Enhancement of Intestinal Recombinant Adeno-Associated Virus Absorption by a Traditional Chinese Medicine Formula

Oral recombinant adeno-associated virus (rAAV) delivery holds significant therapeutic potential but is limited by inefficient intestinal absorption. Traditional Chinese Medicine (TCM), widely used in oral formulations, represents a promising source of absorption enhancers. However, its potential effects on rAAV uptake and transduction remain largely unexplored. The everted gut sac model provides a quantitative ex vivo platform to investigate these interactions. A detailed protocol is presented here utilizing the everted gut sac model to quantitatively assess adeno-associated virus (rAAV) translocation across the intestinal epithelium and subsequent gene transduction within intestinal tissue under controlled experimental conditions. Key steps include harvesting small intestinal segments from laboratory rodents, everting the segments to form sacs, and filling each sac with oxygenated buffer. The everted sacs are immersed in an oxygenated buffer containing rAAV particles and incubated at 37 °C to allow luminal exposure and vector transport. Endpoint analyses involve sampling the internal (serosal) fluid to measure rAAV particles that have crossed the epithelium, as well as analyzing the sac tissue for transgene expression to determine transduction efficiency. By testing multiple intestinal regions, the protocol provides insight into segment-specific differences in rAAV uptake. Moreover, the inclusion of GZNG, a classic TCM formula, during incubation enables evaluation of its impact on vector uptake and transduction. The everted gut sac approach provides a valuable platform for screening rAAV serotypes, delivery enhancers, and interactions between rAAV vectors and TCM components. It's simple and parallelizable design also enables high-throughput screening in preclinical studies.