Development of a human Oct-4 gene-targeted reporter cell line

描述（由申请人提供）：NuPotential, Inc.将使用NIH STTR基金生成并验证人类Oct-4基因靶向报告细胞系，该细胞系将为识别有效的重编程能力的外源性药物提供优越的基因转录预测索引。为了实现这一目标，NuPotential将与爱荷华大学的Gregory Leno博士合作，他的实验室以前曾使用腺相关病毒将报告基因靶向选定的内源性基因位点。用市售外源性药物治疗后，报告细胞活性与内源性Oct-4基因表达之间的显著相关性将得到验证，NuPotential已经显示了Oct-4基因表达的上调。Oct-4基因是人类分化细胞重编程的关键靶标。NuPotential的体细胞重编程平台是基于染色质抑制调节成分的小分子抑制剂体外靶向表观基因组。选择多能相关靶基因的去抑制促进转录和恢复分化细胞的基因组潜力。NuPotential的重点应用包括用于替代疗法的小分子驱动的自体细胞生产和改进的牲畜克隆。新发现的激活Oct-4和分化细胞中其他多能性相关基因的小分子，可以解决现有重编程方法的效率问题。然而，目前基于细胞的高通量药物筛选技术使用随机整合（非靶向）报告基因，这些基因可能缺乏控制内源性基因表达的遗传和/或表观遗传调控元件。这些报告基因的表达也可能受到整合位点未知的染色体背景的控制。因此，在非靶向Oct-4报告细胞系中，报告基因表达的潜在错误调控可能导致鉴定出具有有限或没有细胞重编程潜力的小分子。这项I期计划将产生一种Oct-4基因靶向报告细胞系，该细胞系能够准确地在人二倍体成纤维细胞（HDF）中表达内源性Oct-4基因。报告细胞系将使用小分子进行验证，这些小分子通过抑制HDF细胞中抑制Oct-4表达的表观遗传调控成分来促进Oct-4转录。该报告细胞系将为鉴定调节Oct-4转录的分子提供一种敏感且更具预测性的分析工具。在靶向克隆确认后，报告细胞活性将与人BJ成纤维细胞中Oct-4药物诱导激活前后的内源性Oct-4基因表达进行比较。在II期研究中，这种经过验证的报告细胞系将用于新开发的外源性药物文库的高通量筛选，以鉴定具有重编程潜力的新型小分子。本研究的长期目标是鉴定能够在分化的人体细胞中激活内源性Oct-4转录的新型小分子，而无需强制引入外源基因和逆转录病毒感染。新的Oct-4靶向重编程分子的发现对于建立重编程等基因人体细胞的治疗潜力具有至关重要的意义。通过这项研究获得的知识可能通过为分化细胞的直接重编程提供途径，从而使针对患者患病或受损组织的替代细胞的产生成为可能，从而推进潜在的基于细胞的人类疾病治疗。

DESCRIPTION (provided by applicant): NuPotential, Inc. will use NIH STTR funds to generate and validate human Oct-4 gene-targeted reporter cell lines that will provide superior predictive indexing of gene transcription for the identification of efficient reprogramming-competent epi-drugs. To accomplish this, NuPotential will partner with Dr. Gregory Leno at the University of Iowa whose laboratory has previously used adeno-associated virus to target a reporter gene to a selected endogenous gene locus. Reporter cell lines will be validated by demonstrating a significant correlation between reporter activity and endogenous Oct-4 gene expression following treatment with commercially available epi-drugs with which NuPotential has shown up-regulation of Oct-4 gene expression. The Oct-4 gene is a key target for reprogramming differentiated human cells. NuPotential's proprietary somatic cell reprogramming platform is based on in-vitro targeting of the epigenome with small molecule inhibitors of repressive regulatory components of chromatin. De-repression of select pluripotency-related target genes promotes transcription and restores genomic potential in differentiated cells. NuPotential's focused applications include the small molecule-driven production of autologous cells for replacement therapies and improved livestock cloning. The identification of novel small molecules that activate Oct-4, and other pluripotency-related genes in differentiated cells, could solve efficiency problems associated with existing reprogramming methods. However, current cell-based high-throughput drug screening technology utilizes randomly integrated (non-targeted) reporter genes that may lack genetic and/or epigenetic regulatory elements that govern endogenous gene expression. Expression of these reporter genes may also be governed by the unknown chromosomal context at the site of integration. Hence, the potential mis-regulation of reporter expression in non-targeted Oct-4 reporter cell lines may lead to the identification of small molecules with limited or no cell reprogramming potential. This Phase I proposal will generate an Oct-4 gene-targeted reporter cell line that accurately indexes endogenous Oct-4 gene expression in human diploid fibroblasts (HDF). The reporter cell line will be validated using small molecules that promote Oct-4 transcription by inhibiting epigenetic regulatory components that repress Oct-4 expression in HDF cells. This reporter cell line will provide a sensitive and more predictive analytical tool for identifying molecules that modulate Oct-4 transcription. Following targeted clone confirmation, reporter activity will be compared to endogenous Oct-4 gene expression before and after drug-induced activation of Oct-4 in reporter cells from human BJ fibroblasts. In Phase II studies, this validated reporter cell line will be used for high throughput screening of a newly developed epi-drug library to identify novel small molecules with reprogramming potential. The long-term objective of this research is the identification of novel small molecules that are capable of activating endogenous Oct-4 transcription in differentiated human somatic cells without forced introduction of exogenous genes and retroviral infection. The discovery of novel Oct-4 targeted reprogramming molecules is of paramount importance for establishing the therapeutic potential of reprogrammed isogenic human somatic cells. The knowledge gained through this study may advance potential cell-based therapies for human disease by providing an avenue for the direct reprogramming of differentiated cells making possible the production of replacement cells targeted to a patient's diseased or damaged tissue. PUBLIC HEALTH RELEVANCE: A human Oct-4 gene-targeted reporter cell line will be generated from a BJ diploid fibroblast cell line and validated by demonstrating a significant correlation between reporter activity and endogenous Oct-4 expression before and after epi-drug-induced activation of Oct-4. The characterized reporter line will be used in Phase II studies to screen a novel small molecule library for activators of authentic Oct-4 transcription. The commercial goal is to use this targeted cell line to identify novel small molecules that can improve the efficiency of nuclear reprogramming. Efficiently reprogrammed human cells have viable commercial applications in regenerative medicine.