Measles virus as a tool for iPSC-independent tissue specific reprogramming

麻疹病毒作为独立于 iPSC 的组织特异性重编程工具

• 批准号: 8966897
• 负责人: Patricia DEVAUX
• 金额: $ 19.88万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966897
• 关键词: Adenoviruses; Adult; Biological; C-Peptide; Cardiac; Cardiac Myocytes; Cells; DNA; Development; Diabetes Mellitus; Diabetic mouse; Disease; Ectopic Expression; Fibroblasts; Future; Generations; Genes; Genetic Materials; Genetic Transcription; Genome; Genomics; Glucose; Glycoproteins; Hepatocyte; Human; Hyperglycemia; In Vitro; Insulin; Lead; Left kidney; Lentivirus Vector; Measles; Measles virus; Methods; Modification; Monitor; Mus; Neurons; Paramyxovirus; Pharmaceutical Preparations; Phase; Plasmids; Process; Production; Property; Public Health; RNA; RNA Viruses; Regenerative Medicine; Reporting; Research; SCID Beige Mouse; Sendai virus; Somatic Cell; Staging; Stem Cell Research; Stem cells; Streptozocin; System; Testing; Tissues; Transgenes; Transplantation; Vaccination; Vaccines; Viral; Viral Genome; Viral Vector; base; capsule; cell type; clinical application; diabetic; in vivo; induced pluripotent stem cell; interest; islet; keratinocyte; mouse model; novel; novel strategies; progenitor; public health relevance; recombinant virus; respiratory; reverse genetics; stem cell technology; success; tool; transcription factor; transgene expression; vector

 DESCRIPTION: Induced pluripotent stem cell technology, which allows reprogramming of cell fate through ectopic expression of selected transcription factors, has opened the possibility to reprogram fibroblasts or other somatic cells directly into specific cell types. This direct reprogramming offers an alternative approach for generating lineages of interest without passing through a progenitor state. Similar to the pluripotent reprogramming by the Yamanaka factors, the process to convert fibroblasts directly to neuronal cells, hepatocyte-like cells, insuin producing-cells or cardiomyocyte-like cells requires the delivery of multiple transcription factors into the cells. Several vectors, such as retroviral-, lentiviral-, AAV-vectors or plasmids, have been used for this purpose with different levels of success, but with the potential of integration f genetic material in the host genome. Thus, there is a need for the development of a new viral platform to generate transgene-free direct reprogrammed cells. Here, we propose to develop a new RNA vector system based on a non-integrating human Paramyxovirus, measles virus (MV), to deliver the transcription factors needed for the direct reprogramming of fibroblast into insulin producing cells. We have developed a reverse genetic system allowing the production of a recombinant virus equivalent to the Moraten vaccine strain, which is currently used for vaccination in the US. From this viral genome, we have produced a "one cycle" vector and used it to express the Yamanaka factors for iPSCs reprogramming. We propose to modify these vectors and use them to express the factors required for direct reprogramming. We will test the hypothesis that our replication-deficient measles vector can be modified to express the transcription factors shown to be necessary for the reprogramming of adult somatic cells into functional insulin-producing cells. The cells will be then tested in a diabetic mouse model that will validate the functionality of the reprogrammed insulin producing cells. Achieving the proposed studies outlined here will lead to the development of a new polycistronic vector platform allowing direct trans-differentiation without genomic modification. These studies will open the perspective of using these vectors for future direct reprogramming in vivo.

 描述：诱导多能干细胞技术允许通过选定转录因子的异位表达来重新编程细胞命运，为将成纤维细胞或其他体细胞直接重新编程为特定细胞类型提供了可能性。这种直接重编程提供了一种无需经过祖先状态即可生成感兴趣谱系的替代方法。与山中因子的多能重编程类似，将成纤维细胞直接转化为神经元细胞、肝细胞样细胞、胰岛素产生细胞或心肌细胞样细胞的过程需要传递多种转录因子 进入细胞。几种载体，例如逆转录病毒、慢病毒、AAV 载体或质粒，已用于此目的，取得了不同程度的成功，但具有将遗传物质整合到宿主基因组中的潜力。因此，需要开发新的病毒平台来产生无转基因的直接重编程细胞。在这里，我们建议开发一种基于非整合性人类副粘病毒麻疹病毒（MV）的新RNA载体系统，以传递将成纤维细胞直接重编程为胰岛素所需的转录因子 产生细胞。我们开发了一种反向遗传系统，可以生产与 Moraten 疫苗株相当的重组病毒，该疫苗株目前在美国用于疫苗接种。从该病毒基因组中，我们生产了一种“单周期”载体，并用它来表达用于 iPSC 重编程的山中因子。我们建议修改这些向量并使用它们来表达直接重编程所需的因子。我们将测试这样的假设：我们的复制缺陷型麻疹载体可以被修饰以表达转录因子，这些转录因子对于将成体体细胞重编程为功能性胰岛素产生细胞是必需的。然后这些细胞将在糖尿病小鼠模型中进行测试，以验证重新编程的胰岛素产生细胞的功能。实现此处概述的拟议研究将导致新的多顺反子载体平台的开发，该平台允许直接转分化而无需基因组修饰。这些研究将为未来使用这些载体进行体内直接重编程开辟前景。