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

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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.