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.
Stem cells have enormous potentials to both regenerate themselves and differentiate into different mature cell types, such as lung cells or liver cells. The ability to manipulate 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. The focus of this research project is to develop a genetic tool for gene editing of human embryonic stem (hES) cells with high precision and efficiency. An engineered delivery system has been constructed and tested for their ability to deliver a genetic scissor and/or a donor DNA. We found that this system can accomplish targeted disrupt of desired genes in the genome with high efficiency (30%) and great accuracy. Our experiments also confirmed that such a system can efficiently mediate specific gene addition to a predetermined target site of hES cells. The modified hES cells maintained their self-renewal and pluripotent state of the stem cells. We have also evaluated an mRNA display technique for designing new genetic scissors specific for genome sites of stem cell interests. We showed that such a technique could be adapted to generate scaffold scissors capable of binding to target DNAs. Our experiments further demonstrated the feasibility of utilizing this powerful method to establish a library of ten trillion scissor proteins for selections to identify stem cell-specific binders.
Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We show that a baculoviral vector (BV) system carrying zinc finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and were pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells. We have also validated the mRNA display technology for designing new ZFNs for targeting transcriptional factors involved in controlling hES cell self-renewal and differentiation.
In our previous progress report, we detailed our efforts targeting the human beta-globin gene with zinc finger nucleuses (ZFNs). Our goal was to correct a mutation that results in sickle cell anemia, the most common inherited blood disorder in the United States, affecting ~80,000 Americans. Two recent published reports evaluating the specificity of ZFNs demonstrates the importance of designing highly specific ZFNs to avoid off-target effects. Thus, we have reevaluated our choice of target, our library design, and our mRNA display strategy for designing beta-globin-targeted ZFNs. In addition, we carefully evaluated our protocol for differentiating human embryonic stem cells (hESCs). By combining with growth factors, the undifferentiated hESCs can be directly differentiated into three germ layers through EB formation. The in vivo results also confirmed that hESCs gown in a scaffold could support the growth and differentiation of undifferentiated hESCs into different germ layers by providing physical environment for the interaction of hESCs with host tissues.