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

 描述：诱导多能干细胞技术允许通过异位表达选定的转录因子来重新编程细胞命运，从而打开了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的可能性。这种直接重新编程提供了一种无需经过祖代状态即可生成感兴趣的谱系的替代方法。与山中因子的多能重编程类似，成纤维细胞直接转化为神经细胞、肝细胞样细胞、胰岛素产生细胞或心肌样细胞的过程需要多种转录因子的传递。 进入细胞。几种载体，如逆转录病毒、慢病毒、AAV载体或质粒，已经用于这一目的，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，需要开发一种新的病毒平台来产生无转基因的直接重编程细胞。在这里，我们建议开发一种新的基于非整合人副粘病毒的rna载体系统，麻疹病毒(Mv)，以运送成纤维细胞直接重编程为胰岛素所需的转录因子。 生产细胞。我们已经开发了一种反向遗传系统，允许生产一种相当于Morten疫苗株的重组病毒，这种病毒目前在美国用于疫苗接种。从这个病毒基因组中，我们产生了一个“一个周期”的载体，并用它来表达用于IPSCs重编程的山中因子。我们建议修改这些向量，并使用它们来表示直接重新编程所需的因素。我们将测试这样一个假设，即我们的复制缺陷麻疹载体可以被修改为表达转录因子，这些转录因子被证明是将成年体细胞重新编程为具有功能的胰岛素产生细胞所必需的。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。完成这里概述的拟议研究将导致开发一种新的多顺反子载体平台，从而无需基因组修改即可直接反式分化。这些研究将为将来利用这些载体进行体内直接重编程开辟新的前景。

 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.