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

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一个新的领域。 研究和治疗疾病的途径，特别是有缺陷的骨髓疾病。骨髓衰竭 BMFS综合征的特征在于由于骨髓细胞功能障碍而导致的血细胞减少。 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， 这是由于疾病相关基因的干扰。为了克服这一限制，必须 从根本上纠正异常基因（例如：FANCD 1）在重编程过程期间或之前。在 过去，从这些患者的成纤维细胞获得遗传修饰的iPSC通常涉及多个步骤。 但该领域的最新进展为同时进行重编程和基因靶向铺平了道路， 步骤使用多个附加型载体。在这项研究中，我们提出了转换的多个向量-单步 将该方法转化为单载体一步法以从FA成纤维细胞获得校正的iPSC。我们的单一载体 是基于非整合负链RNA病毒，麻疹病毒（MV）。核心假设是， MV载体可以被设计为在一个基因组中表达所有组分，并导致临床上产生的病毒。 来自FA成纤维细胞的安全、校正和功能性iPSC。拟议研究的理由是， 表达四种重编程因子的“单循环”MV载体MV 4F从人类产生iPSC， iPSC是成纤维细胞的细胞，并且在重编程后迅速稀释并从iPSC中消除。以强为导 根据初步数据，该特定应用的具体目的是生产一套单循环“一体化”MV 载体，含有四种重编程因子加上Cas9-gRNA，并设置方案以同时 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。拟议的工作是 创新，因为它利用了一种新技术，依赖于一个单一的载体表达四个 重编程因子（RFs）用于将体细胞重编程为iPSC;我们的研究小组开发了 技术.最后，将测试校正的iPSC分化成造血干细胞的能力。 细胞建议的工作是有意义的，因为我们开发了一个新的单一载体的生产纠正 iPSC将很快从现有的iPSC中消除，并可以迅速转化为临床， 因为它是基于安全的麻疹疫苗株。最后，建议的研究是相关的， NIH的使命是开发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

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.