The nuclear lamina is important for maintaining nuclear architecture, chromatin organization, and gene expression. Mutations in lamin genes cause a wide variety of diseases called laminopathies. While tremendous gains have been made in understanding the molecular basis of these rare diseases, mechanistic links between the mutation's location and resulting disease phenotype are largely unknown. Furthermore, these gains have been made from relatively few mutations characterized mostly in mouse models. This project aims to increase our understanding of laminopathic mutations by developing a novel, high-throughput platform for characterizing nuclear defects using recent advances in RNA-guided, gene-editing technology and clinically relevant human cardiomyocyte models.
Aim 1 of this proposal focuses on developing a label-free, microarray platform to easily screen the nuclear defects of a subset of mutations linked to dilated cardiomyopathy (DCM) and familial partial lipodystrophy (FPL) using an over-expression human embryonic kidney (HEK) cell model. This platform will set the stage for Aim 2 where a large library of lamin A (LMNA) mutations - generated by error-prone PCR and an inducible gene editable cell line - will be screened for nuclear defects.
In Aim 3, pure cardiomyocytes differentiated from human embryonic stem cells (hESCs) will be used to study adenovirus-mediated overexpression of laminopathic mutations and endogenously generated laminopathic mutations. These cardiomyocyte models will validate results obtained from Aims 1 and 2 and also be used to test several reported therapeutic strategies.
Mechanistic links between laminopathic mutations and cellular phenotypes are largely unknown. This proposal seeks to develop new techniques for analyzing a large number of lamin mutations in human cells and corroborating some of these findings in functional stem cell-derived cardiomyocytes.
