An inducible CRISPR/dCAS9 strategy for directed differentiation of pluripotent stem cells

细胞疗法如造血干细胞移植和红细胞或血小板输注用于治疗许多血液和免疫系统疾病，但这些治疗高度依赖于有限的健康供体供应。此外，尽管对捐献的血液进行了广泛的筛查，但患者总是有可能从捐献的细胞中感染。为了解决这些问题，人们做了很多努力，在实验室中从无限的、无感染的来源产生血细胞。其中一个来源是人类多能干细胞（hPSC），它可以在实验室中作为干细胞无限生长，然后在规定的条件下，可以分化成任何细胞类型，包括血细胞。然而，这一过程的效率非常低，并且不可能产生功能齐全的血细胞。几项研究表明，特定细胞类型的生产和功能可以通过激活作为分子开关的因子的表达来“引导”细胞进入特定细胞类型来改善，以打开特定细胞功能所需的基因的表达。这些研究依赖于使用质粒或慢病毒载体将转基因导入细胞，但它们导致异常高水平的基因表达。我们建议使用一种新的策略，激活细胞自身的遗传网络，使基因表达达到更高的生理水平。我们将首先比较hPSC与成人血细胞的基因谱，这将确定产生功能齐全的成人样血细胞所需的遗传开关，以及可用于跟踪生产过程的新标记。然后，我们将测试这些基因开关在分化hPSC中的激活是否会导致从hPSC生产血细胞的改善。为此，我们将使用一种新的合成生物学策略，通过这种策略，我们选择的因子可以使用称为向导RNA（gRNA）的小分子工具来打开。gRNA被设计为与基因组的调节特定基因表达的区域结合，并且与称为CAS9-SAM的蛋白质复合物一起，可以激活基因的表达。我们的提议的独特方面之一是我们设计并测试了hPSC系，其中蛋白质复合物的活化可以在添加药物后诱导。这种新的iSAM策略代表了该领域的重大进展，因为它使该方法能够同时激活多种因子，这可能是产生全功能血细胞所需的。我们将首先使用我们的iSAM策略来激活单个和组合的遗传因子，并监测它们对血细胞产生的影响。这将产生一个实验管道，使我们能够在一个实验中筛选基因组中的所有基因。我们通过遗传因子编程产生的细胞将使用流式细胞术等方法进行测试，以评估细胞表面标记物的存在，集落形成测定以监测血细胞祖细胞和单细胞RNA测序以分析编程过程的转录后果。该项目将提供对遗传因子编程的更好理解，并可能提供一种生产用于治疗血细胞疾病患者的细胞的途径。我们独特的携带iSAM复合物的iPSC细胞系将与研究其他治疗性细胞类型（如多巴胺能神经元）的编程和生产的研究人员共享，以治疗帕金森病或糖尿病的胰腺β细胞。我们的策略提供了显着的优势，经典的转基因技术，充满了技术困难，如基因沉默和插入诱变，将在临床上提出重大的安全问题。

Cell therapy such as haematopoietic stem cell transplantation and red blood cell or platelet transfusion are used to treat many diseases of the blood and immune systems but these treatments are highly dependent on a limited supply of healthy donors. Also, despite extensive screening of donated blood, there is always a chance that the patient could get an infection from the donated cells. To solve these problems there has been a lot of effort to generate blood cells in the laboratory from a limitless and infection-free source. One such source is human pluripotent stem cells (hPSCs) that can be grown indefinitely in the lab as stem cells and then, under defined conditions, can be differentiated into any cells type including blood cells. However, the efficiency of this process is very low and it has not been possible to generate fully functional blood cells. Several studies have shown that the production and function of specific cell types can be improved by "directing" the cells into specific cell types by activating the expression of factors that act as molecular switches to turn on the expression of genes that are required for a particular cell function. These studies have depended on introducing transgenes into the cells using plasmid or lentiviral vectors but they result in abnormally high levels of gene expression. We propose to use a novel strategy that results in the activation of the cells' own genetic networks and results in more physiological levels of gene expression.We will first compare the gene profile of hPSC- with adult-derived blood cells and this will identify the genetic switches required for the production of fully functional adult-like blood cells as well as novel markers that can be used to track the production process. We will then test whether activation of these genetic switches in differentiating hPSCs will result in the improved production of blood cells from hPSCs. To do this we will use a novel synthetic biology strategy whereby our chosen factors can be turned on using small molecular tools known as guide RNAs (gRNAs). gRNAs are designed to bind to the region of the genome that regulates the expression of a particular gene(s) and, together with a protein complex, known as CAS9-SAM, the expression of the gene can be activated. One of the unique aspects of our proposal is that we have designed and tested an hPSC line in which activation of the protein complex can be induced upon addition of a drug. This novel iSAM strategy represents a significant advance in the field as it makes the approach amenable to the activation of multiple factors at once which is likely to be required for the generation of fully functional blood cells. We will first use our iSAM strategy to activate single and combinations of genetic factors and monitor their effects on the production of blood cells. This will generate an experimental pipeline that will then allow us to screen all the genes in the genome in one single experiment. Cells that we produce by programming with genetic factors will be tested using methods such as flow cytometry to assess the presence of markers on the cell surface, colony-forming assays to monitor blood cell progenitors and single cell RNA sequencing to analyse the transcriptional consequences of the programming process. This project will provide a better understanding of genetic factor programming and could provide a route to producing cells for the treatment of patients with blood cell disorders. Our unique iPSC line carrying the iSAM complex will be shared with researchers studying the programming and production of other therapeutic cell types such as dopaminergic neurons to treat Parkinsons disease or pancreatic beta cells for Diabetes. Our strategy offers significant advantages over the classical transgenic technologies that are fraught with technical difficulties such as gene silencing and insertional mutagenesis that would raise significant safety concerns in the clinic.