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

Abstract

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.

Doxorubicin Improves Cancer Cell Targeting by Filamentous Phage Gene Delivery Vectors

“…The vector is a hybrid [ 5 , 6 ] in which the phage capsid incorporates hybrid genomes of single-stranded human adeno-associated virus (AAV-2) DNA and the fd phage single-stranded genome. Moreover, to allow entry into cancer cells, we used the Nobel Prize-awarded phage display technology to engineer a phage capsid displaying the double-cyclic RGD4C ligand that binds the α v β 3 integrin cell surface receptor overexpressed on tumor cells and tumor vasculature in most cancers but is barely detectable in healthy tissues [ 7 , 8 , 9 ]. Upon cell entry of the hybrid RGD4C/AAV-phage (RGD4C/AAVP), the AAV transgene cassette is released, resulting in gene expression in tumors from a cytomegalovirus, CMV , promoter [ 5 , 6 ].…”

“…Upon cell entry of the hybrid RGD4C/AAV-phage (RGD4C/AAVP), the AAV transgene cassette is released, resulting in gene expression in tumors from a cytomegalovirus, CMV , promoter [ 5 , 6 ]. We have reported that this vector targets numerous cancer models in vivo upon intravenous delivery [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. A study in pet dogs with spontaneous cancers proved the safety of repeated vector dosing, resulting in complete tumor eradication in a few dogs with aggressive cancers [ 20 ].…”

“…All these studies showed dramatic enhancement of gene transfers. Furthermore, we recently reported multifunctional RGD4C/AAVP vectors combining a multiple fusion peptide display on various coated proteins, which confer the ability of RGD4C/AAVP to escape from the endosome-lysosomal degradative pathway, alter the capsid charge and increase the vector stability [ 9 ]. Moreover, to enhance therapeutic gene transcription from the vector in the nucleus, we replaced the CMV promoter with a tumor-activated and chemotherapy-induced promoter of the glucose-regulated protein Grp78 [ 8 , 15 ].…”

In Vitro Assays: Friends or Foes of Cell-Penetrating Peptides

“…In a separate study, a bacteriophage displaying a CPP was used as a shuttle to deliver an adeno-associated virus (AAV) gene. AAV, cloned within the single-stranded genome of the bacteriophage [ 114 , 115 , 116 , 117 ], contained a mammalian transgene cassette encoding the cytomegalovirus (CMV) promoter-driven transgene, such as GFP and luciferase, as well as a polyA region that was flanked by inverted terminal repeats (ITRs) from AAV2 genome [ 116 , 117 ]. In the reported studies, Arg-Gly-Asp (RGD-4C) peptide targeting αv integrin receptors on vascular endothelial cells was displayed at N-terminus of phage PIII protein to facilitate AAV-phage cell entry.…”

The hybrid AAVP tool gets an upgrade

“…The hybrid adeno-associated virus (AAV)/phage (AAVP) has been successfully used in several in vivo models to target solid tumors with greater specificity, facilitating positron emission tomography (PET) imaging and suppressing tumor growth by disrupting tumor angiogenesis. In PNAS, Suwan et al (2) increase the versatility of AAVP by designing 2 phages capable of avoiding nonspecific adsorption and escaping endosomal-mediated degradation, significantly improving the transduction outcome. This work not only will have important implications for phage-mediated cancer gene therapy but may also facilitate similar optimizations in other gene transfer vectors.…”