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

基本信息

批准号：
10462485
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
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10462485
关键词：
Allogenic BRCA2 gene Blood Cells Bone Marrow Bone Marrow Cells CRISPR/Cas technology Cells Clinic Clinical Congenital Abnormality DNA Repair DNA Sequence Alteration Data Degenerative Disorder Development Diamond-Blackfan anemia Disease Failure Fanconi&apos s Anemia Fibroblasts Gene Abnormality Gene Targeting Generations Genes Genetic Diseases Genome Goals Guide RNA Hematological Disease Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hemoglobinopathies Human Immunologic Deficiency Syndromes Inherited Lead Lentivirus Malignant - descriptor Measles Vaccine Measles virus Mendelian disorder Mission Modification Mutation Outcome Pathway interactions Patients Procedures Process Production Protocols documentation RNA RNA Viruses RNA vaccine Regenerative Medicine Research Risk Somatic Cell Stem cell transplant 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 induced pluripotent stem cell technology innovation loss of function new technology next generation novel stem cell gene therapy stem cell therapy tool vector vector-induced

项目摘要

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 (BMFS) are characterized by reduced blood cells due to a dysfunctional bone marrow cells. 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 transform the multiple vector-single step procedure to a single vector-one 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 set of one-cycle “all-in-one” MV vectors, containing the four reprogramming factors plus Cas9-gRNA, and setup the protocol to 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 a 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 their ability to differentiate into hematopoietic stem cells. The proposed work is significant because we develop a new single vector for the production of corrected iPSC, that will be eliminated quickly from the established iPSC and that can be rapidly translated into the clinic, as it is 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, hemoglobinopathies, immunodeficiencies, and other monogenetic disorders to reduce the burden of human disease.

抽象的。诱导多能干细胞（iPSC）和基因治疗工具的最新进展开辟了新的途径研究和治疗疾病，特别是骨髓缺陷性疾病的途径。综合征（BMFS）的特点是由于骨髓细胞功能障碍导致血细胞减少。范可尼贫血 (FA) 是一种骨髓衰竭综合征，其中细胞重编程效率低下，由于疾病相关基因的干扰，有必要克服这一限制。在重编程过程中或之前从根本上纠正异常基因（例如：FANCD1）。过去，从这些患者的成纤维细胞中获取转基因 iPSC 通常需要多个步骤。但该领域的最新进展为同时进行重编程和基因打靶铺平了道路。在本研究中，我们建议将多向量单步转换。单载体程序 - 一步法校正从 FA 成纤维细胞获得的 iPSC。基于非整合负链 RNA 病毒，即麻疹病毒 (MV)。 MV载体可以被设计为表达一个基因组中的所有成分，并导致临床上的产生来自 FA 成纤维细胞的安全、正确且有功能的 iPSC 拟议研究的基本原理是 “单周期”MV 载体 MV4F，表达四种重编程因子，从人类中产生 iPSC 成纤维细胞，并在强重编程后迅速稀释并从 iPSC 中消除。初步数据，该特定应用的具体目标是产生一组单周期“一体式”MV 载体，包含四个重编程因子加上 Cas9-gRNA，并设置协议以同时进行重新编程和编辑携带基因突变的人类成纤维细胞的基因组。创新，因为它利用了一项新技术，该技术依赖于表达四个的单个向量用于将体细胞重编程为 iPSC 的重编程因子 (RF)；我们的团队开发了该因子；最后，将测试校正后的 iPSC 分化为造血干细胞的能力。所提出的工作意义重大，因为我们开发了一种新的单一载体来产生校正的细胞。 iPSC，将从已建立的 iPSC 中快速淘汰，并可快速转化为临床，因为它是基于安全的麻疹疫苗株。最后，拟议的研究与这部分相关。 NIH 的使命是开发针对遗传性骨髓衰竭综合征的新疗法，血红蛋白病、免疫缺陷和其他单基因疾病，以减轻人类的负担疾病。