1st Year Research Reviews – 2015


Dr. Haiyan Fu, Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital

Gene Therapy Treating advanced MPS II via a systemic hIDS gene delivery using AAV serotype 9 vector”


While the experiments are ongoing, our data demonstrate that a single systemic scAAV9-hIDS gene delivery is safe and functionally beneficial for the treatment of neurological and somatic disorders in MPS II mice.

Based on our up-to-date pre-clinical data, we held the pre-IND meeting with the FDA on Sept. 29 2016 for a Phase 1/2 gene therapy clinical trial of systemic scAAV9-mCMV-hIDS delivery in MPS II patients, and the FDA granted us the exemption from the formal GLP-toxicology study.

To read the entire report, click here.



Dr. Brian Bigger, University of Manchester UK, Stem Cell and Neurotherapies Group

“Improving stem cell gene therapy for severe Mucopolysaccharidosis II”

Mucopolysaccharidosis type II is a paediatric neurological lysosomal storage disease caused by genetic deficiencies in the X-linked iduronate-2-sulphatase (IDS) gene whose normal role is to catalyse the degradation of both heparan sulphate and dermatan sulphate. Although enzyme replacement therapy is used to treat attenuated patients, the inability of the enzyme to cross the blood-brain barrier limits its efficacy in the severe form of the disease (Jones et al., 2009). Our goal is to increase the amount of enzyme produced by stem cells after transplant using gene therapy mediated by lentiviral vectors in a mouse model of MPSII. The aim of this study was to develop novel lentiviral vectors that produce IDS enzyme to specifically target the brain and cross the blood-brain barrier (BBB) more effectively.

We developed and cloned 6 novel enzyme-peptide fusion IDS genes into our most effective and clinically relevant lentiviral vector containing the myeloid-specific promoter CD11b (Figure 1). This plasmid aims to overexpress the IDS-peptide fusion enzyme required for the degradation of heparan sulphate and dermatan sulphate.

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Dr. Allison R. Kermode, Simon Fraser University, Department of Biological Sciences

“Validation of small molecule therapeutic leads for treatment of MPS I disease”

The major goal of this research is to advance small molecule “leads” – one of which was previously identified through library screening – as potential therapeutics for MPS I disease. This goal comprises:

Objective 1: To identify new small molecules – pharmacological chaperones (PCs) – for MPS I through screening of new chemical libraries.

Objective 2: To conduct a thorough downstream characterization of select small molecule leads to determine their suitability for treatment of MPS I disease and to discern their mechanism of action.

Objective 1: Identification of new PCs for MPS I through screening of new libraries

Development of a new tool high throughput screening: As noted in our proposal we previously designed a unique screening assay based on post-ER trafficking in plant cells, and used this assay at the Centre for Drug Research and Development (CDRD, Vancouver, BC) to identify a candidate molecule from the NINDS (National Institute of Neurological Disorders and Stroke) library of 1,040 Food and Drug Administration-approved drugs already used to treat humans. Because of the nature of the library compounds, the molecule uncovered has well-characterized pharmacokinetic properties in humans and is known to cross the blood brain barrier. Under Objective 2 we note the progress that we have made in characterizing this primary lead molecule with respect to its selectivity for IDUA and as a putative therapeutic for MPSI disease. The initial discovery of our lead PC was based on the use of a unique plant-based screening system as detailed in our 2015 grant application. To recap in brief, we developed a plant-cell-based screening system based on tobacco BY2 cultured cells to identify putative small molecule therapeutics for MPS I by selecting for library molecules capable of enhancing the post-ER transport of two missense mutant alpha-L-iduronidase (IDUA) proteins, P533R and R383H. Since the recombinant IDUA variants were equipped with a signal peptide, and the expression cells – transgenic tobacco BY2 cells – possess no lysosomes, the assay was based on increased IDUA activity in the secretion media. The principles underlying this screening mechanism are that putative PC candidates for MPS I disease should be capable of shifting the equilibrium of IDUA mutant proteins toward protein folding at the expense of ERAD (ER associated degradation), enabling the folded lysosomal enzyme to pass ER protein quality control (PQC) and traffic beyond this compartment. Stabilizing the ERAD-prone mutant protein thereby allows it to be targeted to the lysosome where it can function. Using the same principles of this powerful system we are developing similar, but potentially more sensitive, means to conduct our library screens. In the upcoming period, we plan to use this modified system to screen a new library – the NCI library of 50,000 terrestrial natural products extract library (Tarling et al., 2008). Our original system requires IDUA enzyme activity assays in primary screens to identify putative PCs, and later activity assays and western blot analyses in downstream validation work. While the IDUA enzyme assay is very sensitive to detect IDUA in the culture media surrounding transgenic BY2 cells (protoplasts), there may be huge potential in using other means of tagging IDUA so that we can identify potentially more small molecule “hits” that can promote post-ER trafficking of mutant IDUAs underlying MPS I disease. Using a 96-well based format, the system under development capitalizes on the FlAsH fluorescent labelling system (Lumio™ Technology) of Invitrogen. Here we a unique 6 amino acid tag (Cys-Cys-Pro-Gly-Cys-Cys) is added onto the target protein of interest – recombinant mutant IDUAs destined for expression in tobacco BY2 cells. When the biarsenical ligand (Lumio molecule) is added to a solution or cell culture containing the tagged protein, it binds to the tetracysteine tag (Fig 1a.). This binding results in a fluorescent emission of a 500-600 nm (max 535 nm) – a wavelength that is easily detected by any standard fluorescent 96-well plate reader. Due to the strong non-covalent binding between the fluorophore and the protein, the tagged IDUA variant protein can be viewed directly on a PAGE gel using a UV trans-illuminator equipped with a standard camera. Not only will this be efficacious for 3 library screening, but in validation work, the system would allow us to directly quantify mutant IDUAs within the secretion medium surrounding the transgenic BY2 cells without the need for western blot analyses. To evaluate the FlAsH-tag-based library screening system to uncover putative PCs for MPS I, the tetracysteine tag has been added to the C-terminus of both the wild-type IDUA, as well as onto the Ctermini of two mutant variants P533R- and R383H- IDUA. The corresponding gene constructs have been generated and used to transform tobacco BY2 cells (Fig 1b), and the high-expressing BY2-cell lines have been identified. We are soon to use our previously identified lead compound, X-372, to validate and optimize the system. This system will then be used in a high-throughput setting to identify lead small molecules from the NCI library in the upcoming period. It is noteworthy that the FlAsH fluorescent labelling system could be added to any soluble protein variant underlying a given lysosomal storage disease in a single PCR reaction; for MPS I it could be used to expand the range of mutant variant IDUAs that are evaluated in PC screening and evaluation efforts.

Objective 2: Downstream characterization of small molecule PC leads for MPS I

We are in the midst of conducting a thorough downstream characterization of select small molecule PC leads to determine their suitability for treatment of MPS I disease and to discern their mechanism of action. For this, we have work underway to analyze several biochemical criteria to verify the efficacy and selectivity of our primary small molecule lead (X372) as well as structurally-related derivatives of X372 (see below). This includes validating the ability of the small molecules to “rescue”/promote the post-ER transport of mutant- and wild-type IDUA proteins (section 2.1), and assessing effects of these molecules on IDUA thermostability (section 2.2). Importantly we have started work to examine if the lead compound X372 can function as a specific non-toxic IDUA modulator in cultured deficient (MPS I patient) fibroblasts bearing relevant disease-causing mutations (section 2.3). Finally, we have made some headway toward elucidating the mechanism of action of our lead small molecule (section 2.4).


From these results, we pose a hypothesis that will be tested over the next period. We postulate that there is a major role for GSH in the maintenance of ER redox homeostasis in association with enhancing the folding and post-ER trafficking of ER-retained folding variants. Thus X-372 may act as a proteostasis regulator (PR), augmenting a general element of proteostasis to enhance protein folding and post-ER transport, by promoting the activity of elements involved in oxidative protein folding in the ER lumen (Thomas, 2016). Studies are underway to identify alterations in cellular levels of GSH and oxidized glutathione (GSSG) in association with X-372 treatment of BY2 cells to test our hypothesis. If such an association can be confirmed, the induction in GSH production as a primary or secondary effect of compound treatment remains to be resolved. Additionally, future work must also be geared towards identifying conserved effects of compound treatment in human derived cell lines which endogenously express the ERAD-prone IDUA missense folding variants. Studies over the next funding period will contribute to further development of our lead compounds as potential therapeutics for attenuated MPS I patients. It is hoped that these small molecules will ultimately be valuable for treatment of MPS I patients afflicted by mutations for which there is no impairment or only slight impairment of the functional domains of the mutant IDUA protein.