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基金来产生和验证人Oct-4基因靶向报告细胞系，该细胞系将提供基因转录的上级预测索引，用于鉴定有效的重编程能力表观药物。为了实现这一目标，NuPotential将与爱荷华州大学的Gregory Leno博士合作，该大学的实验室此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。将通过证明用市售表观药物处理后报告基因活性与内源性Oct-4基因表达之间的显著相关性来验证报告基因细胞系，NuPotential已显示出Oct-4基因表达的上调。Oct-4基因是重编程分化的人类细胞的关键靶点。 NuPotential的专有体细胞重编程平台是基于体外靶向表观基因组的小分子抑制剂的抑制性调控成分的染色质。选择的多能性相关靶基因的去阻遏促进分化细胞中的转录并恢复基因组潜能。NuPotential的重点应用包括小分子驱动的自体细胞生产，用于替代疗法和改善牲畜克隆。在分化细胞中识别激活Oct-4和其他多能性相关基因的新型小分子，可以解决与现有重编程方法相关的效率问题。然而，目前基于细胞的高通量药物筛选技术利用随机整合的（非靶向的）报告基因，其可能缺乏控制内源基因表达的遗传和/或表观遗传调控元件。这些报告基因的表达也可能由整合位点的未知染色体背景控制。因此，非靶向Oct-4报告细胞系中报告基因表达的潜在误调节可能导致鉴定出具有有限或无细胞重编程潜力的小分子。该I期提案将产生Oct-4基因靶向报告细胞系，该细胞系可准确指示人二倍体成纤维细胞（HDF）中的内源性Oct-4基因表达。将使用通过抑制HDF细胞中抑制Oct-4表达的表观遗传调控组分促进Oct-4转录的小分子对报告细胞系进行验证。这种报告细胞系将提供一个敏感的和更有预测性的分析工具，用于识别调节Oct-4转录的分子。在靶向克隆确认后，将报告基因活性与来自人BJ成纤维细胞的报告细胞中药物诱导的Oct-4活化前后的内源性Oct-4基因表达进行比较。在II期研究中，这种经验证的报告细胞系将用于新开发的表观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的同基因人类体细胞的治疗潜力至关重要。通过这项研究获得的知识可以通过提供分化细胞直接重编程的途径，从而可能产生针对患者患病或受损组织的替代细胞，从而推进人类疾病的潜在细胞疗法。 公共卫生相关性：人Oct-4基因靶向报告细胞系将从BJ二倍体成纤维细胞系生成，并通过证明在表观药物诱导的Oct-4活化前后报告活性与内源性Oct-4表达之间的显著相关性进行验证。表征的报告细胞系将用于II期研究，以筛选用于真实Oct-4转录激活剂的新型小分子文库。商业目标是使用这种靶向细胞系来鉴定可以提高核重编程效率的新型小分子。有效重编程的人类细胞在再生医学中具有可行的商业应用。

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.