Generation of Human iPS Cells via Non-integrating Vectors

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DESCRIPTION (provided by applicant): Stem cells have great therapeutic potential for treatment of various diseases, and alternative methods for generation of pluripotent cells are needed. A particularly exciting method for the generation of patient-specific (or isogenic) pluripotent stem cells is somatic cell reprogramming. Three reprogramming approaches have been investigated: i) the somatic cell nuclear transfer (SCNT) approach; ii) cell fusion-based approach; iii) generation of induced pluripotent stem (iPS) cells by reprogramming of somatic cells via the defined transcription factors (Oct3/4 and Sox2 with Klf4 and c-Myc or Nanog and Lin28). The first two approaches are limited by extremely low efficiency and the requirement for fresh human oocytes or formation of somatic/embryonic stem (ES) hybrid cells, thereby limiting their further clinical applications. The last approach does not require embryos or oocytes, and provides a potential alternative to the current source of ES cells, thereby offering the possibility of generating patient- and disease-specific pluripotent stem cells. These cells may be valuable for not only personalized cell transplantation therapy without immune rejection but also for understanding disease mechanisms, drug screening, tissue engineering, and toxicology. iPS cells were generated mainly from mouse somatic cells, and these cells are similar to ES cells in terms of gene expression profile, DNA methylation, and perhaps most significantly self-renewal and differentiation potential. However, these iPS cells are significantly genetically modified with many mutations in the genome, and mice derived from iPS cells develop tumors, mainly through reactivation of c-Myc and inactivation of tumor suppressor genes resulting from viral insertional mutagenesis. Recently, mouse iPS cells were successfully generated from fibroblasts and liver cells via adenovirus-mediated gene delivery of the four transcription factors. However, the efficiency is extremely low, ranging from less than 0.0001% to 0.001%. 'Genetically clean' human iPS cells have not been generated, maybe because of inadequate reprogramming efficiency. We recently improved the generation of iPS cells by forced expression of four reprogramming factors from one single open reading frame (ORF) that was linked by self-cleaving 2A sequences. This improved system significantly increased reprogramming efficiency (up to 100 times) and enabled multiple reprogramming factors to be expressed in a single vector. Therefore, we intend to test whether forced expression of the reprogramming factors from a single ORF via non-integrating vectors enhances generation of 'genetically clean' human iPS cells from somatic cells. To address this question, we propose the following two Specific Aims: Aim 1: To generate human iPS cells from somatic cells by inducible expression of the reprogramming factors from a single ORF via a self-replication episomal vector; Aim 2: To generate human iPS cells from somatic cells by adenovirus-mediated gene transfer of the reprogramming factors in a single ORF. PUBLIC HEALTH RELEVANCE: This project will generate 'genetically clean' human ES-like pluripotent stem cells by somatic cell reprogramming approaches. Therefore, this is an alternative to the use of human embryonic stem cells for regenerative medicine applications.