2nd Year Research Reviews – 2009

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:

Correction of CNS defects in the MPSII mouse model via systemic enzyme replacement therapy.
Polito VA, Abbondante S, Polishchuk RS, Nusco E, Salvia R and Cosma MP. Human Molecular Genetics 2010, Vol 19, No 24.

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, is a devastating disorder associated with a shortened life expectancy. Patients affected by MPSII have a variety of symptoms that affect all organs of the body and may include progressive cognitive impairment. MPSII is due to inactivity of the enzyme iduronate-2-sulfatase (IDS), which results in the accumulation of storage material in the lysosomes, such as dermatan and heparan sulfates, with consequent cell degeneration in all tissues including, in the severe phenotype, neuro- degeneration in the central nervous system (CNS). To date, the only treatment available is systemic infusion of IDS, which ameliorates exclusively certain visceral defects. Therefore, it is important to simultaneously treat the visceral and CNS defects of the MPSII patients. Here, we have developed enzyme replacement therapy (ERT) protocols in a mouse model that allow the IDS to reach the brain, with the substantial correction of the CNS phenotype and of the neurobehavioral features. Treatments were beneficial even in adult and old MPSII mice, using relatively low doses of infused IDS over long intervals. This study demonstrates that CNS defects of MPSII mice can be treated by systemic ERT, providing the potential for development of an effective treatment for MPSII patients.

Dr. Jeffrey D. Esko
University of California, San Diego, CA
Substrate Reduction Strategy for MPS IIIA
The original focus of this grant was to demonstrate whether substrate reduction would prove effective as a treatment for Sanfiippo syndromes. As outlined in our progress report 2010, siRNA knock down of the heparan sulfate biosynthetic gene EXT1 in human MPSIIIa fibroblasts is able to diminish lysosomal storage by ~50% ex vivo. To test substrate reduction therapy in vivo, we have crossed MPSIIIa (Sgsh-/-) mice onto an Ext1+/- background. As shown in figure 1a, Ext1 heterozygosity reduced heparan sulfate chain length by ~25% in mouse embryonic fibroblasts (MEFs) isolated from these mice. This reduction in the amount of cell surface heparan sulfate was sufficient to normalize turnover by ~30% (Fig. 1b), demonstrating the efficacy of this substrate reduction therapy approach.




Currently we are quantifying the amount of heparan sulfate in the tissues ofSgsh-/- mice that are heterozygous forExt1 using mass spectrometry to determine the impact of substrate reduction therapy in different tissues. In addition, the histological markers GFAP and Ubiquitin are being used to determine the impact of substrate reduction on astrocytosis and aberrant autophagy in the brain, respectively. To complement these studies we are also breeding MPSIIIa mice heterozygous for bothExt1 andExt2. Double heterozygosity should result in heparan sulfate chains even shorter than those observed inExt1heterozygotes and may have a greater impact on lysosomal storage and pathology. These studies should be completed in the next 3 – 6 months.

In addition to its ability to reduce lysosomal storage, we hypothesized that substrate reduction therapy may serve as a cost effective way to increase the effectiveness of enzyme replacement therapy. To test this, we compared the dose of recombinant sulfamidase necessary to normalize heparan sulfate turnover in MEFs derived fromSgsh-/- orSgsh-/-;Ext1+/- mice. Importantly, the effective dose (ED50) was improved by more than 2- fold in MPSIIIa MEFs heterozygous forExt1, suggesting that combined substrate reduction therapy and enzyme replacement therapy may provide a more efficient approach to treating Sanfilippo disease. To determine the impact ofExt heterozygosity on the sensitivity ofSgsh-/- mice to enzyme replacement therapy in vivo, we are currently quantifying heparan sulfate levels in different organs ofSgsh-/-,Sgsh-/-;Ext1+/- andSgsh-/-;Ext1+/-;Ext2+/- mice treated with 0, 0.1, 0.3 and 2 mg/kg recombinant sulfamidase using mass spectrometry. These studies should be completed in the 3 6 months.




In summary, we are approaching completion of both aims of the grant. Substrate reduction therapy has been demonstrated ex vivo using siRNA and gene knock out of Ext1 and in vivo studies are underway. In addition to the studies outlined above, funds provided by this grant has allowed us to successfully characterize secondary accumulation of dermatan sulfate in Sanfilippo fibroblasts (Lamanna et al.,J. Biol. Chem. 2011). To determine the impact of secondary dermatan sulfate storage on disease pathology, we are characterizing dermatan sulfate levels in different organs ofSgsh-/- mice as well as the sensitivity of secondary storage to enzyme replacement therapy.

We would like to thank the National MPS Society for this funding opportunity.


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

Cellular trafficking of the chimeric sulfamidase enzymes

Understanding the cellular trafficking of the chimeric sulfamidase enzymes (containing the alternative signal peptide and the ApoB LDLR-BD) is critical to evaluate the clinical efficacy of the engineered sulfamidase and to correctly interpret the results we will obtain from thein vivo studies. We analyzed the capability of the chimeric sulfamidase enzymes to correctly localize with lysosomal compartment in transfected cells and in cells receiving the enzyme upon uptake. The flag tag was replaced with a myc tag, which give more reliable and specific signal in immunofluorescence analysis. The IDSsp-SGSHmyc-ApoB and hAATsp-SGSHmyc-ApoB along with partial modified sulfamidase enzymes (containing only the alternative signal peptides: IDSsp-SGSHmyc and hAATsp-SGSHmyc) and not-modified sulfamidase (SGSHmyc) were transfected in MPS-IIIA MEFs. Immunostaining with anti-myc and anti-LAMP1 antibodies showed a lysosomal localization for both partial and final engineered constructs similar to that observed in cells transfected with not-modified sulfamidase (Fig. 1).

Figure 1. MPS-IIIAMEFcells weretransfectedwith either partial or final engineered constructs or with control not-modifiedSGSHconstruct. All thecontructscontained amyctag. The chimeric constructs display alysosomallocalization as showed byimmunostainingwithanti-mycandanti-LAMP1antibodies.

We then analyzed the capability of chimeric sulfamidase enzyme to be uptaken from MPS-IIIA cells and re-localize to lysosomal compartment. HepG2 cells were transfected with IDSsp-SGSHmyc-ApoB, hAATsp-SGSHmyc-ApoB along with partial modified sulfamidase enzymes (IDSsp-SGSHmyc and hAATsp-SGSHmyc) and non-modified sulfamidase (SGSHmyc). MPS-IIIA MEFs were then incubated with the conditioned medium derived from each transfection. The sulfamidase activity and the subcellular localization of both chimeric and not-modified sulfamidase enzymes were then evaluated in MPS-IIIA MEFs. As shown in figure 2 all the chimeric sulfamidase enzymes display a specific activity in the recipient MPS-IIIA MEF cells thus demonstrating the capability of the chimeric enzymes to be efficiently uptaken (Figure 2). In addition, the chimeric enzymes were also able to correctly re-localize to the lysosomal compartment upon be uptaken (Figure 3).




In vivo large study in MPS-IIIA mice

We expanded the MPS-IIIA colony and have obtained a large number of mice to be used in thein vivo large study.

We systemically injected one-month old MPS-IIIA mice with AAV2/8-TBG vectors harboring cDNAs encoding either AATsp or IDSsp N-terminal-modified sulfamidase enzymes containing the ApoB-LDLR-BD at their C-terminal (AATsp-SGSH-myc-ApoB-BD and IDSsp-SGSH-myc-ApoB-BD). Control mice have been injected with AAV2/8 expressing either the not-modified sulfamidase enzyme (SGSHmyc) or the partially modified sulfamidase enzymes (only containing the signal peptide replacement; AATsp-SGSH-myc and IDSsp-SGSH-myc).

We established three different time points after injections for the evaluation of CNS phenotype rescue (1 month, 3 months and 7 months). We sacrificed the mice corresponding to the first two time points (1 month and 3 months post-injection). These mice are under evaluation for CNS transduction (enzyme activity into the brain) and CNS pathology (storage, autophagy and inflammation).

We obtained very important results by measuring the sulfamidase enzyme activities into the brain of MPS-IIIA mice 3 months post-injection. A stronger sulfamidase activity was observed in the brain of MPS-IIIA mice injected with AAV2/8 encoding the modified sulfamidase enzyme AATsp-SGSH-myc-ApoB-BD when compared to the sulfamidase activity observed in the brain of MPS-IIIA mice injected with not-modified sulfamidase (SGSH-myc) (Figure 4). Moreover, the sulfamidase activity in the brain of MPS-IIIA injected with the modified sulfamidase was also associated to the presence of the enzyme into the brain of injected mice as shown by immunostaining anti-myc (Figure 5). We are now completing the analysis of the first two groups of injected mice corresponding to 1 month and 3 months post-injection (CNS transduction and CNS pathology).The mice a 7 months post-injection will be assessed for behavioural phenotype at the end of September 2011.

Figure 4.SGSHactivity was measured in the brain ofMPS-IIIAmice systemically injected withAAV2/8-TBG-hAATsp-SGSHmyc-ApoBorAAV2/8-TBG-SGSHmyc. The activity in control not- injectedMPS-IIIAbrain was also displayed.


Figure 5.Immunostaining anti-myc in the hippocampus of MPS-IIIA mice systemically injected with either AAV2/8-TBG-hAATsp-SGSHmyc-ApoB or AAV2/8-TBG-SGSHmyc. Control MPS-IIIA hippocampus was also displayed.

Dr. Calogera M. Simonaro
Mount Sinai School of Medicine, New York, NY
Novel Anti-Inflammatory Therapies For The Mucopolysaccharidoses

Although enzyme replacement therapy (ERT) is currently available for three MPS diseases and under development for others, the effects of this therapy on bone and cartilage are very limited. Thus, new approaches are clearly needed to more effectively treat MPS patients, alone or as adjuncts to ERT.

In this research project we comprehensively evaluated the bones and joints of MPS VI rats treated by anti-inflammatory therapy (i.e., anti-TNF-alpha therapy), alone and in combination with ERT. Several anti TNF-alpha drugs (e.g., Remicade, Embrel) are currently used clinically for the treatment of arthritis, inflammatory bowel disease and others common diseases, and the underlying premise of our work is that if these drugs proved effective in MPS animal models, they could potentially be fast-tracked into clinical use for MPS patients. Our previous work also has shown that the anti TNF-alpha inflammatory pathway is activated in many MPS animal models and patients (i.e., the toll-like receptor 4, TLR4, pathway), providing the scientific rationale for this approach.

In our first set of experiments we completed a proof-of-principle experiment in which the TNF-alpha inflammatory pathway (i.e., Toll-like receptor 4 pathway) was inactivated in mice with MPS VII. We found that when this pathway was inactivated from the earliest stages of development, there was a significant improvement in the bone length, bone growth plates, and joint pathology of the MPS VII animals. The paper reporting these findings was published in the Proceedings of the National Academy of Sciences in 2010.

We next turned to experiments evaluating anti TNF-alpha therapy in MPS VI rats, alone and in combination with ERT. In adult MPS VI rats that were treated by anti-TNF-alpha therapy, we found that the circulating levels of many inflammatory molecules in the blood were substantially reduced. Surprisingly, ERT alone also substantially reduced the circulating levels of these inflammatory markers, supporting the concept that the inflammatory pathways in MPS are directly activated by glycosaminoglycan (GAG) storage. We hypothesize that the reduction of circulating inflammatory makers by ERT reflects the delivery and function of the enzyme in readily assessable organs in the MPS animals, such as the liver, spleen, etc.

Animals treated by ERT or anti TNF-alpha therapy alone did not exhibit any significant improvement in bone growth. However, the articular (joint) cartilage of animals receiving anti-TNF-alpha therapy had fewer dying (apoptotic) cells, in contrast to animals receiving ERT that were similar to untreated MPS animals. Importantly, when the two treatments were combined, notable clinical and other improvements were observed. MPS VI rats receiving combined treated exhibited a more normal gait and could remain longer on a rotorod apparatus better than animals receiving either anti TNF-alpha or ERT. Their bones were also slightly longer, and there was much less evidence of inflammation in the joints (e.g., synovial tissue hyperplasia). Perhaps most impressively (and unexpectedly), animals receiving combined treatment had markedly less deformed tracheas, with thinner tracheal walls and wider open spaces. The results of these studies have been summarized in a manuscript that is currently in review.

In conclusion, these animal models studies have suggested that combining anti TNF-alpha therapy with ERT may provide substantial clinical benefits to MPS patients. In addition to a direct effect on inflammation, these therapies could also reduce immune responses against the recombinant enzymes, and improve the accessibility of the enzymes to pathologic sites in vivo. Future studies are planned in the dog and cat models of MPS, and using different anti-inflammatory therapies.