Implementing CRISPR/Cas9 Genomic Screens to Identify Genetic Risk Factors for Lung Radiation Injury
Abstract
Topic Areas: Lung Injury, Pulmonary Fibrosis Genetic factors that predispose to radiation-induced lung injury are poorly understood, and there are currently no genetic tests to identify persons at elevated risk of radiation-mediated lung toxicity. Radiation pneumonitis, a type of lung injury caused by radiation, occurs in approximately 30 percent of patients following treatment of lung cancer with radiation therapy, and approximately 3 to 10 percent of these patients do not survive due to worsening radiation pneumonitis leading to respiratory failure. There is also a long-term risk of radiation-induced pulmonary scarring (pulmonary fibrosis), which can lead to late complications including heart failure and respiratory failure. These risks also exist for occupational and military radiation exposures. Currently, it is not possible to proactively identify these high-risk patients. Modern strategies are urgently needed to identify persons at risk of radiation toxicity so that more effective precautionary measures or radiation treatment strategies may be proactively implemented. To address this goal, we recently validated a genetic screening method (termed CRISPR/Cas9 knockout screening) to identify genes that affect how cells survive following exposure to radiation. The screen successfully identified approximately 250 genes that promote radiation resistance and 200 genes that promote radiation sensitivity (including numerous expected genes), which is a proof of concept for using CRISPR/Cas9 screens as a novel research approach. Over half of these genes have no defined role in the cellular response to radiation, and our preliminary data suggest that most of these candidate genes have a causal role in the cellular response to radiation. Our CRISPR/Cas9 validation screening data indicate that we have the technical capability to employ genome-scale CRISPR/Cas9 screens to identify known and novel genes that affect the cellular response to radiation with high confidence. The overarching goal of this proposal is to conduct genome-scale CRISPR/Cas9 screens on normal human lung-derived cells to identify genes that impact radiation toxicity (using cell survival as an endpoint), which will be utilized to generate a novel radiation toxicity risk algorithm that we will validate on lung tissue-derived cells and clinical samples of patients treated with radiation for lung cancer (from two clinical databases with known incidence of radiation pneumonitis and pulmonary fibrosis). We hypothesize that if we utilize a genome-scale CRISPR/Cas9 knockout screen using radiation as a selective pressure, then we will confidently identify genes that significantly protect or sensitize cells to radiation, which will be used to generate a radiation toxicity risk algorithm. We expect that the radiation toxicity risk algorithm will successfully identify patients at increased genetic risk of radiation-induced lung toxicity (through use of specific biological input data) from the clinical sample datasets. CRISPR/Cas9 genome-scale knockout screens have revolutionized the research landscape by permitting the individual analysis of thousands of genes in a single experiment. This proposal implements an entirely novel utilization of CRISPR/Cas9 screening technology to precisely define the magnitude of impact of individual genes on the radiation response. Our team has unique expertise in successfully applying CRISPR/Cas9 screening technology to radiation as a selective pressure as well as relevant experience in genomic approaches to implement these data into a clinically useful algorithm. Specific Aim 1 will reveal hundreds of genes that affect the risk of cell death following radiation exposure, which has significant importance to the basic research, military research, and clinical research communities. Specific Aim 2 describes converting these data into a radiation toxicity risk algorithm, which will utilize two independent patient datasets w
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010052
Entities
People
- Neil Pfister
Organizations
- Emory University
- United States Army