Proj 1: The discovery of reprogramming somatic cells to induced pluripotent stem cells has opened new dimensions for the study and treatment of human diseases. Since the first description of reprogramming mouse IPS cells by introducing 4 transcription factor genes with retroviral vectors, mouse and human cells of many cell types have been successfully reprogrammed. Although retroviral vectors are still the most proficient vehicles for reprogramming, their property of random integration may cause damage by disrupting vital host gene functions. Many other vehicles for introducing transcription factors have been reported, including plasmids, EB based plasmid vectors, adenoviral vectors, transposons, lentiviral vectors and proteins. Some of these methods are inefficient for reprogramming human somatic cells. Lentiviral vectors also integrate randomly into the genome although they could be removed with cre-lox.
The aim of this project is to investigate novel IPS techniques that can be applied in the future to the treatment of sickle cell disease and B-thalassemia, the 2 most common genetic diseases. We will use 2 strategies. The first is to introduce the 4 transcription factor genes 0CT4, S0X2, FLT4 and cMYC linked by 2A peptides using PhiC31 integrase fpr site-specific integration. The second strategy, directed by the co-investigator Long-Cheng Li, will use his innovation of short activator double stranded RNA to stimulate the expression ofthe endogenous transcription factor genes. He has shown that these saRNAs can stimulate expression of endogenous genes of various kinds. The strategies we propose have the advantage of not disturbing the functions of the host genes. Our project is directed to eventual application to sickle cell disease and thalassemia. We will reprogram skin biopsy cells from patents as well as amniotic fluid and CVS cells which will be useful for early cell therapy after prenatal diagnosis.

Public Health Relevance

This project aims to develop approaches to produce patient-specific IPS (induced pluripotent stem) cells, from patients with sickle cell disease and beta-thalassemia, with the aim of eventual application to their future treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK088760-04
Application #
8710193
Study Section
Special Emphasis Panel (ZDK1-GRB-6)
Project Start
Project End
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
4
Fiscal Year
2014
Total Cost
$381,220
Indirect Cost
$134,768
Name
University of California San Francisco
Department
Type
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Suzuki, Shingo; Sargent, R Geoffrey; Illek, Beate et al. (2016) TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs. Mol Ther Nucleic Acids 5:e273
Beyer, Ashley I; Muench, Marcus O (2016) Comparison of human hematopoietic reconstitution in different strains of immunodeficient mice. Stem Cells Dev :
Ye, Lin; Wang, Jiaming; Tan, Yuting et al. (2016) Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia. Proc Natl Acad Sci U S A 113:10661-5
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Muench, Marcus O; Beyer, Ashley I; Fomin, Marina E et al. (2014) The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny. PLoS One 9:e97312
Xie, Fei; Ye, Lin; Chang, Judy C et al. (2014) Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res 24:1526-33
Ye, Lin; Wang, Jiaming; Beyer, Ashley I et al. (2014) Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A 111:9591-6
Fomin, M E; Togarrati, P P; Muench, M O (2014) Progress and challenges in the development of a cell-based therapy for hemophilia A. J Thromb Haemost 12:1954-65

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