This proposal focuses on the transcriptional control of genes transferred by lentiviral vectors to enhance the properties of beta-cells and to induce the development of stem and progenitor cells into beta-cells. We will examine four types of enhancer/promoters to regulate gene expression: 1. constitutive, 2. lineage-specific (insulin), 3. lineage-determining/auto-regulatory (PDX-1) and 4. chimeric basal/lineage-determining to recruit and direct stem cells into beta-cell differentiation. Expression will be measured in human pancreatic cell lines and primary murine beta-cells and in human ES cells (WAOI) as potential beta-cell progenitors. We will use the eGFP gene as a reporter which can be measured quantitatively by FACS analysis and can be used to isolate cells in which the vector is being expressed by FACS sorting. We will use the PDX-1 gene as a prototypical beta-cell lineage determining gene which also activates its own expression, recognizing that PDX-1 expression alone may not be sufficient to induce differentiation of beta cells to a useful extent. The practical goal of these studies will be to provide useful lentiviral vectors for gene modification of beta-cells and their progenitors to enhance transplantation. Scientifically, these studies will provide information on the activity of these lentiviral vectors for gene expression from beta-cells and human ES and whether PDX-1 can induce beta-cell differentiation of human ES cells. In addition to the uses to modify beta-cells and their progenitors for clinical transplantation purposes, these lentiviral vectors should also have numerous experimental applications, to identify and isolate cells capable of expressing from the insulin promoter (e.g. beta-cells) or the PDX-1 promoter (e.g. beta-cell precursors), or as reporter genes to indicate the transcriptional status of the insulin or PDX-1 genes, e.g. in response to glucose concentrations, hormones, growth factors, genes, or drugs.
| Shaw, K L; Pais, E; Ge, S et al. (2009) Lentiviral vectors with amplified beta cell-specific gene expression. Gene Ther 16:998-1008 |
| Jang, Jung-Eun; Shaw, Kit; Yu, Xiao-Jin et al. (2006) Specific and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors. Stem Cells Dev 15:109-17 |
