Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, accelerated-aging disease that leads to death in patients by their early teens. It is caused by a de novo point mutation in one allele of the LMNA gene. LMNA is typically alternately spliced to produce Lamins A and C which are responsible for stable nuclear envelope structure. The HGPS mutation results in a truncated, farnesylated version of pre-lamin A called progerin which accumulates on the nuclear membrane and leads to abnormal nuclear envelope shape. The primary pathology of HGPS is atherosclerosis ? a buildup of plaque within arteries ? leading to stroke and death. Given this pathology, this project aims to investigate the effects of HGPS on the endothelium that lines arteries, and the potential of genome-editing technology to correct its vascular pathology. Little is known about the effects of HGPS on endothelial cells (ECs) that line the arterial lumen. ECs are sensitive to shear stress exerted on them by the flow of blood, and their gene expression is altered under different levels of shear stress. Additionally endothelial dysfunction is a notable early event in general atherosclerosis. Examining the molecular changes that occur in ECs in the context of HGPS is important to fully understand the disease mechanism. As a result, Aim 1 of this project examines gene expression of HGPS ECs upon exposure to laminar shear stress. ECs derived from iPSCs of HGPS and healthy donors will be exposed to physiologically relevant shear stress using parallel-plate flow chambers for 24 hrs. RNA-seq will be performed to assess differences in gene expression between ECs in static and flow conditions, and between HGPS and healthy ECs under flow. These experiments will give insight into molecular dysfunction of HGPS ECs, leading to the development of atherosclerosis. Because HGPS is a single gene disorder, gene editing presents as a plausible course of treatment. The Lamin A protein is dispensable in mice, and CRISPR/Cas9 editing of LMNA to prevent Lamin A/progerin transcription while preserving Lamin C has recently been shown to decrease progerin and slightly increase life span in mouse models of HGPS. However, these models do not fully recapitulate human disease phenotypes with regards to vascular pathology. It is important therefore to illustrate whether a full knockout of Lamin A is tolerable in a humanized system. Therefore in Aim 2, the efficacy of CRISPR/Cas9 gene editing on human cells for the treatment of HGPS will be tested. Tissue-engineered blood vessels will be constructed using iPS-derived smooth muscle cells and ECs, and CRISPR/Cas9 machinery will be added to the perfusion media.
Aim 3 of this project will then elucidate changes in gene function after HGPS treatment in ECs. RNA- seq will be performed on HGPS ECs that were treated with CRISPR/Cas9 targeting Lamin A transcription, then exposed to flow as in Aim 1. This will give insight into how HGPS treatment corrects EC responses to flow. Overall this project will provide new information about the role of ECs in atherosclerosis development in HGPS and investigate a treatment that may restore vascular function and prolong patient life.
This research will provide insight into the molecular mechanisms and potential treatments of the rare and fatal disease Hutchinson-Gilford Progeria Syndrome (HGPS). This insight has potential to aid in the prolonging of life for HGPS patients, who die due to atherosclerosis by their early teens. This project also has potential to add to the field of knowledge regarding atherosclerosis progression and aging, both of which have a large impact on the wider population.
