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.