Reprogramming iPS Cells with Exogenous and Endogenous Transcription Factor Genes

项目总结（见说明）： 项目1：将体细胞重编程为诱导多能干细胞的发现为人类疾病的研究和治疗开辟了新的维度。自从第一次描述通过用逆转录病毒载体引入4种转录因子基因来重编程小鼠IPS细胞以来，许多细胞类型的小鼠和人细胞已经被成功地重编程。尽管逆转录病毒载体仍然是最有效的重编程载体，但其随机整合的特性可能通过破坏重要的宿主基因功能而造成损害。已经报道了用于引入转录因子的许多其他载体，包括质粒、基于EB的质粒载体、腺病毒载体、转座子、慢病毒载体和蛋白质。这些方法中的一些对于重编程人类体细胞是低效的。慢病毒载体也随机整合到基因组中，尽管它们可以用cre-lox去除。该项目的目的是研究新的IPS技术，可在未来应用于治疗镰状细胞病和B-地中海贫血，这两种最常见的遗传病。我们将使用两种策略。第一种是利用PhiC 31整合酶fpr位点特异性整合，引入由2A肽连接的4个转录因子基因OCT 4、SOX 2、FLT 4和cMYC。 第二种策略，由共同研究者Long-Cheng Li指导，将使用他的短激活剂双链RNA的创新来刺激内源性转录因子基因的表达。他已经证明这些saRNA可以刺激各种内源基因的表达。我们提出的策略具有不干扰宿主基因功能的优点。我们的项目是针对 最终应用于镰状细胞病和地中海贫血。我们将重新编程患者的皮肤活检细胞以及羊水和CVS细胞，这将有助于产前诊断后的早期细胞治疗。

PROJECT SUMMARY (See Instructions): Proj 1: The discovery of reprogramming somatic cells to induced pluripotent stem cells has opened new dimensions for the study and treatment of human diseases. Since the first description of reprogramming mouse IPS cells by introducing 4 transcription factor genes with retroviral vectors, mouse and human cells of many cell types have been successfully reprogrammed. Although retroviral vectors are still the most proficient vehicles for reprogramming, their property of random integration may cause damage by disrupting vital host gene functions. Many other vehicles for introducing transcription factors have been reported, including plasmids, EB based plasmid vectors, adenoviral vectors, transposons, lentiviral vectors and proteins. Some of these methods are inefficient for reprogramming human somatic cells. Lentiviral vectors also integrate randomly into the genome although they could be removed with cre-lox. The aim of this project is to investigate novel IPS techniques that can be applied in the future to the treatment of sickle cell disease and B-thalassemia, the 2 most common genetic diseases. We will use 2 strategies. The first is to introduce the 4 transcription factor genes 0CT4, S0X2, FLT4 and cMYC linked by 2A peptides using PhiC31 integrase fpr site-specific integration. The second strategy, directed by the co-investigator Long-Cheng Li, will use his innovation of short activator double stranded RNA to stimulate the expression ofthe endogenous transcription factor genes. He has shown that these saRNAs can stimulate expression of endogenous genes of various kinds. The strategies we propose have the advantage of not disturbing the functions of the host genes. Our project is directed to eventual application to sickle cell disease and thalassemia. We will reprogram skin biopsy cells from patents as well as amniotic fluid and CVS cells which will be useful for early cell therapy after prenatal diagnosis.