A fiber modified adenovirus vector that targets to the EphrinA2 receptor reveals enhanced gene transfer to ex vivo pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis. To improve treatment options new treatments, such as adenoviral (Ad) gene therapy are necessary. However, low expression of the coxsackie and adenovirus receptor (CAR) in pancreatic cancer cells (PC) limits the therapeutic efficacy of these vectors. The aim of this study was to improve transduction of PC by recombinant adenoviruses by inserting peptides into the HI loop that binds to receptors highly expressed on pancreatic cancer and were shown to target these carcinomas in vivo. We report the successful incorporation into the HI loop of peptide Tyr-Ser-Ala (YSA), a peptide ligand targeting the EphrinA2 (EphA2) receptor, and K237, a peptide targeting to the vascular endothelial growth factor receptor-II (VEGFRII). Subsequently, we showed that both peptides enhanced the transduction of a number of human PC lines that abundantly express the targeted receptor. Additional competition studies confirmed that the YSA peptide redirects Ad-YSA from CAR and specifically targets the EphA2 receptor. Due to this transduction efficiency of Ad-YSA is increased not only in human pancreatic cancer cell lines but more importantly also in pancreatic cancer resection specimens. Since the YSA peptide has been shown to specifically target pancreatic cancer in patients, it may be expected that Ad-YSA will also display increased tropism for this tumour.

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo.

AIM: To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo. METHODS: YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor (CAR) and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS: Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude (nu/nu) mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION: Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.

Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices.

AIM: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS: Human pancreatic tissue samples (malignant and normal) were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% O(2). Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection. RESULTS: With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION: The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

Replacement of native adenovirus receptor-binding sites with a new attachment moiety diminishes hepatic tropism and enhances bioavailability in mice.

The in vivo efficacy of adenoviral vectors (AdVs) in gene delivery strategies is hampered by the broad tissue tropism of the virus and its efficient binding to human erythrocytes. To circumvent these limitations, we developed a prototype AdV lacking native binding sites. We replaced the adenoviral fiber with a chimeric molecule consisting of the fiber tail domain, the reovirus sigma1 oligomerization domain, and a polyhistidine tag as model targeting moiety. We also abolished the integrin-binding motif in the penton base protein. The chimeric attachment molecule was efficiently incorporated onto AdV capsids, allowed efficient propagation of AdV without requirement for complementing fiber and conferred highly specific tropism to the AdV. Importantly, the targeted AdV exhibited markedly reduced tropism for liver cells. In comparison with control AdV with native tropism, the targeted AdV showed 1000-fold reduced transduction of HepG2 cells and 10,000-fold reduced transduction of mouse liver cells in freshly isolated liver slices. After intravenous inoculation of C57BL/6 mice, the targeted AdV exhibited delayed clearance in comparison with the native AdV, leaving approximately 10-fold greater levels in the blood 2 hr after inoculation. For all tissues analyzed, the targeted AdV displayed significantly reduced in vivo transduction in comparison with the native vector. Furthermore, in contrast to the native AdV, the targeted AdV did not bind human erythrocytes. Together, our findings suggest that the targeted AdV design described here provides a promising platform for systemic in vivo gene delivery.