Homologous recombination in human pluripotent stem cells using adeno-associated virus.

Homologous recombination in human pluripotent stem cells using adeno-associated virus.

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02064
Award Value: 
$1,659,043
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
Since their discovery in 1998, human embryonic stem cells (hESCs) have been considered to hold great potential for the treatment of many currently incurable diseases. Possibly the most exciting application of hESC in the clinic is in the arena of regenerative medicine where hESC-derived cell populations are used to replace diseased, damaged or dead tissues. A major safety concern in developing hESC-based cell replacement therapies has been the potential risk of tumor growth, which is due to residual primitive, or undifferentiated, hESCs within the cell graft. Eliminating these undifferentiated and tumor promoting cells has proven to be difficult. In this grant application, we propose to develop a technology to identify and enrich the cells of interest while eliminating undesired and contaminating cell populations. Using an elegant method to introduce genes into hESCs, we will engineer cell lines that will express a marker only when a particular cell type has been produced. Such “marker lines” will be used to develop and optimize protocols to efficiently derive specific mature and specialized cell types suitable for transplantation. Cell purification methods will be applied to enrich the cells of interest and eliminate primitive tumor-promoting cells. Therefore, the proposed research will yield critical tools to overcome safety concerns of tumor growth associated with hESC-based cell replacement therapies. In addition to this crucial contribution to regenerative medicine, this technology is of immense value to basic biologist who wish to dissect developmental processes from undifferentiated to mature and specialized cell types. Such studies lie at the heart of developmental biology and will shed light on the intricate processes that guide a single cell, the fertilized egg, to divide, grow and acquire the thousands of cell types and characteristics of a complex multi-cellular organism. Another application of this technology is to append tags onto genes of interest to facilitate the studies of gene function during cellular growth and differentiation. Scientists studying particular genes and their roles in cellular and developmental biology and biochemistry will benefit tremendously from our engineered hESC lines carrying tagged genes. Finally, this technology will allow us to engineer specific mutations into genes associated with human diseases. HESCs carrying specific gene alterations can then be used to model human diseases in a petri dish, to screen efficacy and safety of drugs, and to devise methods to correct the defects. Together, the proposed technology will yield valuable tools to the stem cell field to overcome multiple roadblocks in basic, translational or clinical stem cell research.
Statement of Benefit to California: 
The rise in life expectancy to over 80 years will likely be associated with a corresponding increase in the number of people suffering from age-related diseases, such as cancer, heart disease and neurodegenerative disorders. Current medical treatments can alleviate symptoms and control, but not cure, such diseases. Human embryonic stem cells (hESC) provide a unique opportunity to develop novel cell replacement therapies for the treatment of many such diseases. Development of cell-based therapies will also overcome the inadequacy of conventional drug-based treatments. A major scientific challenge in the development of hESC-based therapies is the directed differentiation of hESC into functionally mature and pure cell types suitable for transplantation. The technology we propose to develop and disseminate to the greater scientific community utilizes an elegant gene replacement approach to create so-called “marker lines,” which are critical to the derivation and purification of mature cell populations. Such marker lines will be instrumental at gaining insight into the mechanisms that drive directed differentiation. Additionally, this technology will be applied to specifically modify individual genes, thereby enabling analysis of gene and protein functions. The broad stem cell research community will benefit tremendously from the development and streamlining of this technology and from the various engineered hESC marker lines, which will serve as critical building blocks to study and understand human development and disease. Translational and clinical stem cell research will likewise benefit from the tools developed under the proposed research as novel methods for cell isolation and purification will be identified. This research will not only benefit the health of Californians, but also the California economy by creating new reagents, protocols and technologies that will be adopted by existing companies as well as seed and complement novel business ideas. The outcome of this project will contribute to the development of a biotechnology platform that can provide great benefits to the advancement of California biotechnology. The patents, royalties and licensing fees that result from the advances in the proposed research will provide California tax revenues. Thus, the current proposed research provides not only the essential foundation for the scientific advances in regenerative medicine to improve health and quality of life, but also potential technology advancement and financial profit for the people in California.
Progress Report: 

Year 1

Pluripotent stem cell research promises to revolutionize biomedical research by providing a human cell-based model system to study disease progression, test drug efficacy and safety and generate mature cells suitable for cell transplantation. However, progress in this direction is impeded by several deficiencies. First, differentiation of human pluripotent stem cells (hPSCs, both embryonic and induced pluripotent stem cells) is often inefficient and incomplete, often generating only limited numbers of cells with the desired properties surrounded by an excess of undesirable and even tumor-forming cell populations. Second, culture conditions to promote differentiation into specific cell types are often undefined and contain animal products. Third, while specific mature cell populations have been obtained in relatively small numbers sufficient for basic science research, culture conditions for scale-up are limited. The goal of the proposed research is to develop a technology that enables stem cell research and provides solutions to these short-comings. Specifically, we are generating hPSC lines carrying genetic elements that will allow us to readily observe when a cell type has achieved a particular state of differentiation. In addition, this technology will facilitate the development of cell isolation and purification methods. This is especially important so that undesirable cell types—such as tumor forming cells—can be efficiently separated from the cells of interest. To generate the various so-called “marker cell lines” we are using a viral gene transduction method that relies on a genetically modified adeno-associated virus (AAV). This virus can efficiently infect human cells while having no known adverse effects. During the first year of research on this project we have developed a large collection of viral DNA constructs that will be used to target genes encoding fluorescent proteins to specific locations of the genome. The successful completion of this research will provide the scientific community with a large panel of these marker cell lines that can be employed to develop, optimize and define differentiation protocols. In addition, this research will provide a streamlined technology for site-specific alteration and correction of the genome.

Year 2

Pluripotent stem cell research promises to revolutionize biomedical research by providing a human cell-based model system to study disease progression, test drug efficacy and safety and generate mature cells suitable for cell transplantation. However, progress in this direction is impeded by several deficiencies. First, differentiation of human pluripotent stem cells (hPSCs, both embryonic and induced pluripotent stem cells) is often inefficient and incomplete, generating only limited numbers of cells with the desired properties surrounded by an excess of undesirable and even tumor-forming cell populations. Second, culture conditions to promote differentiation into specific cell types are often undefined and contain animal products. Third, while specific mature cell populations have been obtained in relatively small numbers sufficient for basic science research, culture conditions for scale-up are limited. The goal of the proposed research is to develop a technology that enables stem cell research and provides solutions to these shortcomings. Specifically, we are generating hPSC lines carrying genetic elements that will allow us to readily observe when a cell type has achieved a particular state of differentiation. This technology will facilitate the development of cell isolation and purification methods. This is especially important so that undesirable cell types—such as tumor forming cells—can be efficiently separated from the cells of interest. To generate “marker cell lines”—cell lines carrying reporter genes under control of tissue-specific promoters—we are using a viral gene transduction method that relies on a genetically modified adeno-associated virus (AAV). This virus can efficiently infect human cells while having no known adverse effects. We have developed a large collection of viral DNA constructs that we are using to target genes encoding fluorescent proteins to specific locations of the genome. The successful completion of this research will provide the scientific community with a large panel of these marker cell lines that can be employed to develop, optimize and define differentiation protocols. In addition, this research will provide a streamlined technology for site-specific alteration and correction of the genome.

Year 3

Pluripotent stem cell research promises to revolutionize biomedical research by providing a human cell-based model system to study disease progression, test drug efficacy and safety and generate mature cells suitable for cell transplantation. However, progress in this direction is impeded by several deficiencies. First, differentiation of human pluripotent stem cells (hPSCs, both embryonic and induced pluripotent stem cells) is often inefficient and incomplete, generating only limited numbers of cells with the desired properties surrounded by an excess of undesirable and even tumor-forming cell populations. Second, culture conditions to promote differentiation into specific cell types are often undefined and contain animal products. Third, while specific mature cell populations have been obtained in relatively small numbers sufficient for basic science research, culture conditions for scale-up are limited. The goal of the proposed research is to develop a technology that enables stem cell research and provides solutions to these shortcomings. Specifically, we are generating hPSC lines carrying genetic elements that will allow us to readily observe when a cell type has achieved a particular state of differentiation. This technology will facilitate the development of cell isolation and purification methods. This is especially important so that undesirable cell types—such as tumor forming cells—can be efficiently separated from the cells of interest. To generate “marker cell lines”—cell lines carrying reporter genes under control of tissue-specific promoters—we are using a viral gene transduction method that relies on a genetically modified adeno-associated virus (AAV). This virus can efficiently infect human cells while having no known adverse effects. We have developed a large collection of viral DNA constructs that we are using to target genes encoding fluorescent proteins to specific locations of the genome. More recently, we have integrated another method, called CRISPR/Cas9, with the AAV-based method to increase the efficiency of generating marker cell lines. The successful completion of this research will provide the scientific community with a large panel of these marker cell lines that can be employed to develop, optimize and define differentiation protocols. In addition, this research will provide a streamlined technology for site-specific alteration and correction of the genome.

© 2013 California Institute for Regenerative Medicine