Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids

The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many f eatures that make it an attractive vector for gene delivery in vivo. Howeve r, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limite d to a specific organ or cell type. In this study, we explored the possibil ity of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops w ithin the AAV-2 capsid were examined as sites for incorporation of peptides . We tested the effects of deleting these loops and different strategies fo r the incorporation of several targeting peptides. The tumor-targeting sequ ence NGRAHA and a Myc epitope control were incorporated either as insertion s or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, an d transduction in a range of cell lines. Whereas recombinant viruses contai ning mutant capsid proteins were produced efficiently, transduction of seve ral cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NCR, which binds CD13 (a recep tor expressed in angiogenic vasculature and in many tumor cell lines), disp layed an altered tropism toward cells expressing this receptor. Based on th is work and previous studies, possible strategies for achieving in vivo tar geting of recombinant AAV-2 are discussed.