IDBR: Using a Nanopore to Transfect Cells with Single Molecule Precision to Induce Pluripotency Efficiently in Fibroblasts

【摘要】美国圣母大学（University of Notre Dame）开发了一种新工具，该工具使用合成的纳米直径孔（即纳米孔）对特化细胞的遗传记忆进行重编程，诱导它们变得像胚胎干细胞一样。干细胞研究有望为从失明到修复心脏和神经损伤，甚至衰老的所有疾病提供再生疗法。直到最近，临床研究主要集中在两种细胞：胚胎和非胚胎或成体干细胞。除了使用胚胎干细胞带来的伦理困境之外，这两种细胞类型相对于有效治疗所需的水平来说都是稀缺的。另一方面，诱导多能干细胞（iPSCs）现在可能是一种可行的替代方法。诱导多能干细胞是通过强迫四个关键基因的表达而从特化的体细胞中获得的；它们类似于胚胎干细胞。不幸的是，只有大约0.01-0.1%的人类体细胞可以被迫成为多能干细胞。由于缺乏对关键基因的导入和表达水平的控制，体细胞的重编程效率受到削弱。巴黎圣母院的这项研究工作将生产一种工具，该工具使用纳米孔将核酸一次一个分子地转移到单个细胞中，从而严格控制目标基因的表达。合成的纳米孔使用一个紧密受限的电场在细胞膜上形成一个孔，促进核酸甚至整个基因的传递，潜在的单分子精度，同时允许连续或重复的基因传递到同一个细胞中。作为测试该工具的坩埚，编码关键基因的核酸将被引入体细胞以产生多能干细胞。这项研究具有更广泛的意义，不仅仅是开发一种将基因传递到细胞中的工具。该项目还提供了一个通过单分子精确控制基因及其环境来理解和改造生物学的机会。为了利用这一机会，将把这项研究纳入教育课程，并在生物、保健和医学界传播。这个拓展计划有几个组成部分：i.通过每月的系列研讨会和网站互相教学；2。在工程和生物科学的指导下，通过新的实验课程——系统生物学导论——来启发学生；最后，通过与当地一所新科技高中的合作，让公众和高中生了解这种新型纳米技术对生物和医学的影响。

ABSTRACTAn award is made to the University of Notre Dame to develop a new tool that uses a synthetic nanometer-diameter pore (i.e. a nanopore) to reprogram the genetic memory of specialized cells, inducing them to become like embryonic stem cells. Stem cell research promises regenerative therapies for everything from blindness, to the repair of heart and nerve damage, and even aging. Until recently, clinical research has focused mainly on two kinds of cells: embryonic and non-embryonic or adult stem cells. Beyond the ethical dilemma posed by the use of embryonic stem cells, both cell types are scarce relative to the level required for efficacious treatment. On the other hand, induced pluriopotent stem cells (iPSCs) may now represent a viable alternative. iPSCs are derived from specialized somatic cells by forcing the expression of four key genes; they are similar to embryonic stem cells. Unfortunately, only about 0.01-0.1% of human somatic cells can be forced to become iPSCs. The reprogramming efficiency of somatic cells is crippled by the lack of control over the introduction and level of expression of the key genes. This research effort at Notre Dame will produce a tool that uses a nanopore to transfer nucleic acids into a single cell one molecule at a time, affording stringent control over the expression of the target genes. The synthetic nanopore uses a tightly confined electric field to create a pore in the cell membrane that facilitates the delivery of nucleic acids and even whole genes potentially with single molecule precision, while allowing for serial or repeated gene delivery into the same cell. As a crucible for testing the tool, nucleic acids encoding the key genes will be introduced into somatic cells to produce iPSCs.This research has broader implications beyond the development of a tool for gene delivery into cells. This project also presents an opportunity to understand and transform biology through control of the gene and its environment with single molecule precision. To take advantage of this opportunity, the research will be translated into educational curricula and disseminated into the biological, health and medical communities. There are several components to this outreach program: i. teaching one-another through a monthly seminar series and a web-site; ii. inspiring students through novel curricula promulgated through a new lab course, Introduction to Systems Biology, taught under the Engineering and Biological Sciences rubrics; and finally, iii enlightening the public and high school students, in particular, about the impact of this novel nanotechnology on biology and medicine through partnerships with a local New Tech High School.