Recently developed techniques for inserting genes into somatic tissues of animals have provided hope that certain human genetic diseases might in the near future be cured by gene therapy. However, experiments involving gene transfer into hematopoietic tissues have in general been frustrated by low levels of expression. Most of these studies have utilized """"""""minigene"""""""" constructions in which the protein of interest is encoded on a contiguous gene sequence, along with heterologous transcriptional regulatory signals. These arrangements might be considered artificial in the sense that most mammalian genes contain flanking regulatory elements, and also introns which interrupt the coding sequence. In humans, the absence of purine nucleoside phosphorylase (PNP) activity is associated with T-cell immunodeficiency. In this proposal, the human PNP gene is used as a model system to compare the effectiveness of PNP """"""""minigene"""""""" vectors with vectors containing the natural PNP gene in providing expression in hematopoietic tissue of the mouse. One primary advantage of using the PNP gene in this study is its relatively small size (10-13 kb), making it suitable for insertion into a retroviral vector. Initial studies will be undertaken to characterize regulatory elements associated with the human PNP gene (thus facilitating vector construction). Various portions of sequence from the upstream flank of the PNP gene, fused to the chloramphenicol acetyltransferase gene, will be analyzed for gene expression activity in transfection studies. The downstream flanking region from the PNP gene will similarly be tested for regulatory effects. Results from these studies will be used to construct anti-sense retroviral vectors containing the intact human PNP gene. Constructs lacking some or all of the introns and constructs containing a strong promoter will be included for comparison. These vectors will then be used as vehicles for PNP gene transfer into cultured cells and also into hematopoietic tissues of the mouse, testing for gene insertion (by Southern analysis) and expression of electrophoretically distinct human PNP enzyme activity (on isoelectric focusing gels). Results from these experiments will provide an assessment of the importance of natural gene structure in the maintenance of PNP gene expression activity after insertion into somatic tissues and will thus be relevant to the application of gene transfer in the treatment of PNP deficiency and perhaps in the treatment of other genetic diseases as well.
