Year 3

The generation of induced-pluripotent stem (iPS) cells opens the door to promising new medical cell therapy applications and discoveries. However, practically speaking, the generation of pluripotent human iPS cells requires the simultaneous and coordinated expression of a combination of three to five or more reprogramming factors that are often transcription factors. One of the major obstacles in translating the iPS generation technology from basic science discoveries into safe cell based therapies for patients is the risk of acquiring mutations from viral and DNA vectors that are used to generate pluripotent cells. Thus, the risk of genomic mutations from integrated viral or naked DNA vectors used to introduce these transcription factors poses a considerable risk for cancer and represents the major obstacle in translating stem cell therapy into the clinics. To overcome this obstacle, we initially proposed an alternative approach to generating patient-derived stem cell lines using cell-permeable, pluripotent-inducing transcription factor proteins. Importantly, introducing active proteins into cells avoids the long-term risk of genetic mutations caused by DNA based expression vectors of these transcription factors. Our labs pioneered the cell-permeable delivery of proteins, including transcription factors into cells and the overall goal here is to use this epigenetic (no DNA vector) approach to efficiently generate iPS cells from various easily accessible cell types from skin biopsies.

Our first approach was to use our protein delivery technology to generate, produce and purify the fusion proteins of five pluripotent reprogramming factors, namely Oct4, Nanog, Sox2, Klf4, Myc, and Lin28. We engineered these proteins to contain a cell-permeable delivery domain or Protein Transduction Domain (PTD) and produced in large quantities in the lab. However, when we began to extensively test the ability of these proteins to enter the nucleus and to activate specific target genes to induce expression we identified several problems. First, these proteins were not soluble in aqueous solutions and tended to aggregate. This was overcome by the formulations that we used, including addition of salt. Second, with the exception of the PTD-Sox2 transcription factor fusion protein, we were unable to show that the other transcription factor fusion proteins retained their ability to transactive or induce target genes. This was a significant roadblock and while we felt that we could ultimately succeed in making biologically active fusion proteins, similar to the 40+ we have made over the last 10 years, we recognized that this approach was going to take significantly longer to achieve our goal of epigenetic (no DNA) generation of iPS cells.

To circumvent these problems, we decided to take an alternative approach and develop from scratch an efficient and non-cytotoxic, universal mRNA delivery approach. Importantly, mRNAs are not DNA and do not integrate into chromosomes and are therefore still epigenetic. In addition, mRNAs are relatively long lived inside of cells and are repeatedly translated into proteins, thereby amplifying the amount of pluripotent reprogramming factors that would be expressed inside the target cell. Lastly, regardless of what protein the mRNA codes for, mRNAs have similar biophysical properties, making development of a universal mRNA delivery approach infinitely easier. Thus, mRNA delivery is an epigenetic approach to generating iPS cells and has multiple advantages over transcription factor fusion proteins.

First we developed a universal mRNA delivery approach that condensed and compacted the mRNA with one peptide, called a HK peptide, making a small nanoparticle, and then coated that with a second PTD delivery peptide. After extensive development, the result is that we have developed a universal mRNA delivery approach that achieves >90% mRNA delivery into a variety of primary cells in a non-cytotoxic manner and can be repeatedly administered. Second, we developed a long mRNA Replicon that is a single mRNA species that internally encodes four independent pluripotent reprogramming factors. Introduction this RNA three times into primary human cells results in a robust and highly reproducible generation of human iPS cells. We have used two different collection of reprogramming factors to show how efficiently the system works.

Overall, we achieved the goals of this grant to develop an epigenetic approach to generate iPS cells. We envision that the RNA Replicon approach will gather widespread utilization in stem cell research and potentially in generating stem cell therapies down the road.