PracticeUpdate Conference Series - SSIEM 2018

NewCell Reporter Line Enables Drug Screening of NDST1 inPatientsWith Sanfilippo Lysosomal StorageDisorders CRISPR-based genome editing has led to generation of a luciferase-based cell reporter of endogenous NDST1 expression. N ewly generated cell lines can be used to screen libraries of drug- like molecules for compounds

marrow transplantation exhibit low effectiveness for the treatment of mucopolysaccharidosis with neurological complications because enzymes used in these therapies are unable to cross the blood-brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. NDST1 is one of the first modifying enzymes of heparan sulfate precursors. Targeting of NDST1 expression holds strong potential to provide a basis for substrate reduction therapy in some patients with mucopolysaccharidosis. Adding a needed therapeutic strategy that could serve as a standalone or an adjunct, for example, along with enzyme replacement therapy. By extension, it should also improve the efficacy of any form of gene therapy that may emerge in the future for these disorders. “We have decided to work on drug development for Sanfilippo,” Dr. Schulze told Elsevier’s PracticeUpdate, “since this disease is devastating, with fatal outcome in the second or third decade of life and no treatment available that alters the course of the disease. We believe that small molecules can be found that act via substrate reduction to decrease the buildup of storage material that is causing neurodegeneration in affected patients.” Using CRISPR-based homology-directed repair, they achieved in-frame insertion of nanoluciferase at the C-terminus of NDST1 in Hek293, HeLa, and neuronal- like BE2c human cell lines. They also used a co-selection strategy based on Ouabain resistance to select for clones bearing an insertion of the luciferase reporter at the N-terminus of NDST1. Characterization of the tagged NDST1 gene in terms of protein size, intracellular trafficking, Golgi localization, and response to known substrate reduction therapy

(vorinostat, suberoylanilidehydroxamic acid) matched that of the native gene. Dr. Schulze explained that mucopoly- saccharidoses such as the Sanfilippo syndromes represent a group of lysoso- mal storage disorders in which deficiency of a lysosomal degradative enzyme leads to accumulation of the enzyme’s substrate, heparan sulfate, in the patient’s lysosomes. As a treatment modality, substrate reduction therapy with the goal of preventing accumulation of heparan sulfate has been shown to be feasible by reducing the activity of the synthetic enzyme heparan sulfate NDST1, involved in the formation of heparan sulfate. Dr. Schulze concluded that newly generated reporter lines have enabled screening for drugs that target the broader range of transcriptional and translational regulatory mechanisms controlling NDST1 expression. “We have identified a druggable target,” he said, “in NDST1. We are working on designing reporter cells for drug discovery, and after completion, high-content drug screening. Drug candidates will be assessed further in cell systems and finally, in patients’ cells.” He continued, “We hope to identify one or two drug candidates for investigational new drug status. Our approach in using CRISPR/Cas9 to design cell models for drug discovery and confirmatory testing, as exemplified in the present work, is suitable for other rare inborn errors of metabolism. Our research group is also using it, for example, for drug discovery in creatine deficiency syndromes, specifically, guanidinoacetate methyltransferase deficiency. I hope our work and its presentation can contribute to orphan drug development for underserved disorders”.

that reduce the expression of heparan sulfate N-deacetylase/N-sulfotransferase 1 (NDST1) in patients with Sanfilippo lysosomal storage disorders. This conclusion, based on results of a description of the development of the newly generated luciferase-based cell reporter, was presented at the SSIEM 2018.

Andreas Schulze, MD, PhD, o f the Hospital for Sick Children in Toronto, Ontario, Canada , and colleagues used C R I S P R-based genome editing to generate the luciferase-based cell reporter of

Andreas Schulze, MD, PhD

endogenous NDST1 expression. Dr. Schulze explained that the stepwise degradation of glycosaminoglycans is accomplished by 12 lysosomal enzymes. Deficiency in any of these enzymes will result in accumulation of intermediate substrates on the pathway to the complete turnover of GAGs. Accumulation of these undegraded substrates in almost any tissue is a hallmark of all mucopolysaccharidoses. Deficiency in any of these enzymes produces clinical phenotypes that vary frommild to severe forms. Themost severe exhibits progressive delay in cognitive and motor development, aberrations in bone morphogenesis, organomegaly, and cardiovascular abnormality. The more attenuated forms of mucopolysaccharidosis present with milder symptoms and may remain undiagnosed until adulthood. Therapeutics based on enzyme replacement therapy and bone

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SSIEM 2018 • PRACTICEUPDATE CONFERENCE SERIES 19