| By definition, gene therapy is the delivery of genetic material to produce a therapeutic effect. Interest has increased in using embryonic stem (ES) cells as targets for gene therapy. Their properties of self-renewal, clonogenicity, and pluripotentiality give ES cells the potential to be an unlimited source of cells for cell replacement therapy particularly suitable for genetic modification. Recombinant retroviruses are common vehicles for genetic modification, as they accurately and stably integrate the gene of interest into the target cell genome and theoretically offer long-term therapy.;To respond to the nation's desperate need to solve its critical shortage of donated pancreata for transplantation, the long-term goal of current tissue engineering research is to generate physiologically functional insulin-producing cells for alleviation of the complications of type 1 diabetes. However, these efforts have been limited by the inability to generate large numbers of cells suitable for transplantation. The original objective of this proposal was to address this cell source limitation by investigating the genetic modification of murine embryonic stem (mES) cells. We hypothesized that sustained ectopic expression of proendocrine transcription factor neurogenin 3 (ngn3) in mES cells would be an efficient trigger to enhance the differentiation and beta cell functionality of ES cells.;However, genetically modifying ES cells to generate insulin-producing cells and to elucidate the mystery of the endocrine pancreas lineage has proven difficult due to the mechanisms ES cells employ to silence retrovirus gene expression. We found that expression of ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mES cells. Therefore, the objectives of this thesis were redirected to overcome this obstacle and enhance the efficiency of transduction in ES cells and to identify any additional restrictions in gene transfer to ES cells.;We first investigated whether delivering more transgenes would improve retroviral gene transfer to murine embryonic stem (mES) cells. We formed polymer complexes with MSCV-derived ecotropic retroviruses, concentrated them up to 40-fold, and transduced two different murine embryonic stem cell lines, and a mouse fibroblast cell line as a control. The number of integrated transgenes increased more than 50-fold in the embryonic stem cell lines, yet, surprisingly, transgene expression did not increase. Interestingly, the embryonic stem cells had significantly fewer integrated transgenes than the mouse fibroblasts, even though transduction conditions were identical, which suggests that embryonic stem cells may be restricted in more than one step of retrovirus transduction.;We next investigated which steps of the virus lifecycle restrict efficient transduction of ES cells. Using recombinant MMuLV-derived retrovirus and recombinant HIV-1-derived lentivirus, we compared three major steps in the transduction of R1 mES and NIH 3T3 cells: (1) the number of active virus particles that adsorb to cells in a given absorption time; (2) the number of integrated transgenes; and (3) the corresponding level of gene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 cells. R1 mES cells integrated 3-fold fewer retrovirus transgenes than NIH 3T3 cells and showed 11-fold lower retrovirus transgene expression levels. In comparison, R1 mES cells integrated 10-fold fewer lentivirus transgenes than NIH 3T3 cells and showed 8-fold lower lentivirus transgene expression levels. Although silencing remains the biggest obstacle to retroviral gene transfer, these results indicate virus binding and integration in the transduction process are also limiting in ES cells.;We also investigated whether depletion of linker histone 1 in ES cells would alleviate silencing of retrovirus transgenes and improve gene transfer. To study the role of H1 in retroviral gene transfer, we transduced histone H1c, H1d, H1e triple null mouse embryonic stem cells with three different recombinant vectors---murine embryonic stem cell retrovirus (MSCV), and SIV and HIV-1 derived lentiviruses. We found that transduction of mES cells depleted of histone H1 did not improve viral gene transfer, and triple null mES cells had about a 2-fold reduction in the number of eGFP+ cells for all three viruses in comparison to wild-type mES cells.;In summary, we applied methods to overcome the major obstacle of retrovirus gene silencing and identified additional restrictions in retroviral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine. |
