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：
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
项目类别：
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

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）：通过病毒转导外源转录因子的病毒转导证明了将体细胞直接重编程为诱导的多能茎（IPS）细胞。这些最初的演示为体细胞重编程的分子机制提供了宝贵的见解，并提出了可以在工业规模开发替代策略的可能性，以在不使用胚胎或遗传操作的情况下生产多能干细胞。但是，这些IPS方法的临床应用受到了与病毒转导相关的非常低的重编程效率和安全性问题的阻碍。 Nupotential开发了几种方法，通过 *修饰染色质结构来诱导沉默的多能基因的表达来将体细胞重编程为多能状态。这些方法包括：1） *改变培养基中的单个碳代谢途径和限制甲基供体以调节 *甲基化能力； 2）使用干扰RNA技术击倒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基酶）的基因表达； 3）与小分子调节表观遗传 *调节成分； 4）将单个多能性基因C与一个或多个表皮基因的shRNA敲低（Nupotenial诱导的多能干细胞NupipstM）结合在一起。 o这些方法有几个实施方案，Nupitential已向所有方法提交了专利。 2009年10月，Nupitential获得了首个基础专利，这些新颖的重编程F方法是基于的（专利＃7,601,699）。 I作为Nupitential开发了这些方法，这是一种关键途径，似乎是确定了提高重编程效率的键限制步骤（如果不是密钥）。当我们使用SHRNA技术系统地将E击倒各种表观遗传靶标时，我们确定了一种似乎功能n作为补偿的冗余。当将体细胞用小分子t e表毒处理处理时，还观察到这种冗余。尽管Nupitential的战绩表现出具有超过20多个商业上可用的Epi-drugs的I多能基因表达的统计学显着上调，但倍数增加对于有效的重编程而言并不是最佳的，这可能是由于这种补偿性途径所致。这些数据表明，要有效地产生大型，完全编程的多能干细胞（REPSCTM）种群，而无需使用外源 *基因/蛋白质或病毒载体，则需要确定和/或开发该补偿途径的有效目标成分。随后对化学策略的研究 *在我们的实验室中，最近揭示了对冗余表观遗传调节成分的协同抑制作用，并且 *对关键多能基因OCT4的显着上调与epi-Drug和蛋白酶体抑制剂 *处理。这些数据支持以下假设：将小分子Epi-prugs与 *蛋白酶体抑制剂相结合的新型策略可能会通过更有效地靶向 *冗余的Epi-protein来提高体细胞重编程的效率，从而抑制多能性基因和蛋白质，这是重新编程至关重要的，这是该阶段I阶段I建议的基础。该阶段I提案的目标是1）筛选蛋白酶体抑制剂，结合了人类Oct4启动子驱动的GFP报道细胞系中的市售Epi-drugs；和2）通过证明Nupotitial的专有人IP（NupiPSTM）细胞的核重编程的改进，与未经修饰的人类体细胞相比，通过靶向染色质修饰，关键的多能基因和蛋白质表达，集结形成，体外和体内重新分化新细胞类型的能力。为此，Nupotential建议将其在表观遗传细胞重编程中的专业知识与Pennington生物医学研究中心研究人员Elizabeth Floyd博士的专业知识合作，该研究人员专门研究蛋白酶体依赖性蛋白质转移和翻译后修饰。在第一阶段研究中验证的最佳组合策略将在II期中使用，以开发新颖和专有的衍生化合物，用于重新编程，以生成高效，商业上相关的REPSCTM，而无需使用病毒载体转导的IPS细胞。公共卫生相关性：Nupotential将使用STTR基金来开发一种新型的化学方法来通过将Epi-drugs和蛋白酶体抑制剂组合起来，以显着抑制抑制性表观遗传调节组件和上调多能标记。第一阶段的研究将提供关键的数据，该数据将能够开发出新的和专有的衍生化合物库，这些化合物抑制冗余表观遗传调节蛋白的所有可能组合，以显着提高重编程的效率。商业目标是在不使用外源基因，病毒载体，核转移，卵母细胞或胚胎的情况下生产高效的临床和商业重编程多能干细胞（REPSCTM），以进行自体细胞替代疗法。