Production of cell lines with improved reprogramming potential for SCNT

描述（由申请人提供）：NuPotential将使用NIH STTR资金开发和验证一种新的基于表观遗传学的体外系统，用于对牛供体细胞进行重编程，以提高体细胞核移植（SCNT）的效率。为了实现这一目标，NuPotential将改变染色质表观遗传标记，以诱导关键的多能性基因和蛋白质，从而在SCNT之前增加供体细胞的潜力。商业目标是提高家畜克隆的核重编程效率。NuPotential专有的体细胞重编程平台是基于靶向表观基因组，通过抑制抑制性调控成分，在体外诱导多能性基因和恢复分化潜能。为了实现这些STTR研究的目标，NuPotential将通过抑制DNA甲基转移酶（DNMT）和组蛋白脱乙酰酶（HDAC）来恢复SCNT之前牛供体细胞的潜力，以实现多能性基因的转录。在第一阶段，NuPotential将采用shRNA技术来敲低牛供体细胞中DNMT和HDAC的基因表达。其次，我们将通过shRNA处理后的多能基因和蛋白质表达以及染色质修饰来评估供体细胞重编程。第三，NuPotential的重编程系统将通过评估SCNT后的发育潜力来验证。在第二阶段，NuPotential将使用在第一阶段验证的抑制性表观遗传调控靶点的小分子来诱导多能性基因表达，并恢复SCNT牛供体细胞的分化和发育潜力。为了支持这一提议，NuPotential最初在用DNMT 1特异性siRNA处理的牛供体细胞中证明了SCNT后DNA去甲基化和发育潜力的改善。随后，我们证明了上调的多能性基因和恢复分化潜能的牛供体细胞通过改变细胞的甲基化能力，在体外，与全反式维甲酸。最近，NuPotential证明了在用抑制DNMTS或HDAC的shRNA或小分子处理后，在体细胞中诱导与已发表的胚胎干细胞非常相似的表型，包括诱导关键多能性基因/蛋白质和形成胚状体样集落。这些结果清楚地表明，抑制性表观遗传调控组分可以被抑制以恢复体细胞的潜能。NuPotential的体细胞重编程技术用于改进牲畜克隆方法的主要市场包括高遗传价值牲畜、用于大规模生产治疗分子的动物生物反应器以及疾病动物模型的开发。NuPotential将寻求合适的合作伙伴，在这些市场中利用其技术。 公共卫生相关性：NuPotential将开发和验证一种新的基于表观遗传学的系统，以增加供体细胞用于体细胞核移植的潜力。shRNA技术将用于抑制牛供体细胞中的抑制性表观遗传调控组分，以诱导对恢复潜力至关重要的多能性基因的转录。商业目标是提高家畜克隆的核重编程效率。

DESCRIPTION (provided by applicant): NuPotential will use NIH STTR funds to develop and validate a novel epigenetic-based in vitro system for reprogramming bovine donor cells to improve the efficiency of somatic cell nuclear transfer (SCNT). To accomplish this, NuPotential will alter chromatin epigenetic marks to induce key pluripotency genes and proteins to increase potential of donor cells prior to SCNT. The commercial goal is to improve the efficiency of nuclear reprogramming for livestock cloning. NuPotential's proprietary somatic cell reprogramming platform is based on targeting the epigenome by inhibiting repressive regulatory components, in vitro, to induce pluripotency genes and restore differentiation potential. To achieve the goals of these STTR studies, NuPotential will restore potential of bovine donor cells prior to SCNT by inhibiting DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) to enable transcription of pluripotency genes. In Phase I, NuPotential will employ shRNA technology to knock down gene expression of DNMTs and HDACs in bovine donor cells. Secondly, we will assess donor cell reprogramming by pluripotent gene and protein expression and chromatin modifications following shRNA treatment. Thirdly, NuPotential's reprogramming system will be validated by assessment of developmental potential following SCNT. In Phase II, NuPotential will use small molecules that inhibit repressive epigenetic regulatory targets validated in Phase I to induce pluripotency gene expression and restore differentiation and developmental potential of bovine donor cells for SCNT. In support of this proposal, NuPotential initially demonstrated DNA demethylation and improved developmental potential following SCNT in bovine donor cells treated with DNMT1-specific siRNA. Subsequently, we demonstrated up regulation of pluripotency genes and restored differentiation potential in bovine donor cells by altering cellular methylation capacity, in vitro, with all-trans retinoic acid. More recently, NuPotential demonstrated induction of phenotypes remarkably similar to published embryonic stem cells, including induction of key pluripotency genes/proteins and formation of embryoid body-like colonies, in somatic cells following treatment with shRNA or small molecules that inhibit DNMTS or HDACs. These results clearly indicate that repressive epigenetic regulatory components can be inhibited to restore potential of somatic cells. Major markets for NuPotential's somatic cell reprogramming technology to improve livestock cloning methods include high genetic merit livestock, animal bioreactors for large scale production of therapeutic molecules, and development of animal models of disease. NuPotential will seek appropriate partners to exploit its technology in each of these markets. PUBLIC HEALTH RELEVANCE: NuPotential will develop and validate a novel epigenetic-based system to increase potential of donor cells for somatic cell nuclear transfer. shRNA technology will be used to inhibit repressive epigenetic regulatory components in bovine donor cells to induce transcription of pluripotency genes critical for restoring potential. The commercial goal is to improve the efficiency of nuclear reprogramming for livestock cloning.