Retrovirus-Mediated Transduction of Cultured Epidermal Keratinocytes

Retrovirus-mediated gene transfer is an efficient means of introducing and expressing exogenous gene(s) in many cell types including keratinocytes. However, parameters of transduction and gene expression have not been systematically analyzed for keratinocytes. To carry out such a study we have transduced cultures of newborn foreskin cells with retroviral vectors that encode the genes for neomycin resistance (neor) and for beta-galactosidase (B-gal). The neor gene is a dominant selectable marker and the B-gal gene encodes a histochemically detectable product. Our key findings are the following: 1) all keratinocytes that form colonies can be successfully transduced at a viral titer greater than 5 × 106 colony-forming units/ml; 2) transduction is effected by integration of a single copy of retroviral DNA; 3) transduced cells are not at a growth disadvantage and, in fact, single clones of transduced keratinocytes can be expanded to yield over 109 cells, suggesting that stem cells are transduced; 4) whereas most transduced colonies exhibit B-gal staining in a high percentage of constituent cells, some colonies had a mosaic or sectored staining pattern; 5) expression of the non-selectable B-gal gene was somewhat greater in differentiated cells of the culture as compared to nondifferentiated precursors. The ability to transduce stem cells at a high efficiency and to follow expression of transduced genes in clonal progeny will allow lineage mapping in stratified epithelial tissues

Synthesis and Secretion of Apolipoprotein E by Cultured Human Keratinocytes

Non-polar lipids are synthesized by keratinocytes in the epidermis and transported to the extracellular space where they contribute to formation of a permeability barrier. Transport of non-polar lipids in other organs and tissues usually occurs with the lipid complexed to an apolipoprotein. In this study we set out to learn if apolipoprotein E is produced by human epidermal keratinocytes in culture. Analysis of tota' cellular RNA from cultured keratinocytes showed the presence of human apolipoprotein E mRNA at concentrations ranging from 2.5 to 35 molecules/cell. The cells secrete a protein identified as apo E on the basis of molecular weight, isoform pattern, and immunoreactivity. Enzyme linked immunosorbent assay of media from keratinocyte cultures indicated that apolipoprotein E is secreted at a rate of 0.92 ng/h/106 cells

Gene therapy for Type 1 diabetes

In Type 1 diabetes, autoimmunity destroys the insulin-producing -cells of the pancreas, which are located in clusters called 'islets of Langerhans'. Exogenous insulin replacement therapy seldom maintains ideal metabolic control in patients with this disease and consequently, chronic complications progressively develop in many of them. Recently, much of the research concerning the treatment of Type 1 diabetes has been aimed towards the development of gene therapy. The understanding of molecular approaches, such as gene transfer using viral and non-viral delivery systems, are providing novel therapeutic strategies towards a cure for this disease. This review gives an overview of recently filed patents that relate to gene therapy and Type 1 diabetes

Protecting pancreatic beta-cells

Type 1 diabetes mellitus is an autoimmune disorder in which the insulin-producing beta-cells of the pancreatic islets of Langerhans are selectively destroyed. Transplantation of allogeneic islets offers a novel therapeutic approach for type 1 diabetic patients. Primary obstacles to the successful outcome of this treatment are loss of the islets occurring first during the isolation procedure and then immediately following transplantation. The genetic make up of beta-cells contributes to making them particularly vulnerable to apoptosis and necrosis-induced cell death caused by the trauma of the isolation procedure and by non-specific inflammatory events at the transplantation site. In this review we present description of chemical and molecular biology based strategies to confer cytoprotection to beta-cells

Retrovirally Transferred Genes Inhibit Apoptosis in an Insulin-Secreting Cell Line: Implications for Islet Transplantation

The transplantation of pancreatic islets for the treatment of type I diabetes is hindered by the enormous loss of cells due to early apoptotic events. Genetic engineering of islets with cytoprotective genes is an important strategy aimed to enhance the survival of these cells in the transplant setting. The present study was designed to evaluate and compare the effects of five genes on a cell line derived from insulin-producing β-cells, NIT-1. Cells were transduced using a Maloney murine leukemia virus (MLV) vector coding for yellow fluorescent protein (YFP) and for one of the following antiapoptotic genes: cFLIP, FADD-DN, BcL-2, PI-9, and ICAM-2. These genes were able to protect NIT-1 cells from cytokine-induced apoptosis to varying degrees ranging from no protection to significant protection equivalent to an optimal dose of a chemical caspase inhibitor. The data demonstrate that cFLIP, FADD-DN, and PI-9 are significantly more effective in protecting NIT-1 cells than BcL-2 and ICAM-2. Additionally, the data show that despite its weak in vitro inhibition of caspase-3, PI-9 affords significant protection against TNF-α-induced apoptosis in these cells. These genes may be ideal candidates to augment islet survival following transplantation