Improving Epigenetic-based Cell Reprogramming with Proteasome Inhibition

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DESCRIPTION (provided by applicant): Direct reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been demonstrated by viral transduction of exogenous transcription factors. These initial demonstrations have provided valuable insight into molecular mechanisms of somatic cell reprogramming and raised the possibility that alternative strategies could be developed on an industrial scale to produce pluripotent stem cells without using embryos or genetic manipulations. Clinical applications of these iPS approaches are impeded, however, by very low reprogramming efficiencies and safety concerns associated with viral transduction. NuPotential has developed several methods to reprogram somatic cells to a pluripotent state by * modifying chromatin structure to induce expression of silenced pluripotency genes. These methods include: 1) * altering the single carbon metabolism pathway and limiting methyl donors in culture medium to modulate * methylation capacity; 2) knocking down gene expression of repressive epigenetic regulatory proteins (ie., DNA * methyltransferases and histone deacetylases) using interfering RNA technology; 3) modulating epigenetic * regulatory components with small molecules; and 4) Combining over-expression of a single pluripotency gene C with shRNA knockdown of one or more epi-genes (NuPotenial induced pluripotent stem cells, NuPiPStm). o There are several embodiments to these approaches and NuPotential has filed patents on all of them; In n October, 2009, NuPotential was issued its first foundation patent on which these novel reprogramming f methods are based (Patent #7,601,699). i As NuPotential developed these methods, a critical pathway that appears to be a key (if not the key) d rate limiting step to improving the efficiency of reprogramming was identified. As we systematically knocked e down various epigenetic targets using shRNA technology, we identified a redundancy that appears to function n as compensation. This redundancy was also observed when somatic cells were treated with small molecule t Epi-drugs. Although NuPotential has thus-far demonstrated statistically significant up-regulation of i pluripotency gene expression with over 20 commercially available Epi-drugs, fold increases are not optimal for a efficient reprogramming, likely due to this compensatory pathway. These data demonstrate that to efficiently l produce large, fully-reprogrammed pluripotent stem cell (RePSCTM) populations without the use of exogenous * genes/proteins or viral vectors, compounds need to be identified and/or developed that specifically and * efficiently target components of this compensatory pathway. Subsequent investigation into chemical strategies * in our laboratory recently revealed a synergistic inhibition of redundant epigenetic regulatory components and * significant up-regulation of the key pluripotency gene Oct4 with combined Epi-drug and proteasome inhibitor * treatment. These data support the hypothesis that a novel strategy combining small molecule Epi-drugs with * proteasome inhibitors may increase the efficiency of somatic cell reprogramming by more effectively targeting * redundant epi-proteins that repress pluripotency genes and proteins critical for reprogramming, which is the * basis of this Phase I proposal. The goals of this Phase I proposal are to 1) screen proteasome inhibitors in combination with commercially available Epi-drugs in human Oct4 promoter driven-GFP reporter cell lines; and 2) provide proof of principle by demonstrating improved nuclear reprogramming in NuPotential's proprietary human iPS (NuPiPSTM) cells in comparison to unmodified human somatic cells by targeted chromatin modifications, key pluripotency gene and protein expression, colony formation, and in vitro and in vivo capacity for re- differentiation into new cell types. To accomplish this, NuPotential proposes to partner its expertise in epigenetic-based somatic cell reprogramming with that of Dr. Elizabeth Floyd, a Pennington Biomedical Research Center researcher specializing in proteasome-dependent protein turnover and post-translational modifications. Optimal combinatorial strategies validated in Phase I studies will be used in Phase II to develop novel and proprietary derivative compounds for reprogramming to produce highly efficient, commercially- relevant RePSCTM without the use of viral vector transduced iPS cells. PUBLIC HEALTH RELEVANCE: NuPotential will use STTR funds to develop a novel purely chemical approach to somatic cell reprogramming by combining Epi-drugs and proteasome inhibitors to significantly inhibit repressive epigenetic regulatory components and up-regulate pluripotency markers. Phase I studies will provide critical data that will enable development of a library of novel and proprietary derivative compounds that inhibit all possible combinations of redundant epigenetic regulatory proteins in order to significantly improve the efficiency of reprogramming. The commercial goal is to produce highly efficient clinically- and commercially-relevant reprogrammed pluripotent stem cells (RePSCTM) for autologous cell replacement therapies without the use of exogenous genes, viral vectors, nuclear transfer, oocytes, or embryos.