Generation of pluripotent human stem cell lines (iPS) requires the expression of 3-5 transcription factors. However, pre-existing somatic mutations and mutations acquired from integrated viral or naked DNA vectors used to introduce these transcription factors pose a considerable risk for cancer and represent the major obstacle in translating stem cell therapy to the clinic. To overcome this obstacle, we proposed an alternative approach to generating patient-derived stem cell lines using cell-permeable, pluripotent-inducing transcription factors. 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. After mastering the delivery approach in tester human fibroblasts, we will focus our efforts on various easily accessible cell types from skin biopsies.
To use our protein delivery technology for the generation of iPS cells from skin cells, several milestones need to be achieved. First, proteins of five pluripotent transcription factors (Oct4, Nanog, Sox2, Klf4, Myc, and Lin28) were engineered to contain the cell-permeable delivery domain or Protein Transduction Domain (PTD) and produced in large quantities in the lab. Second, to test the activity of each cell-permeable pluripotent transcription factor, we have assessed their ability to enter the nucleus and to activate specific gene targets. We have found several problems, relating primarily to expression levels and retention of transcriptional activity. Our current work suggests that the placement or physical location of the cell-permeable delivery domain may interfere with optimal activity of the transcription factor. To optimize activity, additional fusion proteins have been generated and the intracellular levels and transcriptional activity will be determined. Another possibility for poor activity is the trapping of cell-permeable proteins in endosomal compartments, preventing efficient nuclear trafficking. We have previously utilized additional proteins to facilitate escape of cell-permeable proteins from these compartments and, thus, several peptides and drugs are being screened for their ability to improve targeting by recombinant cell-permeable proteins.
Another area of progress has been the isolation of cells from different compartments of the skin. Fibroblasts from the skin are extremely heterogeneous, thus fibroblasts from different layers of the skin, e.g. papillary vs. reticular dermis, are being isolated and assessed for differences in their efficiency in cell-permeable protein targeting. Current cultures are being assayed to confirm the origin of these cells and will be tested for activity against cell-permeable transcription factors described above. In addition, over the past year, several new adult-stem cell populations have been identified in the hair follicle. Efforts in the upcoming year will be to isolate these populations for similar tests of cell-permeable protein targeting and efficiency in becoming iPS cells.
Another important aspect of iPS generation is the ratio of each pluripotent transcription factor to the other. Using a surrogate approach, we have performed extensive matrices of varying each transcription factor relative to the other and have now identified the optimal ratio. This is a key step in optimize the generation of iPS cell induction. Overall, we have made significant, albeit slower than expected, progress in the design and purification of the PTD-transcription factor fusion proteins. We anticipate that as we perform more experiments with these PTD fusion proteins that work will rapidly accelerate during the second year.
Reporting Period:
Year 2
Public Summary of Scientific Progress (11/01/11)
Grant Number: RL-0644-01
PI Name: Dowdy, Steven F.
Project Title: Protein transduction of transcription factors: a non-genetic approach to generate new pluripotent cell lines from human skin.
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 have found 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.
So the problem was with stability and activity of the transcription factor fusion proteins and the time it would take to resolve these issues. 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.
To develop a universal mRNA delivery approach, we first 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. A recent paper using lipids (liposome transfection agents) has shown that the mRNA approach can work. However, liposomes are cytotoxic to cells and the authors noted that it was extremely difficult to do. We are currently testing our universal mRNA delivery approach to epigenetically generate iPS cells.
Reporting Period:
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.
Grant Application Details
Application Title:
Protein transduction of transcription factors: a non-genetic approach to generate new pluripotent cell lines from human skin.
Public Abstract:
More than 100,000 patients await for organ transplants nationwide this year. The ground-breaking discovery of new pluripotent human stem cell lines (iPS) derived from skin fibroblasts using a core of 3-5 transcription factors opens the door to patient-derived pluripotent stem cells and new approaches to organ and tissue replacement. Patient-derived stem cells could have an immediate impact on hundreds of other medical applications and discoveries. A major bottleneck in translating these breakthroughs into the clinic is that pre-existing mutations in patients and mutations acquired from viral and DNA vectors pose a potential risk for cancer. To overcome this obstacle, we propose an alternative approach to generating patient-derived stem cell lines using cell-permeable, pluripotent-inducing transcription factors. Introducing active proteins into cells avoids the long-term risk of genetic mutations. Over the past 10 years, our labs have pioneered this technology to introduce over 50 functional proteins in a wide spectrum of human and animal cell types. First, we will use this technology to determine the optimal number of cell-permeable proteins necessary to induce pluripotency in human cells. Second, to reduce the risk of pre-existing cancer mutations, we will identify which cells in the human body are the best source to generate mutation-free patient stem cells. These two advances will help us reach our goal of developing new medical therapies for an endless list of diseases using patient stem cells.
Statement of Benefit to California:
Almost 20,000 Californians await organ transplants. The possibility of translating pluripotent stem cell technology to patient-derived stem cells could drastically improve the outlook for organ and tissue replacement therapy in the future. Hundreds of potential medical therapies and inventions using induced-pluripotent stem cells are limited because current approaches rely on DNA technology and cause mutations. Over the past 10 years, our labs have pioneered alternative non-DNA approach, which could introduce pluripotent-inducing transcription factors into cells without causing mutations. We have used this approach to introduce more than 50 active proteins into a broad spectrum of human and animal cell types. In this proposal, we will use this alternative non-genetic approach to induce pluripotent stem cells from human fibroblasts and to identify which human cell types are the best candidates for generating mutation-free patient-derived stem cells. These breakthroughs could have an immediate impact on medical treatments for Californians who are suffering from end-organ damage or have endocrine, neurological, and cardiac disease. Furthermore, these technologies will have an immense economic benefit to California by removing a major obstacle for biotechnology and pharmaceutical companies in California and giving California a leading edge in developing new treatments.
