This proposal describes a detailed plan for the development of an academic research career in cardiovascular disease modeling with induced pluripotent stem cells (iPSCs). The principal investigator (PI) is an Instructor in the Department of Pathology at Stanford University with 85%-15% split between research and clinical attending duties in Molecular Genetic Pathology. The support provided by a K08 award will facilitate the additional training in genomics and stem cell disease modeling that is needed for the PI to fully prepare for a career as an extramurally funded independent investigator. The PI is mentored by Joseph Wu (Director, Cardiovascular Institute, Stanford) and Advisory Committee members Carlos Bustamante (Professor, Genetics, Stanford), Thomas Quertermous (Chief, Cardiovascular Medicine, Stanford) and Elizabeth McNally (Director, Center for Genetic Medicine, Northwestern University). Stanford University, and the Wu laboratory in particular, offer an environment uniquely suited to developing the PI into an independent investigator whose expertise merges the stem cell, cardiovascular, and genomics fields. iPSCs enable direct observation of genetic disease at the single cell level, presenting an opportunity to rapidl phenotype the genome. iPSCs that carry the genetic information of patients with familial hypertrophic cardiomyopathy (HCM) are thus the ideal tools for efficiently characterizing the individual HCM phenotype in vitro. Toward this end, this proposal will determine the genetic, morphological, transcriptional, and functional properties of cardiomyocytes derived from iPSCs (iPSC-CMs) from two families exhibiting heritable HCM. Preliminary data suggest that significant disease can be observed in the morphologic and electrophysiological properties of HCM iPSC-CMs. Targeted DNA sequencing using a novel next generation sequencing platform will elicit the unique variants that may be influencing development of disease in the two families. Furthermore, RNA sequencing of HCM iPSCs during cardiac differentiation from the embryonic stage will identify biological pathways that are unique to early HCM, as well as those pathways that are shared with later stages of HCM and other classes of cardiomyopathies (e.g., dilated cardiomyopathy). Finally, using innovations in genome editing that allow for rational, targeted alterations of genomic DNA in live cells, this proposal will directly address the challenge in discerning true HCM-causing genetic mutations from benign variation. In particular, CRISPR/Cas9 will be used to modify selected variants in iPSCs and determine their individual pathogenicity through a series of functional assays of the resulting iPSC-CMs. In the coming decades, data gained from projects such as this will constitute one of the most promising primary building blocks of cardiovascular precision medicine.
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. Using techniques and methods that have been developed over the past five years, this project will artificially create and study heart cells in a petri dish that carry the same HCM-causing gene mutations as the patients from which they are derived. The goal is to dramatically enhance our understanding of the genetics underlying HCM, and will ultimately lead to novel clinical diagnostics and possibly therapies for this intractable disease.
|Ma, Ning; Zhang, Joe Z; Itzhaki, Ilanit et al. (2018) Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells. Circulation 138:2666-2681|