Statement of Benefit to California:
Year 1Stem 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.
Year 2Human 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.
Year 3In 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.