Measles vectors for genomic modification-free induced pluripotent stem cells

用于无基因组修饰诱导多能干细胞的麻疹载体

• 批准号: 8488790
• 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: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488790
• 关键词: Adenoviruses; Adult; Biological; Cancer Patient; Cardiac; Cell Line; Cells; Child; Clinic; Clinical; Clinical Trials; Complementary DNA; Derivation procedure; Development; Diabetes Mellitus; Disease; Endoderm; Extinction (Psychology); FDA approved; Fibrinogen; Fibroblasts; Future; Generations; Genes; Genetic Transcription; Genome; Genomics; Glycoproteins; Hematopoietic stem cells; Human; Infection; Lead; Lentivirus Vector; Measles; Measles virus; Methods; MicroRNAs; Modification; Molecular; Mus; Paramyxovirus; Patients; Plasmids; Pluripotent Stem Cells; Production; Property; Proteins; Public Health; RNA; Regenerative Medicine; Reporting; Research; Residual state; Safety; Sendai virus; Skin; Somatic Cell; Stem Cell Research; Suicide; System; Techniques; Testing; Toxicology; Transgenes; Translations; Vaccination; Vaccines; Viral; Viral Proteins; Viral Vector; Virus; arm; base; cell type; clinical application; improved; induced pluripotent stem cell; islet; keratinocyte; particle; pluripotency; positional cloning; protein expression; public health relevance; recombinant virus; regenerative; respiratory; stem cell technology; vector; vector genome

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.

描述（由申请人提供）：诱导的多能干细胞（IPSC）技术使患者特异性的多能干细胞从成年体细胞中推导。我们已经证明了使用仙台病毒载体从患者的无基因组修饰IPSC的可重复推导。但是，基于鼠Paramyxovirus的仙台矢量系统从未获得FDA批准用于人类临床试验的使用。在这里，我们建议基于人类帕托病毒的病毒（MV）开发一种新的矢量系统。我们已经开发了一种反向遗传系统，允许生产相当于莫拉滕疫苗菌株的重组病毒，该病毒目前用于美国的儿童疫苗接种。此外，正在生产临床级基因改性的MV来治疗癌症患者，目前正在人类临床试验中进行评估。在AIM 1中，我们将测试可以将麻疹病毒修改为表达四个重编程基因（OCT4，KFL4，SOX2和CMYC）的“一个周期”重编程向量的假设。我们将产生四个表达一个重编程因子（RF）的“一个循环” MV载体，并测试其在IPSC中重编程成年体细胞的能力。在AIM 2中，我们将检验以下假设：我们可以提高“单周” MV载体对IPSC中的成年体细胞的功效和安全性。我们将通过产生单个MV载体来提高功效，该MV向量表达来自单个基因组的四个RF。我们将利用转录的麻疹梯度，以优化四个因素的表达水平，以提高重编程效率。为了提高向量的安全性，我们将使用自杀机制将载体组装起来，这将允许RF表达灭绝以及消除残留基因组和病毒蛋白表达，一旦IPSC的分化开始。我们将插入特异性内胚层中特异性miRNA的目标序列。这将提高我们向量的分化和安全性的效率。这些研究的结果将导致一个新的矢量系统，允许生产无基因组修饰的IPSC，从而使IPSC快速转化为诊所。