Epigenetic-guided studies of AMD pathology and iPSC-RPE transplantation therapy

AMD 病理学和 iPSC-RPE 移植治疗的表观遗传学引导研究

• 批准号: 10057227
• 负责人: Michael Farkas
• 金额: --
• 依托单位: VA WESTERN NEW YORK HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057227
• 关键词: Address; Affect; Age; Age related macular degeneration; Aging; American; Animals; Area; Automobile Driving; Autopsy; Back; Basic Science; Biology; Blindness; Blood; CRISPR/Cas technology; Caring; Cell Cycle; Cell Lineage; Cells; Choroidal Neovascularization; Clinical; Clinical Trials; Code; Collaborations; Complex; DNA; DNA Methylation; Data; Diet; Disease; Early treatment; Elderly; Environmental Risk Factor; Enzymes; Epigenetic Process; Etiology; Exhibits; Eye; Functional disorder; Gene Expression; General Population; Genes; Genetic; Genetic Transcription; Geography; Goals; Hour; Human; Immune response; In Vitro; Individual; Lead; Mediating; Memory; Methodology; Methods; Methylation; Nerve Degeneration; Neurodegenerative Disorders; Nonexudative age-related macular degeneration; Outcome; Oxidative Stress; Parents; Pathogenesis; Pathology; Patients; Phenotype; Pigments; Predisposition; Process; Proteins; Regenerative Medicine; Replacement Therapy; Repression; Research; Retina; Retinal Degeneration; Risk; Risk Factors; Role; Sampling; Smoking; Societies; Stimulus; Structure of retinal pigment epithelium; Testing; Therapeutic; Thymine DNA Glycosylase; Time; Tissues; Variant; Veterans; Vision; base; biological adaptation to stress; case control; cell replacement therapy; cell type; clinically relevant; cost; demethylation; differential expression; disorder risk; effective therapy; epigenetic memory; epigenome; functional improvement; gene therapy; genome wide association study; genome-wide; geographic atrophy; induced pluripotent stem cell; knock-down; methylation pattern; monolayer; novel strategies; replacement tissue; response; retinal progenitor cell; sight restoration; success; therapeutic target; transcriptome sequencing; transplantation therapy; treatment strategy; whole genome

Age-related macular degeneration (AMD) affects nearly 5% of aging veterans, and millions of civilian Americans. AMD is caused by genetic and environmental factors, which lead to central vision loss initially, and can lead to rapid degeneration of remaining vision in a few years. While this is a prevalent disease with costs to both the affected individuals and society as a whole, few treatments, and no cures, currently exist. Contributing to the lack of treatments for AMD, is the fact the underlying genetic factors resulting in pathogenesis remain unsolved. While variants in, or around, 34 genes have been identified as risk factors for disease, none have been demonstrated to be a causative agent. Epigenetics is an area yet to be fully explored in AMD pathogenesis. In collaboration with Dr. Margaret DeAngelis, we have performed DNA methylation studies on RPE from 140 dry AMD donors, identifying over 400 differentially methylated regions. Combining this data with RNA-Seq data from iPSC-RPE under oxidative stress, we have identified 81 genes to be both differentially methylated and differentially expressed. At the top of this list is Thymine DNA Glycosylase, an enzyme that performs the last step of DNA demethylation. We hypothesize that TDG repression results in perturbation of the natural methylation/demethylation cycle that the cell uses to regulate gene expression in response to environmental stimuli, such as oxidative stress. In Aim 1, we will test this hypothesis using CRISPR/Cas9-based knockdown of TDG in induced pluripotent stem cell-derived RPE (iPSC-RPE) under normal and oxidative stress conditions. A second contributing factor to the lack of treatments for AMD is the limited progress observed in clinical trials using cell replacement therapy, a form of regenerative medicine. One hurdle to overcome in regenerative medicine for the eye is the difficulty in producing retinal tissues with high similarity to native tissue. The retinal pigment epithelium (RPE) is a pigmented monolayer at the back of the eye, which has the most regenerative medicine potential, since it can be readily derived from patient-specific iPSCs. Multiple animal and human studies using these iPSC-RPE cells, however, fail to produce lasting results, as the cells either die, produce an immune response, or yield no functional improvement, likely because these iPSC-RPE are only RPE-like and not exact replicas of native RPE. Our group, and others, have shown significant differences in the transcriptional landscape of iPSC-RPE, relative to native RPE. Many of the protein-coding genes that define the RPE are expressed, but at significantly lower levels in iPSC-RPE when compared to native RPE. It has been well documented that reprogramming somatic tissue to iPSCs removes most of the epigenetic memory, but some remains. This memory is passed along during the differentiation process to the target cell type, and can promote dedifferentiation to the original cell type. The exact epigenetic landscape of these cells, however, has yet to be defined. We hypothesize that a retained epigenetic memory in iPSC-RPE is driven, in part, by continued expression of genes that define the parent cell lineage. In Aim 2, we will test this hypothesis by collecting human post-mortem blood and native RPE. The blood will be reprogrammed to iPSC, which will subsequently be differentiated to RPE. From these samples, we will characterize the epigenome to identify the epigenetic marks that are retained from blood (parent cell) to iPSCs to iPSC-RPE (the epigenetic memory) and compare this to the epigenome of native RPE from the same individual. The outcome of these two aims will lead to a better understanding of AMD pathology and iPSC-RPE biology that will have a direct impact on treatment strategies for veterans.

年龄相关性黄斑变性（AMD）影响了近5%的老年退伍军人和数百万美国平民。