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

Abstract

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.

Gene Delivery to Intestinal Epithelial Cells In vitro and In vivo with Recombinant Adeno-Associated Virus Types 1, 2 and 5

“…Previous gene transfer research has demonstrated the protective limitations of the luminal surface of the gastrointestinal tract. Extracellular barriers such as tight junctions, cell surface glycocalyces, mucus and enzymes may prevent vector entry [33,[39][40][41]. Additionally AAV capsid characteristics can also determine if apical or basolateral infection is more optimal [34,42].…”

The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium

“…Given our data and that mice are likely to produce smaller quantities of mucus, application of polycation complexed adenoviruses to larger animal models may not prove as successful. A continuous layer of mucus is well recognized to protect both the lungs 31,32 and the intestinal tract 33,34 from bacterial infection and other damaging agents; its property as a barrier to gene transfer may, therefore, not be very surprising. Efforts to alter the composition or quantity of this layer to facilitate cationic lipid-mediated gene transfer may be important for future clinical studies.…”

In vitro and in vivo asessment of adenovirus 41 as a vector for gene delivery to the intestine

“…40 36 However, pharmaceutical strategies must be employed, or disruption of the membrane performed, to reach the crypts as they are not easily accessible from the lumen. 1,41 The immune response against cells infected by viral gene therapy vectors may also be a major hindrance for gene delivery, resulting in destruction of infected cells, limitations in the length of transgene expression, and elimination of infected cells upon repeat administration of the vector. [42][43][44] The intestine is quite an inhospitable target for viral vectors from this aspect as it has been estimated that 1-2 m of human intestine contain more antibody-secreting cells than do all other tissues combined.…”

“…One of the most compelling is its ease of access via the lumenal route, which would allow direct in vivo gene transfer by oral administration or standard endoscopic approaches. 1 The intestinal epithelium also has a large tissue mass and contains stem cells with known locations in the crypts of Lieberkü hn. 2 Development of cystic fibrosis (CF) mouse models with early mortality due to intestinal obstruction has emphasized the importance of the intestine as a site for gene transfer.…”