Production of cell lines with improved reprogramming potential for SCNT

DESCRIPTION (provided by applicant): NuPotential will use NIH STTR funds to develop and validate a novel epigenetic-based in vitro system for reprogramming bovine donor cells to improve the efficiency of somatic cell nuclear transfer (SCNT). To accomplish this, NuPotential will alter chromatin epigenetic marks to induce key pluripotency genes and proteins to increase potential of donor cells prior to SCNT. The commercial goal is to improve the efficiency of nuclear reprogramming for livestock cloning. NuPotential's proprietary somatic cell reprogramming platform is based on targeting the epigenome by inhibiting repressive regulatory components, in vitro, to induce pluripotency genes and restore differentiation potential. To achieve the goals of these STTR studies, NuPotential will restore potential of bovine donor cells prior to SCNT by inhibiting DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) to enable transcription of pluripotency genes. In Phase I, NuPotential will employ shRNA technology to knock down gene expression of DNMTs and HDACs in bovine donor cells. Secondly, we will assess donor cell reprogramming by pluripotent gene and protein expression and chromatin modifications following shRNA treatment. Thirdly, NuPotential's reprogramming system will be validated by assessment of developmental potential following SCNT. In Phase II, NuPotential will use small molecules that inhibit repressive epigenetic regulatory targets validated in Phase I to induce pluripotency gene expression and restore differentiation and developmental potential of bovine donor cells for SCNT. In support of this proposal, NuPotential initially demonstrated DNA demethylation and improved developmental potential following SCNT in bovine donor cells treated with DNMT1-specific siRNA. Subsequently, we demonstrated up regulation of pluripotency genes and restored differentiation potential in bovine donor cells by altering cellular methylation capacity, in vitro, with all-trans retinoic acid. More recently, NuPotential demonstrated induction of phenotypes remarkably similar to published embryonic stem cells, including induction of key pluripotency genes/proteins and formation of embryoid body-like colonies, in somatic cells following treatment with shRNA or small molecules that inhibit DNMTS or HDACs. These results clearly indicate that repressive epigenetic regulatory components can be inhibited to restore potential of somatic cells. Major markets for NuPotential's somatic cell reprogramming technology to improve livestock cloning methods include high genetic merit livestock, animal bioreactors for large scale production of therapeutic molecules, and development of animal models of disease. NuPotential will seek appropriate partners to exploit its technology in each of these markets. PUBLIC HEALTH RELEVANCE: NuPotential will develop and validate a novel epigenetic-based system to increase potential of donor cells for somatic cell nuclear transfer. shRNA technology will be used to inhibit repressive epigenetic regulatory components in bovine donor cells to induce transcription of pluripotency genes critical for restoring potential. The commercial goal is to improve the efficiency of nuclear reprogramming for livestock cloning.