Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an ironmimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/ phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.

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Human sarcomas are rare but diverse malignant tumors derived from mesenchymal tissue. Clinical response to therapy is currently determined by the modified World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST), but these standards correlate poorly with sarcoma patient outcome. We introduced ligand-directed particles with elements of AAV and phage (AAVP) to enable integration of tumor targeting to molecular imaging. We report drug-response monitoring and prediction in a nude rat model of human sarcoma by AAVP imaging. As a proof-of-concept, we imaged Herpes simplex thymidine kinase in a clinic-ready setting with PET to show that one can a priori predict tumor response to a systemic cytotoxic. Given the target expression in patient-derived sarcomas, this platform may be translated in clinical applications. Sarcoma-specific ligands and promoters may ultimately lead to an imaging transcriptome.H uman sarcomas are rare yet heterogeneous malignant tumors from mesenchymal tissues (1). Monitoring drug responses in soft-tissue sarcoma has long been clinically problematic. Currently, responses are determined by the modified World Health Organization (WHO) criteria (2-4) or the Response Evaluation Criteria in Solid Tumors (RECIST) (5-7), which require marked tumor size decrease for patients to be considered responding to therapy. A major assumption of these criteria is that a solid tumor volume is directly proportional to the cancer cell number. However, in soft-tissue sarcomas, there are reasons to challenge such an assumption (8). First, in addition to tumor cells, sarcomas contain nonmalignant stromal cells and extracellular matrix (ECM) that do not disappear, even if the malignant component is treated. Moreover, when cytotoxics are used against sarcomas, there is often associated necrosis resulting in reduced total cell number but not necessarily overall tumor size changes. Finally, even if a soft-tissue sarcoma is predominantly or entirely composed of cancer cells, its remnant composition may not be fully eliminated when tumor cells are destroyed by therapy, because myxoid-type degeneration is not always promptly removed. Ultimately, the modified WHO criteria and RECIST correlate poorly with drug response and outcome in patients with soft-tissue sarcomas; validation in this setting is sporadic and restricted. In another level of complexity, drug responses in patients with soft-tissue sarcoma are determined through standard methods, such as CT, MRI, or PET scans. However, because systematic quantitative measurements have not been established because of the rarity and diversity of soft-tissue sarcomas, decreases in tumor size and/or density are not accepted as unequivocal evidence of response. Consequently, many conventional soft-tissue sarcoma responses in individual patients are evaluated qualitatively. Thus, new or alternative quantitative imaging criteria to improve management and follow-up of responses were proposed in ref. 9.We have introduced a hybrid vector that ena...