Improving Epigenetic-based Cell Reprogramming with Proteasome Inhibition

描述（由申请方提供）：已通过外源性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已经开发了几种方法，通过修改染色质结构来诱导沉默的多能性基因的表达，将体细胞重编程为多能性状态。这些方法包括：1）* 改变单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）敲低阻遏性表观遗传调节蛋白的基因表达（即，DNA甲基转移酶和组蛋白脱乙酰酶）; 3）用小分子调节表观遗传调节组分;和4）将单个多能性基因C的过表达与一个或多个表观基因的shRNA敲低组合（NuPotential诱导的多能干细胞，NuPiPStm）。这些方法有几个实施方案，NuPotential已经对所有这些方法申请了专利; 2009年10月，NuPotential获得了这些新的重编程方法所基于的第一个基础专利（专利#7，601，699）。i随着NuPotential开发这些方法，确定了一个关键途径，该途径似乎是提高重编程效率的关键（如果不是关键的话）限速步骤。当我们使用shRNA技术系统地敲除各种表观遗传靶点时，我们发现了一种冗余，它似乎起着补偿n的作用。当体细胞用小分子tEpi药物处理时也观察到这种冗余。尽管NuPotential迄今已证明用超过20种市售表型药物对i多能性基因表达的统计学显著上调，但倍数增加对于有效重编程而言不是最佳的，可能是由于这种补偿途径。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的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该补偿途径的组分的化合物。最近，我们实验室对化学策略 * 的后续研究显示，联合Epi药物和蛋白酶体抑制剂 * 治疗可协同抑制多余的表观遗传调控成分，并显著上调关键多能性基因Oct 4。这些数据支持这样的假设，即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的冗余表观蛋白和对重编程至关重要的蛋白质来提高体细胞重编程的效率，这是本I期提案的基础。 该I期方案的目标是1）在人Oct 4启动子驱动的GFP报告细胞系中筛选与市售Epi药物组合的蛋白酶体抑制剂;和2）通过证明NuPotential专有的人类iPS中改进的核重编程来提供原理证明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成新细胞类型的能力。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博士的专业知识合作，后者专门研究蛋白酶体依赖性蛋白质周转和翻译后修饰。在I期研究中验证的最佳组合策略将用于II期，以开发用于重编程的新型和专有的衍生化合物，从而在不使用病毒载体转导的iPS细胞的情况下产生高效的、商业相关的RePSCTM。 公共卫生相关性：NuPotential将利用STTR资金开发一种新的纯化学方法，通过将Epi-drugs和蛋白酶体抑制剂结合起来，显著抑制抑制性表观遗传调控成分，并上调多能性标志物，从而实现体细胞重编程。I期研究将提供关键数据，这些数据将能够开发新型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。

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.