Pan Alexander, Weintraub Neal L, Tang Yaoliang
Vascular Biology Center, Department of Medicine, Medical College of Georgia/Georgia Regents University, 1459 Laney Walker Blvd, Augusta, GA 30912, USA.
Vascular Biology Center, Department of Medicine, Medical College of Georgia/Georgia Regents University, 1459 Laney Walker Blvd, Augusta, GA 30912, USA.
Med Hypotheses. 2014 Dec;83(6):702-5. doi: 10.1016/j.mehy.2014.09.022. Epub 2014 Oct 13.
Ischemic heart disease has remained the number one killer around the world for over the past 20 years. While stem cell therapy has become a promising new frontier to repair the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Strategies to genetically modify stem cells to activate pro-survival and anti-apoptotic and anti-inflammatory pathways, such as Akt and heme oxygenase-1, have been shown to improve the lifespan of transplanted stem cells within the ischemic myocardium, but constitutive overexpression of these pathways at high levels has been shown to have side effects. Therefore, more specific and controlled gene activation would be necessary. Current techniques used for gene regulation include zinc finger and TALE proteins, but there are still disadvantages to each of these methods, such as ease and cost of use. Also, those methods use synthesized promoters to express synthesized cDNA, which lack regulatory elements, including introns and 3' untranslated regions for microRNA mediated post-transcriptional regulation. A new novel technique, the CRISPR/dCas9 system, was recently developed as a simple and efficient method for endogenous gene regulation. With its use of single guide chimeric RNA's (sgRNA's), this system has been shown to provide a high level of specificity and efficiency. When targeting different loci, past studies have found that the CRISPR/dCas9 system can activate gene expression at varying levels. In addition, this system makes use of the genome's endogenous regulatory elements, such as the aforementioned introns and 3' UTR's, which can help provide a safer method of gene activation. If targeted to a gene promoting cellular survival or decreasing cell death, it could potentially improve stem cell longevity in a more efficient and controllable manner. As a result, our hypothesis is to use the CRISPR/dCas9 system to activate expression of an anti-inflammatory and anti-apoptotic gene, such as heme oxygenase-1 (HO-1), to an optimal level to increase transplanted stem cell survival while also mitigating its cytotoxic effects due to lack of internal regulation, thus prolonging its effects within the ischemic myocardium leading to greater therapeutic benefit.
在过去20多年里，缺血性心脏病一直是全球头号杀手。虽然干细胞疗法已成为修复受损心脏的一个充满希望的新领域，但移植后干细胞存活率有限阻碍了该疗法的广泛应用。对干细胞进行基因改造以激活促存活、抗凋亡和抗炎信号通路（如Akt和血红素加氧酶-1）的策略已被证明可提高移植干细胞在缺血心肌中的寿命，但这些信号通路的高水平组成型过表达已被证明有副作用。因此，需要更特异性和可控的基因激活。目前用于基因调控的技术包括锌指蛋白和转录激活样效应因子蛋白，但这些方法都存在缺点，如使用的难易程度和成本。此外，这些方法使用合成启动子来表达合成的互补DNA，缺乏包括内含子和3'非翻译区在内的调控元件，而这些调控元件对于微小RNA介导的转录后调控是必需的。一种新的技术——CRISPR/dCas9系统，最近被开发出来，作为一种简单有效的内源性基因调控方法。由于其使用单导向嵌合RNA（sgRNA），该系统已被证明具有高度的特异性和效率。在针对不同基因座时，以往研究发现CRISPR/dCas9系统可在不同水平激活基因表达。此外，该系统利用基因组的内源性调控元件，如上述内含子和3'非翻译区，这有助于提供一种更安全的基因激活方法。如果靶向促进细胞存活或减少细胞死亡的基因，它可能以更有效和可控的方式提高干细胞寿命。因此，我们的假设是使用CRISPR/dCas9系统将抗炎和抗凋亡基因（如血红素加氧酶-1，HO-1）的表达激活到最佳水平，以提高移植干细胞的存活率，同时减轻由于缺乏内部调控而产生的细胞毒性作用，从而延长其在缺血心肌中的作用时间，带来更大的治疗益处。
