We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which promoter-targeting double-stranded small RNA also known as small activating RNA (saRNA) can induce gene expression. Our approach to iPS cell derivation is through simultaneous activation of stem cell factors including OCT4, NANOG, SOX2 and KLF4 using RNAa. Currently we are focusing on Aim 1 to screen saRNAs that can activate individual stem cell factors as specified in our proposal. To facilitate and expedite such screening process, besides the screening method we originally proposed, that is through expression analysis of endogenous genes, we have adapted three new strategies. They include the use of a lentiviral vector based reporter system, use of a mouse retroviral iPS reprogramming platform and use of established cell lines. We successfully established reporter systems for all four human stem cell factors including OOT4, NANOG, SOX2 and MYC. The reporter systems allow for high throughput screen of saRNAs in primary fibroblast cells. Using a reporter vector for MYC promoter, we identified several activating saRNAs for MYC gene in human fibroblast BJ cells. We are currently using the same system to screen saRNAs for other genes. We are also using the mouse iPS reprogramming platform to screen saRNAs that help reprogramming without first knowing their ability in gene activation and have successfully identified four saRNAs for mouse Myc gene. Replacing Myc retrovirus by the saRNAs significantly increased the number of iPS clones compared to the number of clones from cells infected by the remaining 3 factors (Oct4, Sox2 and Klf4) alone. This result provided proof of principle that saRNAs can be directly screened on an iPS reprogramming platform. We will next move this screening method to human cells and to achieve iPS reprogramming by replacing virus for other stem cell factors particularly OCT4. Since established cancer cell lines are easy to transfect with small RNA, we also used human cancer cell lines to screen saRNAs for KLF4 gene and were able to identify one potent saRNA. RNAa-mediated KLF4 activation caused the modulation of its regulated genes including several cell cycle genes. These results were fully recapitulated by retroviral vector based overexpression of KLF4, thus validating RNAa as a method of restoring endogenous gene function. Ongoing research is identifying saRNAs for additional stem cell factors and maximizing gene activation by optimizing saRNA nucleofection and by combinatorial treatment with epigenetic modifying agents.