Single non-integrating RNA vector for gene editing and reprogramming of Fanconi anemia fibroblasts

用于范可尼贫血成纤维细胞基因编辑和重编程的单一非整合RNA载体

• 批准号: 10009824
• 负责人: Patricia DEVAUX
• 金额: $ 31.8万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009824
• 关键词: Allogenic; BRCA2 gene; Blood Cells; Bone Marrow; CRISPR/Cas technology; Cells; Clinic; Clinical; Congenital Abnormality; DNA Repair; DNA Sequence Alteration; Data; Degenerative Disorder; Development; Diamond-Blackfan anemia; Disease; Fanconi' s Anemia; Fibroblasts; Gene Abnormality; Gene Targeting; Generations; Genes; Genetic Diseases; Genome; Goals; Guide RNA; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Immunologic Deficiency Syndromes; Inherited; Lead; Malignant - descriptor; Measles Vaccine; Measles virus; Mendelian disorder; Mission; Modification; Mutation; One-Step dentin bonding system; Outcome; Pancytopenia; Pathway interactions; Patients; Procedures; Process; Production; Protocols documentation; RNA; RNA Viruses; Regenerative Medicine; Research; Risk; Somatic Cell; Stem cell transplant; Subfamily lentivirinae; System; Technology; Testing; Translating; United States National Institutes of Health; Viral; Viral Vector; Work; alternative treatment; base; bone marrow failure syndrome; c-myc Genes; clinically relevant; combination gene therapy; design; ds-DNA; gene therapy; genome editing; human disease; human pluripotent stem cell; induced pluripotent stem cell; innovation; loss of function; new technology; next generation; novel; stem cell technology; stem cell therapy; tool; vector; vector-induced

Abstract. The recent advances in induced pluripotent stem cells (iPSCs) and gene therapy tools have opened up a new avenue to study and treat diseases, particularly of disorders with defective bone marrow. Bone marrow failure syndromes are usually marked with depleted blood cells caused due to dysfunctional bone marrow compartment. Fanconi anemia (FA) is one such bone marrow failure syndrome where cellular reprogramming is inefficient, owing to interference of the disease-related genes. To overcome this limitation, it is necessary to fundamentally correct the abnormal gene (e.g.: FANCD1) during or prior to the reprogramming process. In the past, obtaining genetically modified iPSC from the fibroblasts of these patients typically involved multiple steps. But recent progress in the field has paved way for simultaneous reprogramming and gene targeting in a single step using multiple episomal vectors. In this study we propose to take the multiple vector-single step procedure to single vector-single step approach to obtain corrected iPSC from FA fibroblasts. Our single vector is based on a non-integrating negative strand RNA virus, Measles virus (MV). The central hypothesis is that a MV vectors can be designed to express all components in one genome, and lead to the generation of clinically safe, corrected and functional iPSCs from FA fibroblasts. The rationale for the proposed research is that the “one-cycle” MV vector, MV4F, expressing the four reprogramming factors, generate iPSC from human fibroblasts and is quickly diluted and eliminated from the iPSC after reprogramming. Guided by strong preliminary data, the specific aim of this particular application is to produce a one-cycle “all-in-one” MV vectors, containing the four reprogramming factors plus Cas9-gRNA, and setup the protocol for concurrently reprogram and edit the genome of human fibroblasts carrying a genetic mutation. The proposed work is innovative, because it capitalizes on a new technology that relies on one single vector expressing the four reprogramming factors (RFs) for the reprogramming of somatic cells into iPSC; and our group developed that technology. Finally, the corrected iPSC will be tested for there ability to differentiated into hematopoietic stem cells. The proposed work is significant because develop a new single vector for the production corrected iPSC, that will be eliminated quickly form the established iPSC and that can be translated into the clinic quickly, as it based on the safe measles vaccine strain. Finally, the proposed research is relevant to that part of NIH's mission that pertains to develop new treatments for Inherited Bone Marrow failure syndromes, haemoglobinopathies, immunodeficiencies, and other monogenetic disorders to reduce the burden of human disease.

抽象的。 诱导多能干细胞（IPSC）和基因疗法工具的最新进展已经开发了一个新的 骨髓衰竭 综合症通常用由于功能失调的骨髓引起的血细胞标记 Fanconi贫血（FA）是一种骨髓衰竭综合征 是由于疾病相关基因的干扰而无效的。 基本纠正异常基因（例如 过去，从这些果皮的成纤维细胞中获得了基因修饰的IPSC，涉及多个步骤。 但是该领域的最新进展已铺平了同时重编程和基因靶向的方法 在本研究中使用多个偶发向量。 单个向量单步进方法，从FA firblasts获得正确的IPSC。 在非整合的阴性链RNA病毒上，无孔病毒（MV）。 可以设计向量以表达一个基因组中的所有组件，并导致临床上产生 安全，正确和功能性的IPSC FA成纤维细胞。 “单周期” MV矢量MV4F，表达了四个重编程因素，从人类产生了ISC 在重新编程后，纤维细胞会迅速稀释并从IPSC中消除 初步数据，这是造成单周的“多合一” MV向量的具体目的， 包含四个重编程因子加上cas9-grna，并为同时重新编程设置协议 并编辑人类成纤维细胞携带的基因组 因为它利用了一项依赖一个表达四个重新编程的新技术的新技术 用于将体细胞复制到IPSC的因素（RF）；我们的小组开发了该技术。 最后，将测试正确的IPSC，以区分造血干细胞 支撑工作是重要的矢量，生产正确的IPSC，它将将 迅速消除成立已建立的IPSC，可以快速转化为诊所，因为它基于它 最终，在安全的疫苗菌株上，拟议的研究与NIH的怀疑有关 开发遗传骨髓衰竭综合症，血红蛋白病的新疗法的持久性， 免疫缺陷和其他单基因疾病，以减轻人类疾病的负担。