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

抽象的。 诱导多能干细胞(IPSCs)和基因治疗工具的最新进展开辟了一种新的 研究和治疗疾病，特别是骨髓缺陷疾病的途径。骨髓衰竭 综合征(BMFS)的特征是由于骨髓细胞功能障碍而导致血细胞减少。 Fanconi贫血(FA)是一种细胞重新编程效率低下的骨髓衰竭综合征， 由于疾病相关基因的干扰。要克服这一限制，有必要 在重新编程过程中或之前，从根本上纠正异常基因(例如：FANCD1)。在 过去，从这些患者的成纤维细胞中获得转基因IPSC通常需要多个步骤。 但该领域的最新进展已经为同时进行重新编程和基因打靶铺平了道路 使用多个上体载体的步骤。在这项研究中，我们提出将多个向量-单步变换 一步法从FA成纤维细胞获得正确的IPSC。我们的单一载体 是基于一种非整合负链RNA病毒--麻疹病毒(MV)。中心假设是一个 MV载体可以表达一个基因组中的所有成分，并导致临床上的 来自FA成纤维细胞的安全、正确和功能正常的IPSCs。建议进行研究的理由是 表达四种重编程因子的一周期MV载体MV4F从人产生IPSC 成纤维细胞，在重新编程后迅速稀释并从IPSC中消除。以Strong为指导 初步数据显示，本申请的具体目的是制作一套单周期的《一体机》MV 载体，包含四个重新编程因子加上Cas9-gRNA，并将协议设置为并发 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。建议的工作是 创新，因为它利用了一种新技术，这种技术依赖于一种表达四个元素的单一载体 重新编程因子(RF)，用于将体细胞重新编程为IPSC；我们的团队开发了 技术最后，将测试校正后的IPSC分化为造血干细胞的能力。 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向量来产生校正后的 IPSC，它将很快从现有的IPSC中被淘汰，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，拟议的研究与以下部分有关 美国国立卫生研究院的使命是开发遗传性骨髓衰竭综合征的新疗法， 血红蛋白疾病、免疫缺陷和其他单基因疾病，以减轻人类负担 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.