Relatively little is known about the regulation of differentiation and development of intestinal epithelium. Cell proliferation in the postnatal animal is confined to the intestinal crypts. As the cells move into the villi they differentiate. Differentiated cells on the villi of suckling and mature animals are functionally distinct. Questions concerning the biology of crypt stem cells are central to the understanding of regulation of differentiation and development of intestinal epithelium. To date the only available information has come from studies using germ line transmission of DNA. The proposed studies seek to develop an alternative means of transferring genes into the intestinal epithelium. The hypothesis is that genetic material administered intraluminally in a suitable vector non- replicating (retroviruses) can be efficiently transferred into the intestinal stem cells and can integrate and be expressed in those cells as well as in their progeny. The proposal will focus on three areas. First is optimization of gene transfer into the intestinal epithelium. Proposed studies involve systematic alterations of variables affecting: (i) Internalization of retrovirus vectors, (ii) integration of proviral DNA into host DNA, and (iii) expression of the transfered genes. Second is the use of retroviral transduction to address two questions relevant to the biology of stem cells: (i) Are stem cells permanent residents of the intestinal crypts? and (ii) are all stem cells pluripotent? The third area is the use of gene transfer as a model system to study the functional behavior of an intestinal promoter. Using constructs in which a proven intestinal promoter, the adenosine deaminase (ADA) promoter, is linked to the chloramphenicol acetyl transferase (CAT) reporter gene, the applicant proposes to determine whether the promoter can cause appropriate expression as assessed by: (i) Vertical (i.e., crypt-villus) distribution, (ii) longitudinal gradients (higher in jejunum than in ileum), and (iii) developmental patterns (low during suckling, increasing at weaning). By generating a new model system for the study of intestinal stem cells, these studies should enable investigation of factors that regulate differentiation and development, provide an alternate system in which to study intestinal gene expression, and develop a better understanding of the possible use of the intestine as a site for somatic gene therapy.
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