PROJECT SUMMARY Familial transthyretin amyloidosis (ATTR) is a devastating multi-systemic protein folding disorder that results from over 100 possible mutations in the transthyretin (TTR) gene. In the disease, TTR misfolds, is secreted from the liver, and aggregates extracellularly in a concentration-dependent manner at downstream target organs such as the heart and/or peripheral nervous system. ATTR exhibits extreme mutation-dependent variation in disease phenotype (e.g. target organ affected and severity) with an average time of diagnosis to death of only 5-10 years. The current standards of care for patients with ATTR, including liver transplantation and small molecule TTR stabilizers, are highly limited; not all patients are candidates for surgery, large donor organ deficits exist, and many patients are refractory to kinetic stabilizers. A better understanding of disease etiology as well as alternative treatment options are necessary to combat systemic amyloid disorders. Problematically, the multi-tissue nature of the disease makes it difficult to study in vitro, while no current animal model accurately recapitulates ATTR pathology. To combat these limitations, our laboratory has developed a novel, induced pluripotent stem cell (iPSC)-based model for studying ATTR. In our platform, patient- derived iPSCs are differentiated into effector cells (hepatocyte-like cells) that produce mutant TTR. Conditioned media is then prepared on these cells to (1) analyze the type and quantity of TTR species secreted and (2) dose target cells (iPSC-derived cardiomyocytes and neurons) to assay resulting toxicity and the efficacy of proposed therapeutics. Using our genetically tractable model, we look to improve the current therapeutic paradigm for ATTR. Importantly, studies show that reducing serum levels of destabilized TTR through liver transplantation or activation of stress-responsive protein folding machinery reduces target organ toxicity. Armed with this insight and our iPSC-based ATTR model, we will test the hypothesis that disruption of aberrant TTR expression or activation of endogenous protein folding machinery will prove therapeutic for ATTR. We propose to evaluate this hypothesis through two Aims. In the first, to overcome limitations of site-specific gene editing approaches for treating ATTR, we will develop a universal gene correction strategy ameliorative of all TTR genetic lesions. In the second, we will activate the ATF6 pathway of the unfolded protein response (UPR) using genomic and pharmacological approaches to selectively decrease production of destabilized, toxic TTR. In both methods, secretion of TTR species and their impact on patient-matched iPSC-derived target cell (neuron and cardiomyocyte) toxicity will be evaluated. Our iPSC-based model, described herein, allows for the unprecedented coupling of protein biochemistry and genomic-based approaches to study novel aspects of systemic amyloidoses. Insight gained here will allow for better understanding of and therapeutics for ATTR and other protein folding disorders.

PROJECT NARRATIVE Systemic amyloidoses represent a large class of protein folding disorders affecting >1 million individuals worldwide. In these difficult to study diseases, which affect multiple organ systems throughout the body, proteins misfold and cause downstream toxicity at peripheral target tissues such as the heart and nervous system. In this project, induced pluripotent stem cells (iPSCs) will be used to better understand systemic amyloid disorders and to develop novel genomic and pharmacologic-based therapeutic options.