New Chromatin Insulators and Enhancers for Gene Therapy of the Hemoglobinopathies

用于血红蛋白病基因治疗的新染色质绝缘子和增强子

• 批准号: 9926304
• 负责人: THALIA STAMATOYANNOPOULOS
• 金额: $ 58.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926304
• 关键词: Advanced Development; B-Lymphocytes; Biology; Cell Line; Characteristics; Chromatin; DNA cassette; Deoxyribonuclease I; Detection; Effectiveness; Enhancers; Environment; Erythroid; Erythroid Cells; Frequencies; Gene Expression; Gene Transduction Agent; Generations; Genes; Genomic approach; Genomics; Globin; Goals; Hemoglobinopathies; Human; Human Genome; Hypersensitivity; Informatics; Insulator Elements; Investigation; Lentivirus Vector; Measurement; Methods; Molecular; Mus; Myeloid Cells; Patients; Phenotype; Research; Resources; Risk; Safety; Sickle Cell Anemia; Site; Specificity; Structure; Thalassemia; Therapeutic; Variant; Viral Vector; base; beta Thalassemia; gamma Globin; gene therapy; genotoxicity; improved; in vivo; learning algorithm; novel; promoter; screening; therapeutic gene; transcription factor; vector

ABSTRACT The goals of this project are to use genomic approaches to identify, in the human genome, powerful chromatin insulators and powerful erythroid specific enhancers. Use of powerful insulators in therapeutic gene therapy vectors will increase safety by decreasing the risks of vector-related genotoxicity. Availability of powerful erythroid specific enhancers will increase the efficacy of gene therapy in patients with severe thalassemia. With the studies of the first specific aim we will discover powerful chromatin insulators in the human genome using the novel genomic screening approaches we have developed. Special emphasis will be given in identifying powerful barrier insulators which also possess powerful enhancer blocking activity. Such insulators are expected to improve both the safety and the efficacy of integrating gene therapy vectors. We will determine the structural characteristics of the new insulators and we will investigate the structural basis of the variation in their potency. In specific aim 2 we will discover new powerful erythroid specific enhancers in the human genome using molecular and informatics methods. We will verify the erythroid lineage specificity of the new enhancers, we will characterize them and will use them to identify the molecular features that determine erythroid lineage specificity. With the studies of Specific Aim 3 we will produce a new generation of globin gene therapy vectors. The most powerful insulators of Specific aim 1 will be used to determine the optimal sizes, the highest titers and the highest protection from genotoxicity required for the gene therapy vectors. The most powerful enhancers of Specific Aim 2 or enhancer cassettes will be used for quantitative assessments of their potency in supporting globin gene expression. The best insulators and enhancers will be incorporated in globin gene vectors and their therapeutic potency will be assessed ex vivo in the erythroid cells of thalassemic patients and in vivo in thalassemic mice.

摘要