Improving Access to Intestinal Stem Cells as a Step Toward Intestinal Gene Transfer

In previous studies exploring the intestinal epithelium as a potential site for somatic gene therapy, we concluded that the mucus lining the intestine constitutes a significant barrier to any attempts at gene transfer via the lumenal route. The mucus problem is aggravated by the fact that the epithelial stem cells, which are the logical target for gene transfer, are located deep in the intestinal crypts. The goals of the current study were to develop procedures that would improve accessibility to the intestinal stem cells and which would effect in vivo mucus removal without damaging the underlying epithelium. Initial experiments involved evaluation of the use of distension to improve accessibility to the intestinal crypts and the use of the mucolytic agents dithiothreitol (DTT) and N-acetyl-cysteine (NAC) versus a control solution of phosphate-buffered saline (PBS) for mucus removal. Catheters were inserted in each end of 3-cm terminal ileal segments in anesthetized rats. Two milliliters of agent was instilled into the clamped segment for 2 min, removed, and repeated. Lumenal distension resulted in shortened villi with wider intervillus spacing, thereby improving crypt access. Both NAC and DTT washes removed significant mucus between the villi but failed to reach the crypt lumen. To enhance mucus release from the crypt lumen, pilocarpine was selected due to its cholinergic properties and preferential binding to muscarinic receptors on crypt goblet cells. Pilocarpine given intraperitoneally 30 min prior to the mucolytic or PBS wash resulted in significant eradication of mucus down into the crypt lumen. This effect was still evident 3–4 hr later provided the intestine remained undisturbed. There are many reasons why the intestinal epithelium may prove to be an attractive site for somatic gene therapy. One of the most compelling is its ease of access via the lumenal route, which would allow direct in vivo gene transfer by standard endoscopic approaches. Since the epithelium turns over rapidly (2–4 days), the ideal targets for gene transfer would be the intestinal stem cells. Accessibility of these cells to lumenally delivered vectors is limited by their deep location and by the mucus that lines the epithelium. In these studies, we have devised conditions under which both of these problems can be minimized. Conditions for mucus removal may also have application in the delivery of genetic material into the respiratory epithelium.