Improving Epigenetic-based Cell Reprogramming with Proteasome Inhibition

通过蛋白酶体抑制改善基于表观遗传的细胞重编程

• 批准号: 8145238
• 负责人: ZELPHA ELIZABETH FLOYD
• 金额: $ 9.99万
• 依托单位: NUPOTENTIAL, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-18 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145238
• 关键词: Autologous; Biological Assay; Bioluminescence; Biomedical Research; Carbon; Categories; Cell Culture Techniques; Cell Line; Cells; Chemicals; Chromatin Structure; Critical Pathways; Culture Media; DNA; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; Data; Dermal; Development; Down-Regulation; Embryo; Employee Strikes; Epigenetic Process; Exhibits; Fibroblasts; Financial compensation; Fluorescence Microscopy; Foundations; Funding; Gene Expression; Gene Proteins; Genes; Germ Layers; Goals; Histone Acetylation; Histones; Human; Immune; Immunochemistry; In Vitro; Injection of therapeutic agent; Investigation; Laboratories; Legal patent; Libraries; Life; Link; Measurement; Messenger RNA; Metabolism; Methods; Methylation; Molecular; Mus; Oocytes; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Pluripotent Stem Cells; Population; Post-Translational Protein Processing; Proteasome Inhibition; Proteasome Inhibitor; Protein Isoforms; Proteins; Publishing; RNA; Replacement Therapy; Reporter; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Safety; Small Business Technology Transfer Research; Somatic Cell; System; Technology; Teratoma; Time; Tissues; Up-Regulation; Valproic Acid; Viral; Viral Vector; Visual; Western Blotting; Work; adult stem cell; base; bisulfite; cell type; chromatin immunoprecipitation; chromatin modification; clinical application; clinically relevant; combinatorial; demethylation; density; genetic manipulation; genetic regulatory protein; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; knock-down; multicatalytic endopeptidase complex; novel; novel strategies; nuclear reprogramming; nuclear transfer; phase 1 study; pluripotency; promoter; protein degradation; protein expression; public health relevance; small hairpin RNA; small molecule; stem; stemness; transcription factor

DESCRIPTION (provided by applicant): Direct reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been demonstrated by viral transduction of exogenous transcription factors. These initial demonstrations have provided valuable insight into molecular mechanisms of somatic cell reprogramming and raised the possibility that alternative strategies could be developed on an industrial scale to produce pluripotent stem cells without using embryos or genetic manipulations. Clinical applications of these iPS approaches are impeded, however, by very low reprogramming efficiencies and safety concerns associated with viral transduction. NuPotential has developed several methods to reprogram somatic cells to a pluripotent state by * modifying chromatin structure to induce expression of silenced pluripotency genes. These methods include: 1) * altering the single carbon metabolism pathway and limiting methyl donors in culture medium to modulate * methylation capacity; 2) knocking down gene expression of repressive epigenetic regulatory proteins (ie., DNA * methyltransferases and histone deacetylases) using interfering RNA technology; 3) modulating epigenetic * regulatory components with small molecules; and 4) Combining over-expression of a single pluripotency gene C with shRNA knockdown of one or more epi-genes (NuPotenial induced pluripotent stem cells, NuPiPStm). o There are several embodiments to these approaches and NuPotential has filed patents on all of them; In n October, 2009, NuPotential was issued its first foundation patent on which these novel reprogramming f methods are based (Patent #7,601,699). i As NuPotential developed these methods, a critical pathway that appears to be a key (if not the key) d rate limiting step to improving the efficiency of reprogramming was identified. As we systematically knocked e down various epigenetic targets using shRNA technology, we identified a redundancy that appears to function n as compensation. This redundancy was also observed when somatic cells were treated with small molecule t Epi-drugs. Although NuPotential has thus-far demonstrated statistically significant up-regulation of i pluripotency gene expression with over 20 commercially available Epi-drugs, fold increases are not optimal for a efficient reprogramming, likely due to this compensatory pathway. These data demonstrate that to efficiently l produce large, fully-reprogrammed pluripotent stem cell (RePSCTM) populations without the use of exogenous * genes/proteins or viral vectors, compounds need to be identified and/or developed that specifically and * efficiently target components of this compensatory pathway. Subsequent investigation into chemical strategies * in our laboratory recently revealed a synergistic inhibition of redundant epigenetic regulatory components and * significant up-regulation of the key pluripotency gene Oct4 with combined Epi-drug and proteasome inhibitor * treatment. These data support the hypothesis that a novel strategy combining small molecule Epi-drugs with * proteasome inhibitors may increase the efficiency of somatic cell reprogramming by more effectively targeting * redundant epi-proteins that repress pluripotency genes and proteins critical for reprogramming, which is the * basis of this Phase I proposal. The goals of this Phase I proposal are to 1) screen proteasome inhibitors in combination with commercially available Epi-drugs in human Oct4 promoter driven-GFP reporter cell lines; and 2) provide proof of principle by demonstrating improved nuclear reprogramming in NuPotential's proprietary human iPS (NuPiPSTM) cells in comparison to unmodified human somatic cells by targeted chromatin modifications, key pluripotency gene and protein expression, colony formation, and in vitro and in vivo capacity for re- differentiation into new cell types. To accomplish this, NuPotential proposes to partner its expertise in epigenetic-based somatic cell reprogramming with that of Dr. Elizabeth Floyd, a Pennington Biomedical Research Center researcher specializing in proteasome-dependent protein turnover and post-translational modifications. Optimal combinatorial strategies validated in Phase I studies will be used in Phase II to develop novel and proprietary derivative compounds for reprogramming to produce highly efficient, commercially- relevant RePSCTM without the use of viral vector transduced iPS cells. PUBLIC HEALTH RELEVANCE: NuPotential will use STTR funds to develop a novel purely chemical approach to somatic cell reprogramming by combining Epi-drugs and proteasome inhibitors to significantly inhibit repressive epigenetic regulatory components and up-regulate pluripotency markers. Phase I studies will provide critical data that will enable development of a library of novel and proprietary derivative compounds that inhibit all possible combinations of redundant epigenetic regulatory proteins in order to significantly improve the efficiency of reprogramming. The commercial goal is to produce highly efficient clinically- and commercially-relevant reprogrammed pluripotent stem cells (RePSCTM) for autologous cell replacement therapies without the use of exogenous genes, viral vectors, nuclear transfer, oocytes, or embryos.

描述（由申请人提供）：通过外源转录因子的病毒转导，已经证明体细胞直接重编程为诱导多能干细胞（iPS）。这些初步的演示为体细胞重编程的分子机制提供了有价值的见解，并提出了在工业规模上开发替代策略以生产多能干细胞而不使用胚胎或基因操作的可能性。然而，由于重编程效率极低以及与病毒转导相关的安全性问题，这些iPS方法的临床应用受到了阻碍。NuPotential已经开发了几种方法，通过修饰染色质结构来诱导沉默多能基因的表达，将体细胞重编程为多能状态。这些方法包括：1)改变单碳代谢途径，限制培养基中的甲基供体来调节甲基化能力；2)抑制表观遗传调控蛋白的基因表达。（DNA *甲基转移酶和组蛋白去乙酰化酶）；3)用小分子调控表观遗传调控成分；4)将一个多能性基因C的过表达与shRNA敲低一个或多个表基因结合（nupotential induced pluripotent stem cells, NuPiPStm）。o这些方法有多个实施例，NuPotential已经为所有这些方法申请了专利；2009年10月，NuPotential获得了其第一个基础专利，这些新颖的重编程方法就是基于该专利（专利# 7601699）。在NuPotential开发这些方法的过程中，发现了一条关键途径，它似乎是提高重编程效率的关键（如果不是关键）限速步骤。当我们使用shRNA技术系统地敲除各种表观遗传靶标时，我们发现了一种冗余，它似乎起到了补偿的作用。当用小分子t - epi药物治疗体细胞时，也观察到这种冗余。尽管NuPotential公司迄今为止已经证明了20多种市售的epi药物对i多能性基因表达的统计学显著上调，但由于这种代偿途径的原因，对有效的重编程来说，倍倍的上调并不是最理想的。这些数据表明，为了在不使用外源基因/蛋白或病毒载体的情况下有效地产生大型、完全重编程的多能干细胞（RePSCTM）群体，需要鉴定和/或开发特异性和有效地靶向这一补偿途径成分的化合物。我们实验室对化学策略的后续研究最近发现，epi药物和蛋白酶体抑制剂联合治疗可协同抑制冗余表观遗传调控成分，并显著上调关键多能基因Oct4。这些数据支持了一个假设，即结合小分子epi药物和蛋白酶体抑制剂的新策略可以通过更有效地靶向抑制多能性基因和重编程关键蛋白的冗余epi蛋白来提高体细胞重编程的效率，这是本I期建议的基础。该I期计划的目标是：1)在人类Oct4启动子驱动的gfp报告细胞系中筛选蛋白酶体抑制剂与市售epi药物联合使用；2)通过靶向染色质修饰、关键多能性基因和蛋白质表达、集落形成以及体外和体内再分化为新细胞类型的能力，证明NuPotential专有的人类iPS （NuPiPSTM）细胞的核重编程优于未经修饰的人类体细胞，从而提供原理证明。为了实现这一目标，NuPotential提议将其在基于表观遗传学的体细胞重编程方面的专业知识与Elizabeth Floyd博士合作，Elizabeth Floyd博士是Pennington生物医学研究中心专门研究proteasome依赖性蛋白质周转和翻译后修饰的研究员。在I期研究中验证的最佳组合策略将在II期中用于开发新的和专有的衍生化合物，用于重编程，以生产高效的、商业相关的RePSCTM，而无需使用病毒载体转导的iPS细胞。