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 immune response following oral administration of rAAV2. Similarly, Hara et al.^3,5, demonstrated that oral rAAV/Aβ can transduce gastrointestinal mucosa and reduce brain amyloid deposition, whereas Steel et al.⁶ found that oral vaccination demonstrated superior survival and anti-tumor immunity compared with intramuscular injection. The same study reported that rAAV6 exhibited stronger intestinal tropism than rAAV5. Beyond vaccine applications, therapeutic potential has been achieved in other disease models. Ma et al.⁷ and Hao et al.⁸ proved that oral rAAV led to tumor suppression and anti-fibrotic therapy in the liver and stomach, respectively, following gastrointestinal transduction. Huang et al.⁹ identified the engineered serotype Rec2 as selectively transducing brown adipose tissue after oral administration, in contrast to the predominant liver tropism observed with intravenous delivery¹⁰. Despite these pioneering findings, research on oral rAAV delivery remains limited, in part due to the lack of in vitro screening systems for large-scale evaluation.

The everted gut sac model, developed by Wilson and Wiseman in 1954¹¹, remains a valuable in vitro platform for investigating intestinal drug absorption mechanisms. Unlike other absorption simulator apparatus, this system uniquely accounts for multiple physiological processes beyond simple passive diffusion¹². This characteristic makes it particularly advantageous for studying TCM¹³. Using this system, it is documented that TCM compounds often exhibit segment-dependent intestinal absorption patterns throughout the gastrointestinal tract. For example, one study demonstrated that multiple alkaloids from Wuwei Qingzhuo San are selectively absorbed in different intestinal segments, with the jejunum and ileum showing the most active uptake¹⁴. Additionally, co-administration of TCM constituents can modulate intestinal absorption. Liao et al.¹⁵ reported that co-administration with Radix Angelicae dahuricae extract significantly enhanced the intestinal absorption of puerarin, highlighting the influence of TCM interactions on segmental uptake. Therefore, the everted gut sac model serves as an ideal tool for elucidating the complex absorption behaviors and synergistic interactions inherent in TCM formulations and other drugs.

Selecting an appropriate model for rAAV absorption research requires a nuanced understanding of the trade-offs among physiological accuracy, technical complexity, and throughput. The table below summarizes the primary experimental models used to study intestinal absorption.

This study provides proof of concept that the everted gut sac model can be used to investigate the intestinal absorption of recombinant adeno-associated virus (rAAV) vectors. It provides valuable insights into how co-administration of TCM influences the intestinal absorption of rAAV vectors, underscoring the need for further characterization of their mechanistic interactions under physiologically relevant conditions. Optimizing TCM-enhanced oral delivery strategies through this approach may facilitate the development of non-invasive rAAV therapeutics, thereby improving patient compliance and treatment accessibility.

Researchers should evaluate the everted gut sac model based on the following criteria to ensure it aligns with their experimental objectives. Appropriate applications include rapid screening of serotypes, adjuvant/enhancer evaluation, and regional kinetics, while inappropriate applications and limitations include long-term expression studies (>48 h) and systemic pharmacology.

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-EGFP to the 37 °C Tyrode's solution to achieve a final concentration of 1 x 10¹¹ vg/mL (Control condition).
   2. For the experimental condition, supplement the AAV9-CMV-EGFP preparation with a TCM formula GZNG (Table 2) dissolved in vehicle to a final concentration of 200 mg/mL.
   3. For the GZNG-only control, add GZNG (200 mg/mL) to Tyrode’s solution without AAV9 CMV-EGFP.
      NOTE: Prepare viral dilutions immediately before use to preserve infectivity. The pH of the GZNG solution is approximately 5.0–6.0.

2. Animal preparation and intestinal tissue harvesting

1. Fasting: Fast the 8-week-old Sprague–Dawley rats for 24 h prior to surgery. Ensure free access to water during this period to maintain hydration.
   NOTE: Fasting helps minimize gut content and ensures accurate measurement of intestinal absorption during ex vivo experiments.
2. Check animal health before fasting and ensure that they are in good condition. Exclude animals with signs of distress or illness from the study.
3. Administer sodium pentobarbital (200 mg/kg, intraperitoneally) to induce deep anesthesia. Verify that the animal is fully anesthetized by checking for a lack of reflexes and steady respiration.
   CAUTION: Pentobarbital is a hazardous controlled substance; handle with gloves and PPE in a fume hood to avoid skin contact/inhalation and follow institutional guidelines for safe disposal and emergency response.
4. Perform euthanasia by cervical dislocation once deep anesthesia has been confirmed.
5. Swab the abdominal and thoracic areas with 75% ethanol to disinfect the skin and reduce microbial contamination during surgery.
6. Make a midline incision along the abdominal region using sterile surgical scissors to expose the peritoneal cavity.
7. Gently separate and expose the stomach and intestines. Use a sterile drape to protect surrounding tissues and organs during the procedure.
8. Carefully transect the pyloric sphincter to separate the stomach from the proximal duodenum. Ensure the duodenum remains intact for subsequent procedures.
9. Dissect the distal colon at the recto-anal junction while avoiding any damage to the surrounding tissues.
10. Gently free the mesenteric attachments from the small intestine using fine forceps or scissors. Be cautious not to stretch or tear the mesentery.
11. Immediately transfer the intact small intestine to an ice-cold solution of oxygenated Krebs-Ringer Buffer (Tyrode's solution, 4 °C).

3. Ex vivo everted gut-sac preparation

1. Select intestinal segments as follows: an 8 cm segment located 10 cm distal to the pylorus was designated the duodenal segment.
2. Collect an 8 cm jejunal segment starting 16 cm distal to the duodenal segment.
3. Obtain an 8 cm ileal segment starting a further 16 cm distal to the jejunal segment.
4. For the colon, collect an 8 cm segment located 5 cm proximal to the cecal blind end.
   NOTE: Ensure the tissue is elastic and intact, suitable for inversion during preparation.
5. Rinse the intestinal lumen using a 2 mL syringe filled with ice-cold, oxygenated Tyrode's solution.
6. Dispose of the effluent following institutional biohazard waste protocols to maintain sterility and prevent contamination.
   CAUTION: Handle biological waste using gloves and appropriate protective equipment to avoid exposure to potentially infectious material. Dispose of all biological waste and effluent following institutional biohazard safety guidelines to prevent environmental contamination.
7. Insert the proximal end of the intestinal segment onto a 4 mm polished glass rod. Ensure the rod surface is smooth to prevent mucosal tearing.
8. Tie the distal end of the segment with a 2-0 silk suture to secure the tissue onto the rod. Confirm that the tissue remains stable during inversion.
9. Gently evert the intestinal segment by sliding the tissue along the rod until the mucosa is completely exposed. Avoid excessive stretching or twisting to maintain tissue integrity.
10. Instill 2 mL of ice-cold Tyrode's solution into the lumen to check for leakage. Acceptable leakage should be <5% within 1 min. If leakage is detected, adjust the sutures and verify complete inversion of the mucosal surface.
    NOTE: Perform the eversion in a cold environment to minimize enzymatic degradation and preserve tissue viability.
11. Insert a 5 cm length of 3 mm glass capillary into the open end of the everted segment to serve as the inlet for perfusion. Advance the cannula gently to avoid mucosal tears.
12. Secure the cannula using 2-0 silk sutures. Ensure that the cannula remains fixed in position without collapsing or obstructing fluid flow.
13. Apply gentle tension to the sutures to maintain the proper shape of the gut sac. Prevent kinking or twisting, as these may disrupt perfusion and alter absorption results.
14. Avoid excessive tightening, as this could damage the tissue or deform the sac. Maintain uniform tension throughout the experiment. Regularly examine the everted gut sac for leakage or structural damage. If leakage exceeds 5%, repair or replace the segment before continuing.

4. AAV transduction and incubation

1. Place each everted sac into a 7 mL conical tube containing 4 mL of 37 °C oxygenated Tyrode's solution.
2. Add viral solution as follows: Control: AAV9-CMV-EGFP alone; Experimental: AAV9-CMV-EGFP + GZNG.
3. Incubate statically at 37 °C for 2 h, during which a gas mixture of 95% O₂/5% CO₂ was continuously bubbled into the solution.

5. Sampling, endpoint processing, and analysis

1. Time-course sampling
   1. Withdraw 50 µL of serosal fluid from each gut sac at 0, 30, 60, 90, and 120 min using a calibrated microsyringe. Ensure accurate sampling without introducing air bubbles.
   2. Immediately replace the withdrawn fluid with 50 µL of prewarmed (37 °C) Tyrode's solution to maintain osmotic balance and internal volume. Minimize the time between withdrawal and replacement to preserve consistent experimental conditions.
      NOTE: When using a microsyringe (e.g., Hamilton syringe), maintain sterility and handle gently to prevent damage to the gut sac membrane.
   3. Perform sampling promptly at each time point to minimize disturbance of the incubation environment.
2. Virus titer determination
   1. Extract total DNA from the serosal fluid using a commercially available Animal Genomic DNA Extraction Kit according to the manufacturer's instructions.
   2. Perform absolute quantification of the rAAV vector genome copy number by qPCR. Use a SYBR Green Kit with ITR primers. Carry out the qPCR amplification in a standard 96-well plate using a thermal cycler with the following cycling conditions: Initial denaturation: 95°C for 10 min; Denaturation: 95°C for 15 s; Annealing and extension: 60°C for 1 min; 40 cycles in total
   3. To quantify the genome copy number, generate a standard curve using serial dilutions of plasmid rAAV-CMVp-gfp (1 ng, 0.1 ng, and 0.01 ng). Use these dilutions to calculate the viral genome copy number based on their corresponding cycle threshold (Ct) values.
   4. Determine the vector genome copy number in the collected serosal fluid using the standard curve. The results were reported as genome copies per 100 ng of extracted DNA.
      NOTE: Ensure that all reagents and equipment are sterile to prevent contamination during DNA extraction and qPCR analysis. Use appropriate controls to verify qPCR accuracy, including no-template controls (NTC) and plasmid standards.
   5. Perform qPCR in triplicate for each sample to ensure reproducibility and reliability of the results.
3. Terminal tissue processing
   1. Aspirate all residual luminal fluid from each gut sac using a sterile pipette. Handle gently to avoid rupturing the tissue.
   2. Rinse each sac three times with phosphate-buffered saline (PBS) containing 1% penicillin–streptomycin (P/S) to remove residual medium, bacteria, and impurities. Ensure thorough washing to maintain sterility for downstream applications.
   3. Cut the intestinal tissue into 2 mm³ (2 mm * 2 mm * 0.5 mm) fragments using sterile surgical scissors or blades. Keep the fragments uniformly sized for consistent incubation.
   4. Incubate the tissue fragments in 10 mL of DMEM supplemented with 10% FBS and 1% (v/v) P/S at 37 °C for 48 h in a humidified incubator with 5% CO₂. The culture medium should be refreshed every 12 h during incubation.
      NOTE: This step maintains tissue viability and allows cell recovery post-experiment. Ensure the mucosa remains intact during cutting and washing. Perform three complete PBS washes to eliminate debris and microbial contaminants. Maintain constant temperature (37 °C) and high humidity to support metabolic activity and tissue health.
      CAUTION: Handle tissue fragments with sterile tools and avoid mechanical stress to preserve epithelial integrity.
4. Post-incubation analyses
   1. Aliquot A (Histological analysis):
      1. Fix the tissue samples in 4% paraformaldehyde (PFA) for 24 h at 4 °C to preserve cellular architecture.
      2. After fixation, embed the tissues in OCT compound and perform Hematoxylin and Eosin (H&E) staining to assess tissue integrity and morphology.
         CAUTION: Paraformaldehyde is a hazardous fixative; handle it within a fume hood using appropriate personal protective equipment
   2. Aliquot B (Fluorescence analysis and genome quantification):
      1. Snap-freeze the remaining tissue in liquid nitrogen and store at -80 °C until further processing.
      2. Use the frozen samples for fluorescence microscopy to detect EGFP expression, assessing transfection efficiency or viral uptake.
      3. Perform DNA extraction followed by qPCR to determine viral genome copy number or transgene expression.

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.