Measles vectors for genomic modification-free induced pluripotent stem cells

描述（由申请人提供）：诱导多能干细胞（iPSC）技术能够从成人体细胞中衍生出患者特异性多能干细胞。我们已经证明了使用仙台病毒载体从患者中可再现地衍生出无基因组修饰的iPSC。然而，基于鼠副粘病毒的仙台载体系统从未被FDA批准用于人类临床试验。在这里，我们建议开发一种新的载体系统，基于人类副粘病毒，麻疹病毒（MV）。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儿童疫苗接种。此外，临床级转基因MV正在生产用于治疗癌症患者，目前正在人体临床试验中进行评估。在目标1中，我们将检验麻疹病毒可以被修饰成表达四种重编程基因（OCT 4、KFL 4、SOX 2和cMYC）的“一个循环”重编程载体的假设。我们将产生表达一种重编程因子（RF）的四种“一个周期”MV载体，并测试它们在iPSC中重编程成体体细胞的能力。在目标2中，我们将测试我们可以提高“单循环”MV载体在iPSC中重编程成体体细胞的功效和安全性的假设。我们将通过从单个基因组产生表达四种RF的单个MV载体来提高功效。我们将利用麻疹的转录梯度，优化四个因子的表达水平，以获得最高的重编程效率。为了增加载体的安全性，我们将用自杀机制武装载体，一旦iPSC开始分化，这将允许RF表达的消失以及残留基因组和病毒蛋白表达的消除。我们将插入在定形内胚层中特异性表达的miRNA的靶序列。这将提高我们载体的分化效率和安全性。这些研究的结果将导致一种新的载体系统，允许生产无基因组修饰的iPSCs，使iPSCs能够快速转化为临床。

DESCRIPTION (provided by applicant): The induced pluripotent stem cells (iPSCs) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells. We have demonstrated reproducible derivation of genomic modification-free iPSCs from patients using Sendai viral vectors. However, the Sendai vector system, which is based on a murine Paramyxovirus has never been approved by the FDA for the use in human clinical trial. Here, we propose to develop a new vector system, based on a human Paramyxovirus, measles virus (MV). We have developed a reverse genetic system allowing the production of recombinant virus equivalent to the Moraten vaccine strain, which is currently used for children vaccination in the US. Additionally, clinical grade genetically modified MV is being produced to treat cancer patients and is currently being evaluated in human clinical trials. In aim 1, we will test the hypothesis that measles virus can be modified into "one cycle" reprogramming vectors expressing the four reprogramming genes (OCT4, KFL4, SOX2 and cMYC). We will produce four "one cycle" MV vectors expressing one reprogramming factor (RF) and test their ability to reprogram adult somatic cells in iPSCs. In aim 2, we will test the hypothesis that we can improve the efficacy and safety of the "one-cycle" MV vectors to reprogram adult somatic cells in iPSCs. We will increase efficacy by producing a single MV vector expressing the four RFs from a single genome. We will take advantage of the measles gradient of transcription, to optimize the expression levels of the four factors for the highest reprogramming efficiency. In order to increase the safety of the vector, we will arm the vector with a suicide mechanism, which will allow extinction of the RF expression as well as the elimination of the residual genome and viral protein expressions as soon as differentiation of the iPSCs starts. We will insert the target sequence of a miRNA specifically expressed in the definitive endoderm. This will increase the efficiency of differentiation and safety of our vectors. The result of these studes will lead to a new vector system allowing the production of genomic modification-free iPSCs, enabling rapid iPSCs translation into the clinic.