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

基本信息

批准号：
7999718
负责人：
ZELPHA ELIZABETH FLOYD
金额：
$ 22.92万
依托单位：
NUPOTENTIAL, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-18 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7999718
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 Histone Deacetylase 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

项目摘要

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）：已经通过外源转录因子的病毒转导证明了体细胞直接重编程为诱导多能干（iPS）细胞。这些初步演示为体细胞重编程的分子机制提供了宝贵的见解，并提出了在工业规模上开发替代策略以在不使用胚胎或遗传操作的情况下生产多能干细胞的可能性。然而，这些 iPS 方法的临床应用受到极低的重编程效率和与病毒转导相关的安全问题的阻碍。 NuPotential 开发了多种方法，通过修改染色质结构以诱导沉默的多能基因的表达，将体细胞重编程为多能状态。这些方法包括： 1) * 改变单一碳代谢途径并限制培养基中的甲基供体以调节 * 甲基化能力； 2) 使用干扰RNA技术敲低抑制性表观遗传调节蛋白（即DNA*甲基转移酶和组蛋白脱乙酰酶）的基因表达； 3）用小分子调节表观遗传*调控成分； 4)将单个多能性基因C的过表达与一种或多种表观基因的shRNA敲低相结合（NuPotenial诱导多能干细胞，NuPiPStm）。 o 这些方法有多种实施方式，NuPotential 已针对所有这些实施方式申请了专利； 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（专利号：7,601,699）。随着 NuPotential 开发这些方法，确定了一条关键途径，该途径似乎是提高重编程效率的关键（如果不是关键）速率限制步骤。当我们使用 shRNA 技术系统地敲低各种表观遗传靶点时，我们发现了一种冗余，它似乎起到了补偿作用。当用小分子 Epi 药物处理体细胞时，也观察到这种冗余。尽管 NuPotential 迄今为止已证明 20 多种市售 Epi 药物对 i 多能性基因表达具有统计显着上调作用，但倍数增加对于有效重编程而言并不是最佳选择，这可能是由于这种补偿途径所致。这些数据表明，为了在不使用外源基因/蛋白质或病毒载体的情况下有效地产生大量的、完全重编程的多能干细胞（RePSCTM）群体，需要鉴定和/或开发专门且有效地靶向以下成分的化合物：这条补偿途径。我们实验室对化学策略的后续研究最近揭示了表观遗传药物和蛋白酶体抑制剂联合治疗对冗余表观遗传调控成分的协同抑制，以及关键多能性基因 Oct4 的显着上调。这些数据支持这样的假设：将小分子表观药物与蛋白酶体抑制剂相结合的新策略可以通过更有效地靶向抑制多能性基因和对重编程至关重要的蛋白质的冗余表观蛋白来提高体细胞重编程的效率，这是*第一阶段提案的基础。该第一阶段提案的目标是 1) 在人 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-药物的组合； 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能力，证明 NuPotential 专有的人类 iPS (NuPiPSTM) 细胞与未修饰的人类体细胞相比，核重编程得到改善，从而提供原理证明用于再分化成新的细胞类型。为了实现这一目标，NuPotential 建议将其在基于表观遗传的体细胞重编程方面的专业知识与彭宁顿生物医学研究中心研究员伊丽莎白·弗洛伊德 (Elizabeth Floyd) 博士的专业知识合作，伊丽莎白·弗洛伊德博士专门研究蛋白酶体依赖性蛋白质周转和翻译后修饰。在第一阶段研究中验证的最佳组合策略将在第二阶段使用，以开发新颖且专有的衍生化合物，用于重编程以产生高效、商业相关的RePSCTM，而无需使用病毒载体转导的iPS细胞。公共健康相关性：NuPotential 将利用 STTR 资金开发一种新颖的纯化学方法来体细胞重编程，通过结合 Epi 药物和蛋白酶体抑制剂来显着抑制抑制性表观遗传调控成分并上调多能性标记。第一阶段研究将提供关键数据，使开发新的专有衍生化合物库成为可能，这些化合物抑制冗余表观遗传调节蛋白的所有可能组合，从而显着提高重编程的效率。商业目标是生产高效的临床和商业相关的重编程多能干细胞（RePSCTM），用于自体细胞替代疗法，而不使用外源基因、病毒载体、核移植、卵母细胞或胚胎。