5-NAGLU and delivery of NAGLU-IGF2 to brain parenchyma in Naglu-/- mice. A. Robust expression of NAGLU-IGF2 (brown stain) in 4th ventricle choroid plexus epithelia of wild type P2 mice after E15 administration.  An anti-IGF2 antibody was used for immunohistochemical detection. Negative control slide (anti-IgG) shows no staining. B. Robust enzyme activity observed in brains from treated Naglu-/-  mice. Dashed line indicates activity in Naglu+/- heterozygotes.      C. Neuronal detection of NAGLU-IGF2 in basal amygdala and stria terminalis of an adult-treated NAGLU-/- mouse.



Prof Vito Ferro
School of Chemistry & Molecular Biosciences, the University of Queensland
Small molecule chaperones for EET for MPS II

Final Report: National MPS Society Research Initiative 2011-2013 (extension to 2014)
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 Enzyme Enhancement Therapy (EET; aka “Pharmacological Chaperone Therapy”). 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 yet to be tried for the mucopolysaccharidoses such as MPS II. This is thus the first time that this promising approach has been attempted for MPS II. EET 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 EET 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 8 test compounds are now in hand and preparations are in progress for their testing using purified enzyme and cell preparations from MPS II patients. The testing will be conducted by our collaborators at the Lysosomal Diseases Research Unit (LDRU) in Adelaide. Our collaborators have established a fluorometric assay for determining the affinity of the compounds for the enzyme. In addition, they have identified MPS II patient mutations that are likely to respond to chaperone therapy. Skin fibroblast cells from these patients will be used to test the compounds for chaperone activity, once Human Ethics Committee approval has been obtained. These studies should demonstrate cellular uptake of chaperone molecules and a reduction of cellular mucopolysaccharide levels, and should identify which mutations respond (best) to chaperone therapy.

We anticipate that the biological testing will identify the most promising candidate chaperones for further optimization and provide proof of concept that this is a viable approach for MPS II treatment. This project continues under funding from an MPS Society Grant (for 2013-2015) with the aim of generating sufficient preliminary data to obtain more significant research funding to accelerate this program towards clinical candidates.


General Grants

Alberto Auricchio  
Fondazione Telethon, Naples, Italy
Gene therapy of MPS VI

Adriana M. Montaño
Saint Louis University
Role of inflammation in pathogenesis of MPS IVA

Morquio A disease (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive disorder, caused by the  deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Patients with Morquio A disease have accumulation of the glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate, mainly in bone and cartilage, causing systemic skeletal dysplasia.  The broad goals of this research were to characterize the  immune  profile  of  Morquio  A  mouse  model  and  to  elucidate  the  role  of  cartilage  and  bone inflammatory reactions in the pathogenesis of Morquio A disease through investigation of secreted inflammatory factors involved in cartilage destruction and bone remodeling.

1.   Characterization of the immune profile of the Morquio A mouse model.

Immune response to enzyme replacement therapy (ERT) is the principal limitation in the effectiveness of the treatment. The first step in the characterization of the immune profile of the knock-out Morquio A mouse model was to investigate the immune response after ERT.

We have found that Morquio A mice undergoing ERT have: i) the highest immune humoral response towards the recombinant human GALNS enzyme at 14 weeks of treatment, and ii) the highest cellular response at 16 weeks of treatment. This is consistent with previous observations where age of the mice and length of treatment play a role in the levels of immune response.

To ameliorate both humoral and cellular responses in Morquio A mice we are looking at oral tolerance as an alternative. This will to not only decrease the need of administration of immune suppressors to avoid immune responses but also will improve the efficacy of enzyme by avoiding presence of neutralizing antibodies to the enzyme used in ERT.

2.   Characterization of expression profiles of genes associated to the pathogenesis of Morquio A disease.

The  current study  sought  to  understand  if  and  how  inflammation  impacts  the  autophagic pathway  in cartilage tissue of a Morquio A mouse model.

We performed expression analysis of over 60 genes using qRT-PCR in Morquio A and wild type mice at 1, 3,

9 and 12 months of age. We have found that inflammatory, apoptotic and autophagic markers were up- regulated  in  12-month-old  MKC  mice  compared  to  age-matched  wild-type  controls  and  compared  to younger mice.  These findings suggest that inflammatory receptors modulate autophagy and apoptosis in cartilage tissue of Morquio A mice.  These data could be used in developing future treatment modalities for bone growth abnormalities in Morquio A patients.


Richard Steet, Ph.D.
University of Georgia, Athens, GA
Blockade of cathepsin activity and TGF-beta signaling as a therapeutic approach for LSDs

We continue to take advantage of our ML-II zebrafish model to explore the relevance of cathepsin and matrix metalloproteases in both the cartilage and cardiac pathogenesis of this disease.  Our published work in this area has demonstrated that inhibition of cathepsin K by genetic and pharmacological means leads to substantial correction of the cartilage defects in ML-II embryos as well as an unexpected reduction in the activity of other proteases.  Using multiple approaches, we have also shown that this protease is hypersecreted from zebrafish chondrocytes and subject to abnormal proteolytic activation to its mature form.

Over the last year, we have begun to assess the role of other proteases (cathepsin L and MMP13) in the cartilage phenotypes.  Using a specific inhibitor to MMP-13 (whose activity is increased 10-12-fold in ML-II zebrafish), we showed that a maximal inhibition of 70% could be achieved in embryo lysates.  Despite this level of inhibition, no phenotypic correction of the cartilage phenotype was observed in the embryo, suggesting that this protease is not a central contributor to the developmental defects in this particular tissue.  MMP13’s contribution to later stages of disease and to other tissues (such as the cardiac defect) warrants further exploration.  More recently, we have begun investigating the pathogenic contribution of cathepsin L and have shown that this protease also appears to undergo abnormal proteolytic activation in ML-II embryos.  Since four homologous cathepsin L genes are now known to exist in zebrafish, we are currently characterizing the expression and localization of their individual transcripts.  These analyses, which serve as a prelude to genetically manipulating cathepsin L expression, have revealed that one previously unidentified gene, ctsL1b, is the only isoform with increased transcript levels in the ML-II model.  This is important, as our original transcript analyses did not distinguish between the four isoforms. This is also consistent with the finding that morpholino-knock down of the cathepsin L1a isoform did not reduce total cathepsin L activity.  In light of these new expression studies, we are continuing to manipulate cathepsin L expression, with cts1b as the main gene targeted.

Another goal of the proposed research was to address the relevance of altered TGFbeta signaling in ML-II pathogenesis.  Investigation of this pathway and its role in various ML-II phenotypes is underway, and has lead to the identification of several molecular hallmarks for both the cartilage and cardiac defects. We are now positioned to determine whether directly manipulating TGFbeta signaling impacts the phenotypes.

The demonstration that a reduction in cathepsin activity can provide some therapeutic benefit suggests that further study into small molecule protease inhibitors for the treatment of MPS and MPS-related disorders is warranted.  We thank the MPS Society for their continued support of this research and remain hopeful that the avenues of research that stem from this work can be translated into therapies for MPS and MPS-related disorders.