Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno‐associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the α v β 3 integrin receptor. Second, genes are expressed from a tumor‐activated and temozolomide (TMZ)‐induced promoter of the glucose‐regulated protein, Grp78 . Here, we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP‐ Grp78 . Firstly, in vitro we showed that TMZ increases endogenous Grp78 gene expression and boosts transgene expression from the RGD4C/AAVP‐ Grp78 in human GBM cells. Next, RGD4C/AAVP‐ Grp78 targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP‐ Grp78 targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage’s minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.

Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNFα) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses.

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.Versican/PG-M (1, 2) is a large chondroitin sulfate proteoglycan of the extracellular matrix (ECM) 2 that exhibits two distinct expression patterns. In various developing embryonic tissues (3), including the nervous system, versican is transiently expressed and plays important roles in cell adhesion (4), migration (5), proliferation, and differentiation (6). In some adult tissues, such as the heart, blood vessels, and brain, it is constitutively expressed and serves as a structural macromolecule of the ECM.In developing cartilage, versican is transiently expressed at a high level in the mesenchymal condensation area and rapidly disappears during cartilage development (7,8). Recent immunohistochemical studies on developing limb bud cartilage revealed that an area positive for versican gradually shifts out of the diaphysis and is replaced by an area positive for aggrecan. Differentiating chondrocytic N1511 and ATDC5 cells showed similar expression patterns of these molecules (9). These reciprocal patterns of versican and aggrecan expression suggest that versican serves as a temporary framework in developing cartilage matrix. Although the aggrecan aggregate (10) is the major component of cartilage ECM and versican has not been detected by immunohistochemical studies (3), constitutive low level transcription of the versican gene is observed in cartilage (11) and chondrocytes (11,12). In addition, extracts of human adult articular cartilage contain versican (13), suggesting its distinct role there.The core protein of versican consists of N-and C-terminal globular domains (G1 and G3) and two chondroitin sulfate (CS) domains (CS-␣ a...