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Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.

Satellite phage P4 directs the capsid proteins of its helper phage, P2, to form a head which is only one-third the size of the normal P2 head. The P2 head contains a genome of molecular weight 22 x 10(6), while the small P4 head contains a genome with a molecular weight of only 7 x 10(6). We have used in vitro DNA packaging to test whether P2 and P4 phage head sizes are determined by DNA size. The small DNA of satellite phage P4 added to a P2-infected cell extract was packaged primarily into particles containing three copies of the P4 genome. This process occurred with approximately the same efficiency as P2 DNA packaging in the same cell extract. In contrast, the large DNA of P2 was packaged 300-fold less efficiently than the small DNA of P4 in an extract derived from P4-infected, P2-lysogenic cells. These results suggest that DNA size is not sufficient to determine head size. The results are compatible with DNA packaging via the filling of preformed empty capsids.

Replicating DNA molecules of adenovirus-associated virus (AAV) were selectively extracted from KB cells coinfected at 39.5 detrees with a DNA minus, temperature-sensitive mutant of adenovirus 5 (ts125) as helper. Under these conditions AAV DNA replication proceeds normally, but there is little, if any, adenovirus DNA synthesis. An analysis of the replicating molecules in sucrose density gradients reveals that there are AAV DNA intermediates which consist of covalently linked plus and minus DNA strands. Under denaturing conditions, these concatemers are linear single strands whose lengths can reach at least four times the size of the AAV genome. The most abundant concatemeric species is a dimer which presumably exists in vivo as a unit length hairpin. Unit length linear duplexes appear to be immediate precursors of plus and minus progeny strands. These findings are compatible with a self-priming mechanism for the synthesis of AAV DNA.

Preinfection of chick embryo fibroblasts with an avian tumor virus does not eliminate the requirement of early DNA synthesis for other avian tumor viruses superinfecting the same cells several days after the primary inoculum. The function served by the early DNA in avian tumor virus infection either fails to persist into late infection or is genetically specific for each viral type.