Funding opportunities

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

Funding Type: 
Tools and Technologies II
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 993 586
Funding Recommendations: 
Grant approved: 
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.
Review Summary: 
The goal of this proposal is to establish a robust technology pipeline for homologous recombination (HR) in human embryonic stem cells (hESC) utilizing a novel molecular approach, to disseminate use of this method to the scientific community and to generate a panel of cell lines carrying gene-specific modifications. Three specific aims are described to achieve this goal. Specific aim 1 is to generate a panel of targeting vectors, designed to introduce a variety of sequences encoding fluorescent and other marker proteins or protein tags into various loci to facilitate analyses of cell fate and protein function. Additional vectors for modifications that create specific mutations in disease-associated genes to develop models of human disease are planned. Specific aims 2 and 3 are focused on the generation and characterization of hESC lines carrying some of the targeted gene modifications. These genetically modified hESC lines will be employed for studies in the applicant’s laboratory as well as those of several collaborators, and all resulting hESC lines will be shared with the research community. HR allows the precise, targeted modification of individual genes, and reviewers agreed that a major roadblock in the study of hESCs is the lack of a robust technology for efficient HR. Thus, the development of such a technology, although not particularly novel, would represent a significant advance in the field. The PI hopes to overcome current technical difficulties by using a relatively novel strategy, and reviewers were enthusiastic about the chosen approach for HR. Another major strength of this proposal lies in the goal of producing a panel of hESC lines with targeted reporter knock-ins that will be very valuable for exploring the development of mature and specialized cell types from undifferentiated hESC and for studying disease phenotypes. Many investigators will be able to use these cells, and the list of collaborators requesting modifications of various genes illustrates the strong interest within the stem cell community and the large potential impact of the proposed study. The proposed studies are generally well designed, and the underlying rationale was judged to be logical and scientifically sound. Preliminary data indicated success with targeting of two genes in hESC using the proposed approach, although some of that data will require further verification. Overall, reviewers were fairly confident that this approach would overcome most of the issues that other targeting strategies have encountered. Reviewers cautioned that many of the proposed knock-in constructs may still be difficult to generate, especially those in which the targeted genes are not expressed in hESC. Also, the details of vector design were vague, and the targeting strategy for each gene could not be evaluated directly. Reviewers appreciated that the principal investigator (PI) recognized that the funding mechanism of this RFA cannot support the modification of all the proposed genes, and they agreed with the applicant’s plan in aim 2 to limit the generation of hESC lines to a subset of those targeted by the panel of vectors produced in Aim 1. The characterization of the derived hESC lines in Aim 3 was considered straightforward and adequate as proposed, but the cellular microarray analysis was judged to be underdeveloped. This criticism was mitigated, however, by reviewers’ opinion that the point of the proposed work is to develop reagents and not to perform a detailed analysis of gene-modified hESC lines. The PI is well trained and possesses appropriate expertise for the proposed hESC work. Numerous outstanding stem cell scientists will contribute valuable expertise to the project, and these collaborations were well documented by strong letters of support. Reviewers were particularly appreciative of the involvement of a relevant vector core facility. Overall commitment to this project and the requested budget were considered appropriate. Overall, reviewers agreed that this proposal addresses an important bottleneck in hESC research. They expressed strong enthusiasm about the relatively novel approach for accomplishing HR in hESC and felt confident that this research team has a good chance to be successful. If so, this work will provide an important technology and a valuable collection of genetically modified hESC lines to the broader stem cell community.

© 2013 California Institute for Regenerative Medicine