Gene transfer to the gastrointestinal tract after peroral administration of recombinant adeno-associated virus type 2 vectors.
OBJECTIVE
The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment.


METHODS
The stability of rAAV2 vectors carrying beta-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the beta-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays.


RESULTS
The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (beta-galactosidase) was below the level of detection of the histochemical assay.


CONCLUSIONS
Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract after peroral rAAV2 administration remained low.
Shao
Guohong
G
Center for Gene Therapy, Columbus Children's Research Institute, OH, USA.
Greathouse
Kristin
K
Huang
Qin
Q
Wang
Chiou-Miin
CM
Sferra
Thomas J
TJ
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001709:Biotechnology; D000229:Dependovirus / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005744:Gastric Acid / Q000378:metabolism; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D016133:Polymerase Chain Reaction; D017693:Sodium Bicarbonate / Q000494:pharmacology; D013047:Specific Pathogen-Free Organisms; D001616:beta-Galactosidase / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*
D017693:Sodium Bicarbonate; D007611:Aprotinin; D001616:beta-Galactosidase