Supplemental Methods

Cell culture and AAV treatment

The colon-derived human epithelial cell lines HT29 (ATCC HTB-38), T84 (ATCC CCL-248), Caco2 (ATCC HTB-37) were used between passages 8 and 35. HT29 and T84 cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (Cellgro by Mediatech; Herndon, VA) supplemented with 4.0 mM L-glutamine (Sigma Chemical; St Louis, MO), 1% antibiotic-antimycotic (ABAM) solution (Atlanta Biologicals; Norcross, GA), and 5% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals; Norcross, GA). Caco2 cells were grown in Eagle’s minimum essential medium (EMEM) (Cellgro by Mediatech; Herndon, VA) supplemented with 4.0 mM L-glutamine, 1.0 mM sodium pyruvate (Cellgro by Mediatech; Herndon, VA), 0.018 mM sodium bicarbonate (Fisher Scientific; Pittsburgh, PA), 1.0% nonessential amino acid 100X solution (Cellgro by Mediatech; Herndon, VA), 1% ABAM and 20% FBS. Cells were grown to 75% confluency and detached by trypsinization, washed by gentle centrifugation and resuspended in their respective media. For each transduction experiment 1x105 cells were allowed to adhere overnight, in 24 well tissue culture plates (BD; Franklin Lakes, NJ). The fully concentrated media was then replaced with 200 ml of diluted media, which was then treated with AAV-GFP multiplicity of infection (MOI) of 100 or 1000 DNase-resistant physical particles. Simultaneous transduction experiments were done with wt AdV5 co-infection (MOI 1). Control transductions were done simultaneously using wt AdV5 (MOI 1) alone or a mock transduction with PBS alone. Cells and vector were incubated up to 8 hrs (37oC, 5% CO2), after which full media was added.

Transductions were done at 24, 48 and 96 hr cell growth times along side controls and performed at least in four separate experiments. Transgene expression was confirmed by observing live cellular GFP fluorescence using a Leica DM ILT fluorescence microscope (Leica Microsystems; Wetzlar, Germany) or a MRC 1024 ES Confocal Laser Scanning Microscope (Bio Rad; Hercules, CA), using a FITC filter. Images were captured with a Spot RT color camera using Spot software version 3.4 (Diagnostic Instruments; Sterling Heights, MI) or Laser Sharp software (Bio Rad, Hercules, CA), respectively.

To assess sustained transgene expression 5x105 HT29 cells were transduced with the protocol described above using AAV2/2 (particle MOI 10,000), in 6 well tissue culture plates, without AdV co-infection. Transgene expression or GFP fluorescence was confirmed by microscopy. A concentrated population of GFP positive cells was created by sorting out GFP positive cells using a FACSort flow cytometer. Cells were detached by standard trypsinization, washed with PBS for sorting. In a sterilized cytometer biparametric plots, (FSC vs. SSC), presorting gates were set to exclude debris and cell groups. Negative control transductions were used to set sorting parameters such that the GFP positive quadrant in the intensity plot (FSC vs. log green fluorescence) selected for only fluorescent events. Cells were sorted at a rate of 1000 events per second, washed in sterile PBS and transferred to a T75 flask for maintenance.

Cells were detached following treatment using standard trypsinization, washed with gentle centrifugation, and fixed in sterile filtered 1% paraformaldehyde (in 1X PBS) solution (pH 7.4) for 20 minutes at 4oC. The cells were then washed, resuspended in cold 1X PBS and analyzed by flow cytometry within 2 hrs.

Flow cytometric analysis

Quantification of vector transduction efficiency or GFP expression was determined by flow cytometric analysis using a FACSort flow cytometer with an argon laser tuned to 488 nm (Becton Dickinson Franklin Lakes, NJ) within the ICBR Flow Cytometry Core at the University of Florida; Gainesville, FL. A minimum of 10,000 fluorescent events were analyzed using Cell Quest 3.3 software. GFP positive cells were determined by gating a biparametric plot, forward scatter (FSC) vs. side scatter (SSC), to a one-parameter green fluorescence intensity plot, FSC vs. log green fluorescence. Background green fluorescence or auto-fluorescence was controlled by negative control mock transductions and/or by AdV5 infections and limited to 0.5%. Transduction efficiency was calculated as the percentage of GFP positive cells (R2) over the total cell number (R1+R2). Three flow analysis parallels were performed for every experiment.

Human colon biopsy fixation

The specimens were removed after 24-36 hrs post infection, fixed in fresh 4% paraformaldehyde in 1X PBS (pH 7.4) 4 hrs, rinsed in 1X PBS and cryopreserved in fresh 30% sucrose (pH 7.4) overnight at 4oC. The fixed tissue was then further processed through the Molecular Pathology Core at the University of Florida, Gainesville, FL; where it was embedded in OCT, snap frozen in isopentane and 4 µm thick frozen sections were cut on a cryostat. Slides were air dried for 30 minutes at room temperature and then washed twice with distilled water before mounting with Vector Shield plus DAPI (Vector Labs, Burlingame, CA).

GFP immunohistochemistry

Paraffin sections (4 mm) were deparaffinized and blocked for endogenous peroxidase activity with 3% H2O2 in methanol for 10 minutes. Serial sections were incubated with rabbit anti-GFP, 1:800, (Abcam, Cambridge, MA) or normal goat IgG (for negative control) for 1hr at room temperature. Antibody binding was visualized by incubation with a biotinylated goat anti-rabbit secondary followed by streptavidin-biotin-peroxidase complex technique, Vectastain Elite (Vector Laboratories, Burlingame, CA) with diamino-benzidine (DAB) as the chromagen. Hematoxylin was used as counterstain.

Primers

Standard PCR primers: GFP 452 bp product, sense: GAACTGGATGGCGATGTGAA, antisense: TGCTTGTCGGCCATGATGTA; mouse beta-Actin 246 bp product, sense: GTGGGCCGGTCTAGGCACCA; antisense: GGTTGGCCTTAGGGTTCAGG. Real Time PCR primers: GFP 196 bp product, sense: TGGAACTGGATGGCGATGTG; antisense: TGCTGCTTCATATGGTCTGG. The same mouse beta-Actin primer set was used as above.

Real Time PCR

Mouse beta-Actin was used as a reference gene to normalize the GFP DNA. To allow comparison between groups the GFP CT value was normalized to the resulting beta-Actin CT values for the animals in that group, through the following calculations: lowest Actin CT value/each individual Actin CT value to provide a normalization ratio per sample. Each corresponding GFP CT value was then multiplied by the normalization ratio to provide a normalized GFP CT value. This GFP CT value was then used to extrapolate the GFP DNA copy number from the standard curve.

The standard curve was prepared as outlined in the ABI SDS 7000 bulletin. Sham DNA was spiked with serially diluted concentrations of GFP DNA, 200 pg/ml or 2.5x107 copies was serially diluted, 1:2, down to 0.012 pg/ml or 1547 copies and each dilution amplified by the same PCR protocol. DNA copy number was calculated using the known concentration of DNA with the following formula: (6.023x1023 no. of copies/mol x DNA concentration)/(660 g/mol [MW of one bp] x genome size [bp]). A linear regression equation for obtained CT values was calculated as a function of known GFP DNA copy numbers to provide an adequate standard curve equation, y = -3.8048x + 49.959 and R2 = 0.9974.