Measles vectors for genomic modification-free induced pluripotent stem cells

描述(申请人提供)：诱导多能干细胞(IPSCs)技术能够从成人体细胞中衍生出患者特有的多能干细胞。我们已经证明了使用仙台病毒载体可从患者身上获得无基因组修饰的IPSCs。然而，基于小鼠副粘病毒的仙台病毒载体系统从未被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)。我们已经开发了一种反向遗传系统，允许生产相当于Morten疫苗株的重组病毒，目前在美国用于儿童疫苗接种。此外，临床级别的转基因MV正在生产中，用于治疗癌症患者，目前正在进行人体临床试验评估。在目标1中，我们将检验这样一个假设，即麻疹病毒可以被改造成表达四个重编程基因(OCT4、KFL4、SOX2和cMYC)的“一周期”重编程载体。我们将生产四个表达一个重编程因子(RF)的“一周期”MV载体，并测试它们在IPSCs中对成体细胞重编程的能力。在目标2中，我们将检验这样一种假设，即我们可以提高“一周期”MV载体在IPSCs中重新编程成体细胞的有效性和安全性。我们将通过生产一个表达来自单个基因组的四个RF的单一MV载体来提高效率。我们将利用麻疹的转录梯度，优化这四个因子的表达水平，以实现最高的重编程效率。为了增加载体的安全性，我们将用自杀机制武装载体，一旦IPSCs开始分化，就可以消除RF的表达以及残留的基因组和病毒蛋白的表达。我们将插入在最终内胚层中特异表达的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能够快速转化到临床。

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.