Adenovirus Neutralizing Antibody Assay

Supplementary Figure Legends

Supplementary Figure 1: Detection of human IGF-IR mRNA in the liver following HD-CBsIGFIR injection. Total RNA was extract from frozen liver fragments 72 hrs following virus inoculation as described in the legend to Fig 3 and analyzed by RT-PCR using the primers (forward): 5’-TTGCCCGAAGGTCTGTGAG-3’ and (reverse) 5’-GGAGCAGTAATTGTGCCGG-3’ that amplify a fragment of 1025 bp specific to human IGF-IR. The GAPDH transcript was used as an internal control and amplified using the primers (forward) 5’-GGTGAAGGTCGGTGTGAACGGATTT-3’ and (reverse) 5’-AATGCCAAAGTTGTCATGGATGACC-3’. cDNA amplification conditions were as previously described (1) and the DNA fragments analyzed by electrophoresis on 1% agarose gels without further purification. Mouse colon carcinoma MC-38 and human colon carcinoma KM12SM cells that express high levels of the respective IGF-IR (2) were used as controls for primer specificity. Shown is a representative gel of 2 separate analyses.

Supplementary Figure 2: Lack of virus neutralizing activity in mice sera following a single injection of virus particles. Virus neutralizing activity was analyzed using a fluorescence-based assay (3). Sera collected from mice 1, 7 and 21 days following the injection of 1-4X1011 (days 1 and 21) or 1.2X1012 (day 7) HD-CBsIGFIR particles were heat inactivated, serially diluted and AdV HCA-GFPCBLacZ particles (4) were added at a MOI of 250. The mixture was added to 104 A549 cells in a 96-well plate for a 24 hr incubation at 370C. The A549 cells were fixed using 2% formaldehyde and fluorescence was measured at an excitation wave length of 488nm a using a plate reader. Results are expressed as fold change (±SD) relative to normal (uninfected) mouse serum used at the same dilution and are based on sera obtained from 3 different mice.

* -p<0.1 as analyzed by the student’s t-test.

REFERENCES

1.Burnier JV, Wang N, Michel RP, Hassanain M, Li S, Lu Y, et al. Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis. Oncogene. 2011;30(35):3766-83.

2.Wang N, Fallavollita L, Nguyen L, Burnier J, Rafei M, Galipeau J, et al. Autologous bone marrow stromal cells genetically engineered to secrete an igf-I receptor decoy prevent the growth of liver metastases. Mol Ther. 2009;17(7):1241-9.

3.Sprangers MC, Lakhai W, Koudstaal W, Verhoeven M, Koel BF, Vogels R, et al. Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors. Journal of clinical microbiology. 2003;41(11):5046-52.

4.Robert MA, Lin Y, Bendjelloul M, Zeng Y, Dessolin S, Broussau S, et al. Strength and muscle specificity of a compact promoter derived from the slow troponin I gene in the context of episomal (gutless adenovirus) and integrating (lentiviral) vectors. J Gene Med. 2012;14(12):746-60.