Reprogramming iPS Cells with Exogenous and Endogenous Transcription Factor Genes

使用外源和内源转录因子基因重编程 iPS 细胞

基本信息

批准号：
8917047
负责人：
YUET Wai KAN
金额：
$ 38.14万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8917047
关键词：
Adenovirus Vector Amniotic Fluid Bacterial Chromosomes Bacteriophages Biopsy Cell Culture Techniques Cell Therapy Cell physiology Cells Chemicals Chorionic Villi Sampling Chromosomes Complementary DNA Coupled Development Diagnosis Dimensions Disease Double-Stranded RNA Early treatment Ensure Enzymes Epigenetic Process FLT4 gene Fibroblasts Future Gene Expression Generations Genes Genetic Recombination Genome Hematopoietic Hemoglobinopathies Hereditary Disease Homologous Gene Human Human Genome Instruction Integrase Legal patent Lentivirus Vector Link Location Mediating Methods Mus Patients Peptides Plasmid Cloning Vector Plasmids Prenatal Diagnosis Procedures Property Proteins RNA RNA Interference Reporting Research Personnel Retroviral Vector Sarna Sequence Homologs Sickle Cell Anemia Site Skin Small Interfering RNA Somatic Cell Stem cells System Techniques Tetracyclines Thalassemia Time Transcription factor genes Transgenes Virus base beta Thalassemia c-myc Genes cell type combinatorial gene function high throughput screening human disease improved induced pluripotent stem cell innovation integration site mammalian genome mouse genome novel programs promoter site-specific integration small molecule stem transcription factor vector

项目摘要

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.

项目摘要（参见说明）：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。自从首次描述通过用逆转录病毒载体引入 4 个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类细胞已被成功重编程。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会通过破坏重要的宿主基因功能而造成损害。已经报道了许多其他用于引入转录因子的载体，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。其中一些方法对于重新编程人类体细胞来说效率低下。慢病毒载体也会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未来可用于治疗镰状细胞病和 B 地中海贫血（两种最常见的遗传病）的新型 IPS 技术。我们将使用两种策略。首先是使用 PhiC31 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 0CT4、S0X2、FLT4 和 cMYC。第二个策略由共同研究员 Long-Cheng Li 指导，将利用他的短激活双链 RNA 创新来刺激内源转录因子基因的表达。他证明这些 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。我们的项目旨在最终应用于镰状细胞病和地中海贫血。我们将从专利中重新编程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。