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Development of a human Oct-4 gene-targeted reporter cell line

Award Information

Agency:
Department of Health and Human Services
Branch:
N/A
Award ID:
Program Year/Program:
2010 / STTR
Agency Tracking Number:
R41RR028143
Solicitation Year:
2010
Solicitation Topic Code:
NCRR
Solicitation Number:
PA09-081
Small Business Information
NUPOTENTIAL, INC.
340 East Parker Street BATON ROUGE, LA -
View profile »
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No
 
Phase 1
Fiscal Year: 2010
Title: Development of a human Oct-4 gene-targeted reporter cell line
Agency: HHS
Contract: 1R41RR028143-01
Award Amount: $310,768.00
 

Abstract:

DESCRIPTION (provided by applicant): NuPotential, Inc. will use NIH STTR funds to generate and validate human Oct-4 gene-targeted reporter cell lines that will provide superior predictive indexing of gene transcription for the identification of efficient reprogramming-competent epi-drugs. To accomplish this, NuPotential will partner with Dr. Gregory Leno at the University of Iowa whose laboratory has previously used adeno-associated virus to target a reporter gene to a selected endogenous gene locus. Reporter cell lines will be validated by demonstrating a significant correlation between reporter activity and endogenous Oct-4 gene expression following treatment with commercially available epi-drugs with which NuPotential has shown up-regulation of Oct-4 geneexpression. The Oct-4 gene is a key target for reprogramming differentiated human cells. NuPotential's proprietary somatic cell reprogramming platform is based on in-vitro targeting of the epigenome with small molecule inhibitors of repressive regulatory components of chromatin. De-repression of select pluripotency-related target genes promotes transcription and restores genomic potential in differentiated cells. NuPotential's focused applications include the small molecule-driven production of autologous cells for replacement therapies and improved livestock cloning. The identification of novel small molecules that activate Oct-4, and other pluripotency-related genes in differentiated cells, could solve efficiency problems associated with existing reprogramming methods. However, current cell-based high-throughput drug screening technology utilizes randomly integrated (non-targeted) reporter genes that may lack genetic and/or epigenetic regulatory elements that govern endogenous gene expression. Expression of these reporter genes may also be governed by the unknown chromosomal context at the site of integration. Hence, the potential mis-regulation of reporter expression in non-targeted Oct-4 reporter cell lines may lead to the identification of small molecules with limited or no cell reprogramming potential. This Phase I proposal will generate an Oct-4 gene-targeted reporter cell line that accurately indexes endogenous Oct-4 gene expression in human diploid fibroblasts (HDF). The reporter cell line will bevalidated using small molecules that promote Oct-4 transcription by inhibiting epigenetic regulatory components that repress Oct-4 expression in HDF cells. This reporter cell line will provide a sensitive and more predictive analytical tool for identifying molecules that modulate Oct-4 transcription. Following targeted clone confirmation, reporter activity will be compared to endogenous Oct-4 gene expression before and after drug-induced activation of Oct-4 in reporter cells from human BJ fibroblasts. InPhase II studies, this validated reporter cell line will be used for high throughput screening of a newly developed epi-drug library to identify novel small molecules with reprogramming potential. The long-term objective of this research is the identification of novel small molecules that are capable of activating endogenous Oct-4 transcription in differentiated human somatic cells without forced introduction of exogenous genes and retroviral infection. The discovery of novel Oct-4 targeted reprogramming molecules is of paramount importance for establishing the therapeutic potential of reprogrammed isogenic human somatic cells. The knowledge gained through this study may advance potential cell-based therapies for human disease by providing an avenue for thedirect reprogramming of differentiated cells making possible the production of replacement cells targeted to a patient's diseased or damaged tissue. PUBLIC HEALTH RELEVANCE: A human Oct-4 gene-targeted reporter cell line will be generated from a BJdiploid fibroblast cell line and validated by demonstrating a significant correlation between reporter activity and endogenous Oct-4 expression before and after epi-drug-induced activation of Oct-4. The characterized reporter line will be used in Phase IIstudies to screen a novel small molecule library for activators of authentic Oct-4 transcription. The commercial goal is to use this targeted cell line to identify novel small molecules that can improve the efficiency of nuclear reprogramming. Efficientlyreprogrammed human cells have viable commercial applications in regenerative medicine.

Principal Investigator:

Gregory Leno
225-615-8954
gregory-leno@uiowa.edu

Business Contact:

Rachel Power
225-615-8954
rapnupotential@cox.net
Small Business Information at Submission:

NUPOTENTIAL, INC.
340 East Parker Street BATON ROUGE, LA -

EIN/Tax ID: 157120314
DUNS: N/A
Number of Employees: N/A
Woman-Owned: No
Minority-Owned: No
HUBZone-Owned: No
Research Institution Information:
University Of Iowa
UNIVERSITY OF IOWA
IOWA CITY, IA 52242-
Contact: