Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer

Bacteriophage (phage) have attractive advantages as delivery sys-tems compared to mammalian viruses, but have been consideredpoor vectors because they lack evolved strategies to confrontand overcome mammalian cell barriers to infective agents. Wereasoned that improved efficacy of delivery might be achievedthrough structural modification of the viral capsid to avoid pre-and post-internalization barriers to mammalian cell transduction.We generated multifunctional hybrid AAV/phage (AAVP) particlesto enable simultaneous display of targeting ligands on the phage’sminor pIII proteins and also degradation-resistance motifs on thevery numerous pVIII coat proteins. This genetic strategy of directedevolution, bestows a next-generation of AAVP particles that fea-ture resistance to fibrinogen adsorption or neutralizing antibodies,and ability to escape endolysosomal degradation. This results insuperior gene transfer efficacyin vitroand also in preclinicalmouse models of rodent and human solid tumors. Thus, the uniquefunctions of our next-generation AAVP particles enable improvedtargeted gene delivery to tumor cells

Bacteriophage-guided cancer immunotherapy

Over the past decades, gene delivery has become a powerful tool for treating many diseases; therefore, a wide range of delivery systems, simply categorised into viral and non-viral vectors, have been developed and applied in both research and clinical translation. In spite of high transduction efficacy, viral vectors encounter a number of limitations regarding their native entities. A hybrid bacteriophage vector has been successfully developed by our Phage Virotherapy group by integrating AAV elements into the filamentous bacteriophage genome. In doing so, the new vector, named adeno-associated phagemid-based vector (PAAV), is able to overcome limitations associated with AAV native tropism by using advantages of bacteriophages that lack native tropism for mammalian cells and tissues. PAAV vector has been applied in a few research areas including cancer therapy. To systemically target solid tumours, the vector is displayed cyclic RGD4C (CDCRGDCFC) ligands on the pIII capsids allowing it to specifically target the αVβ3 integrin receptors overexpressed on tumour cell surface and tumour blood vessels. In this study, PAAV vector has been further refined to display the endosomal escape peptide (H5WYG) on the recombinant pVIII capsids to enhance endosomal escape and transgene expression. The PAAV displaying H5WYG peptide showed great buffering capacity at mild acidic pH relating to the situation that occurs during endosomal maturation and subsequently enhanced luciferase reporter gene expression in numerous human cancer cell lines, including lung carcinoma (A549), melanoma (M21) and meduloblastoma (UW228). Importantly, the modified vector remained safe for normal cells. H5WYG peptide facilitated endosomal escape through a proton sponge effect mechanism as PAAV-mediated gene expression decreased when applying the vascuolar ATPase inhibitor (bafilomycin A1) during vector transduction. Furthermore, displaying H5WYG peptide on the PAAV capsid augmented the secreted TNFα gene expression resulting in a greater cell death of A549, M21 and UW228 cells. The PAAV gene delivery vector was tested in various applications in this thesis. Firstly, the vector was applied as a delivery tool to express the tumour associated antigens (MUC1 and PSMA) which are common target antigens for CAR T cell therapy. PAAV vector augmented MUC1 and PSMA expression in A549, Suit2 and UW228 cells. Although CAR T cells were not applied here, the strategy based on PAAV vector to enhance antigen expression can make the tumours more visible for CAR T cells and might be useful for CAR T cell therapy in solid tumours. Secondly, PAAV was applied on cancer vaccination in combination with a malaria vaccine. The vector was used to deliver a malarial epitope, Pb9, to present on tumour cell surfaces via restricted H2-Kd MHC class I molecules while adenoviral vector encoding a malaria sequence (ME.TRAP) was used to induce an immune response against Pb9. It is shown here that PAAV vector mediated Pb9 expression in EF43.fgf4 breast tumours. The ME.TRAP vaccine stimulated immune response according to ex vivo investigations of specific effector cell proliferation, activation and function. Pb9-specific tumour cell killing was also found both ex vivo and in vivo. The tumour cell death was mediated through caspase-3 dependent apoptosis. All these findings indicate the efficiency and numerous applications of PAAV-mediated gene delivery for cancer treatment.Open Acces

TROLLOPE: A novel sequence-based stacked approach for the accelerated discovery of linear T-cell epitopes of hepatitis C virus.

Hepatitis C virus (HCV) infection is a concerning health issue that causes chronic liver diseases. Despite many successful therapeutic outcomes, no effective HCV vaccines are currently available. Focusing on T cell activity, the primary effector for HCV clearance, T cell epitopes of HCV (TCE-HCV) are considered promising elements to accelerate HCV vaccine efficacy. Thus, accurate and rapid identification of TCE-HCVs is recommended to obtain more efficient therapy for chronic HCV infection. In this study, a novel sequence-based stacked approach, termed TROLLOPE, is proposed to accurately identify TCE-HCVs from sequence information. Specifically, we employed 12 different sequence-based feature descriptors from heterogeneous perspectives, such as physicochemical properties, composition-transition-distribution information and composition information. These descriptors were used in cooperation with 12 popular machine learning (ML) algorithms to create 144 base-classifiers. To maximize the utility of these base-classifiers, we used a feature selection strategy to determine a collection of potential base-classifiers and integrated them to develop the meta-classifier. Comprehensive experiments based on both cross-validation and independent tests demonstrated the superior predictive performance of TROLLOPE compared with conventional ML classifiers, with cross-validation and independent test accuracies of 0.745 and 0.747, respectively. Finally, a user-friendly online web server of TROLLOPE (http://pmlabqsar.pythonanywhere.com/TROLLOPE) has been developed to serve research efforts in the large-scale identification of potential TCE-HCVs for follow-up experimental verification

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP

Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer

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