Improving Epigenetic-based Cell Reprogramming with Proteasome Inhibition

描述(由申请人提供)：通过病毒转导外源转录因子，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宝贵的见解，并提高了在不使用胚胎或遗传操作的情况下，可以在工业规模上开发替代策略来生产多能干细胞的可能性。然而，由于与病毒转导相关的低重编程效率和安全性问题，这些iPS方法的临床应用受到阻碍。NuPotential已经开发出几种方法，通过修改染色质结构来诱导沉默的多能性基因的表达，从而将体细胞重新编程为多能性状态。这些方法包括：1)改变单一的碳代谢途径并限制培养基中甲基供体以调节甲基化能力；2)利用干扰RNA技术抑制表观遗传调节蛋白(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节成分；以及4)将单个多能基因C的过度表达与一个或多个表观基因(NuPotenial诱导的多能干细胞，NuPiPStm)的shRNA敲除相结合。O这些方法有几个实施例，NuPotential已经为所有这些方法申请了专利；2009年10月，NuPotential获得了其第一项基础专利，这些新的重新编程方法基于这些基础专利(专利#7,601,699)。当NuPotential开发这些方法时，发现了一条关键途径，它似乎是提高重新编程效率的关键(如果不是关键的话)d速率限制步骤。当我们使用shRNA技术系统地击倒各种表观遗传靶点e时，我们发现了一个冗余，它似乎起到了补偿n的作用。当用小分子药物处理体细胞时，也观察到这种冗余。尽管到目前为止，NuPotential已经在统计上显著上调了20多种商用表面药的I多能性基因表达，但折叠增加对于有效的重新编程并不是最佳的，这可能是由于这种代偿途径。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高效地产生大量完全重新编程的多潜能干细胞群体，需要识别和/或开发能够特异性和*高效地靶向这一补偿途径的组件的化合物。我们实验室随后对化学策略的研究*最近发现，通过联合使用表观药物和蛋白酶体抑制剂，可以协同抑制多余的表观遗传调节成分，并显著上调关键的多能性基因Oct4的表达。这些数据支持这样一种假设，即结合小分子表观药物和蛋白酶体抑制剂的新策略可能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表观蛋白来提高体细胞重编程的效率，这是这一第一阶段提案的*基础。这项第一阶段建议的目标是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商业上可用的Epi药物；以及2)通过有针对性的染色质修饰、关键的多潜能基因和蛋白的表达、克隆形成以及体外和体内重新分化为新细胞类型的能力，证明NuPotential的专有人类iPS(NuPiPSTM)细胞的核重新编程能力与未经修饰的人体细胞相比有所改善，从而提供原理证明。为了实现这一目标，NuPotential建议将其在基于表观遗传学的体细胞重新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·弗洛伊德博士合作，后者专门从事蛋白酶体依赖的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用于开发新的和专有的衍生化合物，用于重新编程，在不使用病毒载体转导的iPS细胞的情况下生产高效、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来重新编程体细胞，方法是将表观药物和蛋白酶体抑制剂结合起来，显著抑制抑制性表观遗传调节成分，并上调多能性标记。第一阶段的研究将提供关键数据，使开发新的和专有的衍生化合物库成为可能，这些化合物可以抑制多余的表观遗传调节蛋白的所有可能组合，以便显著提高重新编程的效率。商业目标是生产高效的临床和商业相关的可重编程多能干细胞(RePSCTM)，用于自体细胞替代治疗，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.