Identifying New Therapeutics and Molecular Mechanisms in Congenital Disorders of Glycosylation. - Glycosylation is an essential biological pathway that involves the post- or co-translational addition of sugar moieties to proteins. Congenital Disorders of Glycosylation (CDGs) are rare developmental disorders caused by inborn errors of metabolism in glycosylation pathways. CDGs lack good treatment options, and this is typically due to poorly understood mechanisms and difficulty in establishing clinical trials in small patient populations. My long-term objectives are to determine mechanisms of CDGs and identify new therapeutics for CDG patients. One example is DPAGT1-CDG - a CDG caused by mutations in the gene DPAGT1 which encodes for the first enzyme used in N-linked glycosylation. Recently, I identified many modifier genes which can be perturbed to rescue a model of DPAGT1-CDG, but their mechanisms are not yet known. In Aim 1, I propose to determine the mechanisms of these modifier genes using human cell culture in order to characterize new therapeutic targets for this disorder. I will use a DPAGT1-CDG cell model to determine how these rescuing modifier genes affect patient-related health metrics of proliferation, stress, and their glycoproteome. In Aim 2, to identify new drugs that can rescue this disorder, I will use an in vivo Drosophila DPAGT1-CDG model to perform a repurposed drug screen using 1,500+ small molecules (98% FDA/EMA-approved). Using an in vivo model will ensure these drugs are safe during development, and this repurposed drug screen will help expedite the clinical trial process for new CDG therapies. In Aim 3, I will characterize a new finding that suggests that genes underlying CDGs ( CDG genes ) themselves represent an enriched set of modifier genes for treating CDGs. Perturbation of CDGs can rescue models of DPAGT1-CDG, as well as a model of the most common CDG, PMM2-CDG. I will use RNA interference to perturb all 150+ CDG genes to identify any that are capable of rescuing both DPAGT1- and PMM2-CDG human cell models (with the same health metrics as in Aim 1). The discovery of new CDG gene modifier genes capable of rescuing these models could have the potential to translate into future therapies for many other CDGs. Finally, in Aim 4, I will synthesize the above Aims to test therapeutic drugs from Aim 2 in human cell culture models and CDG modifier genes from Aim 3 in Drosophila models. I will use high-throughput tools in cell culture and in vivo stress markers in Drosophila to determine the mechanisms of these new therapies. This multi-species approach will ensure a better transition from preclinical models into therapies for patients. In addition to the above, completion of this proposal will provide me with training to complete my career goals. I will learn new techniques in cell culture and small molecule screens while also taking formal courses in mentorship and writing. I have an outstanding mentor, co-mentor, and advisory committee consisting of faculty with expertise in CDGs, cell culture, drug screening, genetics, and molecular biology. I also have state-of-the-art facilities and staff at the University of Utah available to me. With my plan, committed faculty members, and excellent institution, completing this proposal will help me successfully transition to an independent research career.
