S. Liu1, J. Lee3, J. Yu1, R. Sanchez1, M. Van Dam3, E. Rozengurt2, F. Brunicardi1 1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Division Of Digestive Disease,Los Angeles, CA, USA 3David Geffen School Of Medicine, University Of California At Los Angeles,Crump Institute For Molecular Imaging,Los Angeles, CA, USA
Background: Recombinant adeno-associated virus type 2 (AAV2) vectors transduce a wide variety of tissues in vivo and provide long-term gene expression with minimal immune responses and no pathological responses to the host, thus making AAV2 an attractive vector for gene delivery in vivo. However, its broad host range limits its usefulness in which transgene expression needs to be limited to a specific organ or cell type, such as cancer. Furthermore, low efficiency of wild type AAV2 (AAV2wt) transfection of pancreatic cancer (PDAC) cells further limits its effectiveness. In this study, we explored the possibility of directing rAAV2 transduction by incorporating a panel of tumor homing peptides to target human PDAC cells and tumors in mice.
Methods: 18 tumor homing peptides (TumorHoPe) were selected from literature and TumorHoPe database for AAV2 capsid display. Synthetic oligos were inserted into the rAAV2 capsid at R588. scAAV-eGFP and scAAV2-Gaussia Luciferase (GLuc) reporters were used. Real time-PCR was used to quantify the AAV2 titers. AAV infection of human PDAC cell lines (PANC-1, Mia PaCa2, Capan-2 and AsPC-1) and benign HPDE cells were performed, followed by peptide competition assays. GLuc secretory activity was determined by bioluminescence assay. In vivo imaging was performed in PANC-1 subQ xenograft using systemic delivery of AAV2TumorHoPe-GLuc imaging vector via tail vein.
Results: 18 TumorHoPe displayed AAV2 and one mutated AAV2 were created (AAV2TumorHoPe). The greatest viral infection was obtained in PANC-1 by AAV2RGD (CDCRGDCFC), Mia PaCa2 by AAV2RGR (CRGRRST), Capan-2 by AAV2RGD and AsPC-1 by AAV2LGL (RGDLGLS), with no infection of benign HPDE cells (Fig. Upper). There was no significant GFP expression in AAV2WT transfections of PDAC cells. To further identify whether the TumorHoPe display virus targeted PDAC cells and enhancing the infection efficiency, competition assays were performed on each cells infected by virus in the presence of excess exogenous TumorHoPe peptides; infection efficiency dropped to 42.5%, 34.3%, 25.6% and 36.9% in PANC-1, Mia PaCa2, Capan-2 and AsPC-1 cells, respectively. Systemic delivery of AAV2TumorHoPe-GLuc imaging vector in PANC-1 tumor model resulted in highly specific imaging of tumors without toxicity (Fig. Bottom A), whereas control AAV2 vector showed only non-specific transfection with none seen in the PANC1 tumor (Fig. Bottom B).
Conclusion: This study demonstrates that TumorHoPe display on AAV2 surface enhances viral targeting of PDAC cells and tumor, but not benign HPDE cells. The unique type of TumorHoPe on the AAV2 surface contributes to specific efficiency in PDAC cells. These preclinical data suggest that TumorHoPe AAV2 gene delivery could be used for targeted imaging of PDAC.
