Tools and Technologies I
Genetic modification of human embryonic stem (hES) cells offers an invaluable tool to address issues in stem cell development experimentally and provides a key element for potential hES cell clinical applications. Current methods to deliver genes into hES cells are hampered by the low rates of transgene insertion into the hES cell genome. Even in the rare cases stable hES cell clones are established, expression of the transgene very often is not under proper regulation. The main reason is because random insertion of the transgene gene places it under the influence of different local chromatin environment, leading to the observed differential expression of the transgene. The problem of low gene delivery efficiency can be overcome by lentiviral vector as this vector system can infect up to 50% of hES cells in culture. However, like these other non-viral gene delivery methods, lentiviral vector randomly integrates its genome into the chromosome of hES cells, therefore suffers the same problem of aberrant transgene expression as other methods. Random vector integration may also alter the expression of host genes and increase the likelihood of altering hES cell properties such as their capacity to proliferate or differentiate. To avoid the problem of aberrant gene expression, we propose in this application to evaluate a novel strategy for site-specific gene insertion into hES cells. This strategy involves a two-step process. In the first step, we will use lentiviral infection to deliver a reporter gene into hES cells. Based on the proper expression regulation of the reporter gene, we will establish stable hES cell clones with the vector integrating into different positions in the host genome. We will identify these vector integration sites and determine whether vector integration affects the pattern of hES cell gene expression and the capacity of hES cell to proliferate and differentiate. Those hES cell clones without any noticeable change in these properties will be picked for site-specific gene insertion. The reporter gene in the lentiviral vector is designed to be flanked on both sides by DNA sequences termed loxP which can be recognized by Cre, a sequence-specific recombinase. In the presence of Cre, the integrated reporter gene can be replaced by any DNA fragment flanked by the same loxP sites. Based on this principle, the second step of the strategy involves the use of the Cre/loxP system to facilitate a cassette exchange reaction between the integrated reporter gene and an incoming gene in the presence of Cre. This strategy will enable the establishment at high frequencies of hES clones with predictable transgene expression patterns. This feature would facilitate the studies of understanding how hES cell proliferation and differentiation are regulated. Site-specific gene insertion also reduces the biosafety concern of using genetically modified hES cells in clinical application to treat human diseases.
Statement of Benefit to California:
Genetic modification of human embryonic stem (hES) offers an invaluable tool to address issues in stem cell development experimentally and provides a key element for potential hES cell clinical applications to treat human diseases. However, the current methods for gene delivery into hES cells suffer two limitations; (1) low gene delivery efficiency; (2) aberrant gene expression due to random DNA insertion. Utilizing the lentiviral vector circumvents the limitation of low gene delivery but won’t solve the problem of aberrant gene expression. We propose to perform a proof-of-principle study to demonstrate the feasibility of establishing hES cell clones for site-specific gene insertion. The success of this study would enable the establishment at high frequencies of hES clones with predictable transgene expression patterns. Site-specific gene insertion also reduces the biosafety concern of using genetically modified hES cells in clinical application. Since California supports advance stem cell research and regenerative medicine, a success in our strategy would impact on the development of using hES cells for cure, therapies and diagnostics.
Like the enormous contributions to basic biomedical research by transgenic animals, establishment of transgenic human embryonic stem cell (hESC) lines will undoubtedly have major impacts on basic and potentially translational research. DNA introduced into cells typically integrates randomly into the genome. hESCs with pre-determined integration sites will be very valuable, provided it is clear that the integration site does not interfere with a wide range of genes involved in differentiation and has no effect on the growth characteristics of the cell. There are three approaches one might use: (1) homologous recombination, (2) AAV-mediated insertion which occurs at known genomic locations, or (3) random viral insertions, followed by screening the resulting clones for those with innocuous insertion sites. This project proposes to use the latter approach and will involve the construction of a lentiviral vector containing a target site (called loxP) for the Cre recombinase, so that, in a second step, any desired gene may be introduced specifically into that target site via Cre-mediated recombination. This is a proposal to enable engineering of hESC, which, if successful, will facilitate research in a significant way given the fact that homologous recombination in hESC has proven difficult. Reviewers felt that the proposed random integration / screening approach was cleverly designed but they also raised concern that, no matter how well characterized the selected cells are in vitro, applications in more complex or in vivo situations will require the activity of genes not examined for perturbation and that unwanted effects might therefore arise. Furthermore, one reviewer pointed out that it would be challenging to ensure that select integration sites are not silenced upon differentiation into certain cell types, whereas another reviewer felt that although any given site in the genome may not be universally good for all purposes, the cells generated under this proposal will be very useful for many projects. Reviewers commented that this is a well thought out and well-described project with a feasible timeline, and that the principle behind the experimental design is sound. In particular, the inclusion of both positive and negative selection markers will enhance the selection of desired clones; and the use of different loxP sites will reduce aberrant recombination. However, reviewers identified several weaknesses of the proposal. They were concerned about the choice of lentivirus for gene transfer, since this vector tends to integrate into multiple sites of the genome, diminishing the chances of identifying suitable single integrants. The principal investigator (PI) should consider this and modify the experimental design accordingly. Furthermore, reviewers questioned the choice of the Oct4 promoter as a tool to screen for integrants that allow faithful gene expression. Although it serves to identify appropriate expression at the ESC stage, it does not allow examination of transgene expression in differentiated cells. Also, the first proposed gene insertion, though providing a proof of principle, only generates an Oct4 reporter line, which is available. Overall, it would be much more desirable to test the possibility of expressing a gene with a reporter that functions in differentiated cells. The PI has extensive experience in virology and gene therapy and reviewers were confident that s/he could perform the proposed work on genetic modification. The lack of any ES cell expertise by the PI is a concern, but one that is addressed to some extent by evidence of success growing iPS cells from humans and the inclusion of a collaborator with hESC experience. The proposed budget was considered appropriate. Overall, this proposal generated considerable interest because of the feasibility of the proposed genetic manipulations in hESC, and the clever approach. However, enthusiasm was dampened by some inherent limitations of the proposed system and by some of the molecular tools chosen for this study.