The use of stem cells or stem cell-derived cells to treat disease is one important goal of stem cell research. A second, important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. Existing means of generating genetically modified stem cells are not ideal, as they do not preserve the normal gene regulation, are inefficient, and do not permit removal of foreign genes.
We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. We are adapting this approach for use with human embryonic stem cells, so that these cells can be better understood and harnessed for modeling, or even treating, human diseases. We will use this approach to create a human stem cell model of Charcot-Marie-Tooth (CMT) disease, an inherited neuropathy. How gene dysfunction leads to nerve defects in CMT is not clear, and there is no cure or specific therapy for this neurological disease. Thus, we will use our genetic tools to investigate how gene function is disrupted to cause CMT. By developing these tools and using them to gain understanding of CMT, we will illustrate how this system can be used to gain insight into other important diseases.
Although human stem cells hold the potential to generate new understanding about human biology and new approaches to important diseases, the inability to efficiently and specifically modify stem cells currently limits the pace of research. Also, there is presently no safe means of changing genes compatible with the use of the stem cells in therapies. We are developing new genetic tools to allow for the tractable manipulation of human stem cells. By accelerating diverse other stem cell research projects, these tools will enhance the scientific and economic development of California.
We will use these tools to create cellular models of Charcot-Marie-Tooth (CMT), a neurological disease with no cure that affects about 15,000 Californians. This model will facilitate understanding of the etiology of CMT, and may lead to insights that can be used to develop specific therapies.
Beyond gaining insight into CMT, the ability to engineer specific genetic changes in human stem cells will be useful for many applications, including the creation of replacement cells for personalized therapies, reporter lines for stem cell-based drug screens, and models of other diseases. Thus, our research will assist the endeavors of the stem cell community in both the public and private arenas, contributing to economic growth and new product development. This project will also train students and postdoctoral scholars in human stem cell biology, who will contribute to the economic capacity of California.
Historically, genetic modification of human embryonic stem cells (hESC) has proven challenging. This proposal aims to further develop a different technique than that typically used in the field to alter DNA. Using this technology, the applicant has derived an hESC line for modeling a specific inherited neurological disorder, Charcot-Marie-Tooth disease (CMT), and proposes to use it for the investigation of the molecular mechanisms of disease. The applicant also proposes to develop a resource to enable other investigators to take advantage of this technology. This resource will consist of a large collection of hESC lines, each altered at a specific gene locus, yielding a set of genetic tools that can be used to modify those gene loci. These tools will enable investigators to perform detailed studies of gene function and will be made available to the scientific community for use in studies such as disease modeling and further understanding of hESC biology.
Significance and Innovation
- The applicant proposes to develop a very novel resource that would be of great value to the scientific community. The proposed set of genetic tools contains a very comprehensive array of useful reagents.
- The proposed resource is based on technology developed for mouse ESC, where it has proven extremely useful. The technology developed here for hESC is state-of-the-art and takes into account improvements informed by years of experience with the corresponding mouse ESC work.
- Although another gene modification technology for hESC, termed TALEN, is rapidly improving and gaining use, the proposed resource offers unique features that will provide additional value to the research community.
- Although the proposed size of the genetically altered hESC collection is substantial, it will only provide partial coverage of the genome.
Feasibility and Experimental Design
- The PI has successfully developed the proposed technology for the mouse system. The preliminary results in the hESC system are strong, and most importantly show that the applicant’s laboratory can handle hESCs.
- The study design is strong, and the feasibility of generating the proposed resource in three years is convincing. However, it is unclear whether it will be possible to validate pluripotency and normal karyotype in all the proposed cell lines within the period of the award.
- The hESC line with a modified allele of the gene mutated in CMT has been generated already and is an excellent candidate for modeling that disease. An alternative approach to creating a disease causing allele is well conceived.
- Plans for hESC differentiation into Schwann cells in order to model CMT are very simplistic and rely on an approach that has been poorly validated. This work may prove to be much more challenging than anticipated.
Principal Investigator (PI) and Research Team
- The PI’s productivity is outstanding and includes a strong publication and grant record.
- The PI has considerable expertise with the technology proposed in this application as well as a highly significant track record of murine disease modeling.
- The team is excellent and has all the expertise necessary to accomplish the proposed aims. However, this project would benefit from a collaboration with an expert in directed differentiation of pluripotent stem cells to Schwann cells and neural fates.
Responsiveness to the RFA
- The application is reasonably responsive to the RFA. It includes quite extensive tool building activities, which is not a focus of this RFA, but those tools are justified and should impact specifically hESC research. Furthermore a disease-relevant component was included, though that component is not well developed in the proposal.