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

Summary

Here, we present a protocol for establishing an everted gut sac model to evaluate interactions between Traditional Chinese Medicine (TCM) and recombinant adeno-associated virus (rAAV) during intestinal absorption, providing an ex vivo platform for optimizing oral gene delivery strategies.

Abstract

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.

Introduction

Recombinant adeno-associated virus (rAAV) represents a promising platform for systemic gene therapy, with three primary delivery routes currently being explored: intravenous injection¹, intraperitoneal injection², and oral administration³. While routes of intravenous and intraperitoneal injections have been extensively studied, oral administration, a non-invasive approach, remains comparatively underexplored. The feasibility of oral rAAV delivery was first demonstrated by Xin et al.⁴, who reported successful transduction of intestinal mucosa and induction of an anti-HIV imm....

Protocol

All animal procedures were conducted in strict accordance with the Naval Medical University Animal Ethics Committee–approved guidelines (approval CHEC (A.E)-2025011).

1. Preparation of Tyrode's solution and viral solutions

1. Tyrode's solution preconditioning:
   1. Prepare two 500 mL aliquots of Tyrode's solution (pH 7.4) (See Table of Materials).
   2. Cool one aliquot to 4 ℃ and warm the other to 37 °C.
   3. Bubble each aliquot with 95% O₂/5% CO₂.
2. Viral solution preparation:
   1. Add recombinant AAV9-CMV-EGF....

Results

This protocol was used to assess the efficiency of AAV9-mediated gene delivery across different segments of the small intestine, as well as the impact of the GZNG treatment on enhancing gene transfer and expression. Several key outcomes were observed, demonstrating both the successful application of the protocol and some variability that can arise under certain conditions.

The assessment of AAV9-mediated EGFP expression by histological (H&E staining) and fluorescence (EGFP) analysis across.......

Discussion

In this study, a novel in vitro approach to evaluate intestinal rAAV absorption by adapting the classic everted gut sac model was established. This method allows segment-specific analysis of AAV transduction efficiency across the small intestine under controlled conditions and evaluation of the synergistic effects between TCM and rAAV. Here, the critical steps, troubleshooting and modifications, limitations, and their broad applications in gene therapy and drug delivery are discussed.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	WISENT	319-005-CL	Liquid, with 4.5 g/L D-Glucose, with L-Glutamine, phenol red and sodium pyruvate
EGFP CDS region qPCR primers	TSINGKE		F: 5‘-ACTTCTTCAAGTCCGCCATG-3’; R: 5‘-TGTCGGCCATGATATAGACG-3’
Fetal Bovine Serum	WISENT	086-550	Superior quality, primary cell culture grade
Penicillin-Streptomycin(P/S)	WISENT	450-201-EL	This solution contains 10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin.
PrimeScript RT Master Mix (Perfect Real Time)	TaKaRa	RR036A
TaKaRa MiniBEST Universal RNA Extraction Kit	TaKaRa	9767
TSINGKE TSE401 ArtiCanCEO SYBR qPCR Mix	TSINGKE	TSE401	This product is suitable for fluorescence quantitative experiments using the SYBR Green dye method and is compatible with various real-time fluorescence quantitative PCR instruments.
TSINGKE TSP202-200 Trelief  Hi-Pure Animal Genomic DNA Kit	TSINGKE	TSP202-200
Tyrode's solution	Yuan Ye	R20268-500ml	It mainly consists of sodium chloride, potassium chloride, magnesium chloride, phosphates, calcium chloride, glucose, and HEPES.