Stem cells are powerful undifferentiated cells that are able to both regenerate themselves and differentiate into different mature cell types, such as lung cells or liver cells. The ability to edit stem cell genomes is useful for both understanding stem cells at a fundamental level as well as for practical and therapeutic purposes, such as regenerative medicine. However, there is a current lack of tools available for targeting these cells in a specific and efficient way. Our proposal is to utilize a genetic scissor, which has the ability to target specific sites in the genome, and an engineered delivery vehicle, to site-specifically alter genes of interest in stem cells. This would enable us to correct genetic mutations or deficiencies at known and targeted points in the stem cell genome, which is much more efficient and safer than current methods, which involve random insertion. The random placement of a gene into the stem cell genome could potentially disrupt the production of necessary cellular proteins, which could result in death of the stem cell, or even lead to the development of a cancerous stem cell. Thus, targeting DNA to a specific site has an important advantage over random gene insertion and should be developed and studied further. The success of this proposal has many implications. The methods developed can be used correct inherited genetic diseases such as severe combined immunity disorder (SCID) and sickle cell anemia (SCA) in a safe and efficient way. They can also be used in cell transplantation therapies for diseases such as diabetes, Parkinson’s disease, and cardiovascular diseases. Besides these practical applications, the techniques developed can also be used to qualitatively and quantitatively study the molecular mechanisms of stem cells.
We propose to develop a novel and general technology capable of rationally and precisely manipulating the genome of human stem cells (both human embryonic stem cells and induced pluripotent stem cells). This gene targeting tool will provide a unique opportunity to advance our understanding of the role that genetic factors play in controlling the pluripotency and lineage differentiation of human stem cells. This proposal will significantly improve our ability to qualitatively and quantitatively study the molecular regulators in stem cells. The establishment of such a tool in California will enable California to become a world leader in many aspects of stem cell research. In fact, we are determined, in the future, to establish a core facility in California, to help disseminate the technology derived from this study to California stem cell researchers for solving their own challenging problems. This study provides a new approach to safely and efficiently correct the inherited genetic mutations in stem cells that cause many devastating diseases such as severe combined immunity disorder (SCID) and sickle cell anemia (SCA); significant numbers of Californians are afflicted by these types of diseases. This study would also allow us to identify robust conditions to direct the differentiation of pluripotent stem cells to tissue-specific cells, which will benefit many people of California who need cell transplantation therapies for the treatment of diabetes, Parkinson’s disease, cardiovascular disease, etc. This new technology can also be employed to genetically modify stem cells for modeling various human diseases, by which new therapies and drugs may be discovered. These new therapies and drugs will not only benefit many individuals in California who bear the corresponding diseases, but they will also inspire and fuel California’s biotech industry and benefit general Californians economically.
The goal of the proposed study is to develop a method for genetic modification of human embryonic stem cells (hESC) with high precision and efficiency. The approach involves development of a baculoviral vector (BV) system, which can accommodate large DNA inserts, together with specifically engineered zinc-finger nucleases (ZFN) that can stimulate site-specific DNA integrations. In a first aim, conditions will be optimized for using BV-mediated DNA delivery to target the CCR5 locus in hESC. In a second aim, a ZFN capable of targeting the Oct-4 locus will be designed, using a novel display technique. In aim 3, the applicants will use the developed tools to create an Oct4 reporter hESC line.
The proposed research could have an important impact on stem cell research. The ability to genetically manipulate hESC by delivering large genomic fragments to specific genetic loci would be a significant advancement that could facilitate marking genes or making precise changes in stem cell genomes. This will aid the study of molecular regulation of stem cell differentiation and facilitate the development of hESC-based disease models. The approach could also enable the generation of genetically corrected patient-specific induced pluripotent (iPS) cells from patients with genetic disorders.
Reviewers found the rationale for the proposed study to be solid and compelling. Research plans were appropriately supported by previous work in the investigators’ labs. Reviewers judged the aims as straightforward, proposed experiments well described, and alternative strategies outlined clearly. Very convincing preliminary data supported feasibility of the approach. Although the project was viewed as ambitious and somewhat risky, reviewers felt that the potential benefits as well as the solid investigative plan portend good prospects for success. One minor criticism related to the choice of Oct4 as a reporter locus, as such a reporter hESC line already exists. Furthermore, an attempt should have been made to explore the strength of the BV delivery system, its capacity to deliver large DNA fragments.
The applicant is a productive scientist with expertise in viral vector technology and polymer chemistry, and the co-investigator brings key expertise with the novel display technology to the project. The assembled research team appears substantial and well qualified to carry out the proposed investigation.
Overall, this is a well-designed proposal for creating a valuable new tool for stem cell research. Reviewers were enthusiastic about the approach and its likelihood for success.