Testing Substrate-Flux Therapies for Glycosylation Disorders using Zebrafish

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DESCRIPTION (provided by applicant): For two years, four investigators will each hire a new full-time employee to identify and validate a novel therapeutic strategy for a group of rare inherited metabolic disorders, the Congenital Disorders of Glycosylation-Type I (CDG-I). These disorders result in underglycosylation of proteins and associated multisystem pathologies, morbidity, and mortality are common since the vast majority of CDG-I patients cannot be treated. Currently, 13 Type-I CDGs are clinically and genetically defined. Most have deficiencies in transferases and substrates necessary to synthesize the precursor of N-linked glycoproteins, glucose3mannose9GlcNAc2-P-P-dolichol, also known as "lipid-linked oligosaccharide" or "LLO". These deficiencies result in underglycosylation of proteins and associated multi-system pathologies, morbidity, and mortality. In many CDG-I patients, the primary defect impairs mannose metabolism. CDG-I patients all have hypomorphic alleles, and we propose to take advantage of the residual enzyme activities by a novel therapeutic strategy we term "substrate-flux". For example, in CDG-Ia (the most common form of CDG), we will use various chemical agents to "coax" more of the patients' own limited metabolite pool (in this case mannose- 6-phosphate) toward the defective enzyme (phosphomannomutase), divert the pool away from competing enzymes (mannose phosphate isomerase), and/or alter associated pathways (providing dolichol-P or regulating protein synthesis) so that the limited substrate pool is used more effectively. Our research team has three known drugs and five experimental compounds in-hand, all of which have given promising results in preliminary cell culture experiments and are ready for animal testing. However, a severe limitation of CDG research is the absence of representative animal models. Knockout mice are embryonic lethal and although CDG-gene hypomorphic mice are just now emerging, they are not yet ready as a model to evaluate therapies. Thus, to test substrate-flux therapy immediately, we are harnessing the power and speed of zebrafish genetics to generate new vertebrate CDG-I zebrafish models with morpholino oligonucleotides (essentially a "knockdown" approach). These are being validated both pathologically and biochemically. Recent preliminary data support the successful generation of pmm2 morphant zebrafish as a model of CDG-Ia; they have an appropriate LLO defect and phenotypes recapitulating aspects of CDG patients. Positive drug results with zebrafish models will rapidly determine whether flux-altering agents affect CDG-I phenotypes, and will then be confirmed with authentic CDG-I patient cell cultures. Concurrent with these studies, the mechanisms of the agents in question will be tested directly by mannose flux experiments in both normal and CDG-I human cell cultures and zebrafish models. Efficacious compounds are confirmed by flux studies in normal mice. Screening of the next generation of flux-enhancing molecules is underway. Within two years, we will identify fluxmodifying agents and demonstrate real potential for CDG treatment. At this point the known drugs would be appropriate for off-label clinical evaluation, and the experimental drugs will then be tested in emerging mouse models of CDG-I. PUBLIC HEALTH RELEVANCE: Very few patients with "Congenital Disorders of Glycosylation" (CDG) can be successfully treated. These patients suffer multi-system pathology, morbidity, and mortality because they do not glycosylate proteins normally. Our four-investigator team will hire four scientific staff to test a new treatment strategy called "substrate-flux" therapy. Since mouse models do not yet exist we will evaluate therapy in a series of novel zebrafish CDG models that we've created.