Gene therapy requires safe and efficient delivery systems that can target a specific cell or tissue. Bacteriophage-mediated gene therapy is now possible, due to their adaptability. Transgene delivery typically employs eukaryotic viruses, as they transduce cells with high efficiency. However, their native tropism for mammalian cells severely limits their use, as it leads to unwanted side effects. Bacteriophages represent a safer option as they can be re-engineered to transduce specific eukaryotic cells or tissues.
The double cyclic RGD peptide (CDCRGDCFC, RGD4C) that targets αvβ3 integrin, a marker of cancer vasculature and tumor tissue, was successfully exploited as a targeting moiety (by the research group of Pasqualini and Arap) to build up a hybrid adeno-associated virus (AAV)/M13 phage (P) system termed AAVP. This vector carries an eukaryotic AAV cassette in the intergenomic region of the RGD4C phage, which is packaged within the viral capsid, together with phage DNA [
52]. An AAVP vector engineered to include the
GFP gene under the
CMV promoter was shown to be efficiently internalized by cancer cells. When systemically administered to human Kaposi sarcoma xenografted mice, RGD4C-AAVP accumulated in tumor vasculature by a ligand-directed mechanism. Similarly, an AAVP modified with a luciferase reporter gene for bioluminescence imaging showed specific accumulation in tumors and no accumulation in normal organs. To potentially translate this system into the clinic, a Herpes simplex virus-1/thymidine kinase (
HSVtk) expression cassette was introduced to be exploited (i) as a suicide gene upon administration of ganciclovir (GCV) and (ii) as a positron emission tomography (PET) imaging platform, in the presence of the radiolabeled nucleoside analog 20-[
18F]-fluoro-20-deoxy-1-β-D-arabino-furanosyl-5-ethyluracil ([
18F]FEAU). With this system, tumor response to GCV treatment was monitored across cohorts of human-cancer-cell-xenotransplanted mice showing reduction of tumor volume and destruction of tumor vasculature at histopathological analysis. In another work by the same group, a similar approach was used to treat and image nude rats bearing human sarcoma tumors with the purpose of creating a preclinical model to predict drug response, when the overall tumor-size change does not represent a good read-out of therapy outcome [
53]. An implementation of these AAVPs was designed by including an alternative promoter and a different targeting moiety. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum protein chaperone, whose expression is induced in a variety of tumors, in conditions of glucose deprivation and hypoxia. Therefore, the
GRP78 promoter, besides being specifically activated in cancer cells, is expected to improve gene expression compared to the
CMV promoter, also because it is not silenced in eukaryotic cells [
54]. Of note, upon a single systemic administration of either AAVP-RGD4C/
GRP78-
HSVtk or AAVP-RGD4C/
CMV-
HSVtk in L9 gliosarcoma-xenotransplanted nude mice, followed by GCV treatment, tumors showed similar responses. However, repeated administration of GCV in large tumors showed efficacy only in AAVP-RGD4C/
GRP78-
HSVtk treated mice, further confirming transcriptional delivery of the transgene in a stress-permeated setting [
54]. A number of other promoters were assessed in vitro and in vivo to optimize the delivery efficiency of suicide genes, suggesting that some of them—namely, carcinoembryonic antigen (
CEA) [
55,
56], epithelial cell adhesion molecule (
EpCAM) [
57], human telomerase reverse transcriptase (
hTERT) [
58]—might as well be suitably included in AAVP for cancer targeting. In several cancer settings, the GRP78 protein is not only overexpressed, but is also relocated from the cytoplasm to the cell surface, becoming suitable for ligand-targeted approaches. For example, moderate to strong levels of GRP78 are seen in inflammatory breast cancer (IBC), an aggressive disease characterized by difficult-to-detect micrometastasis at presentation. AAVPs displaying the GRP78-targeting peptide motif WIFPWIQL were employed to image IBC tumors in vivo with NIR-conjugated dyes. The positive outcome of these preliminary studies was translated into a theranostic platform to deliver the
HSVtk suicide gene via a GRP78-targeted AAVP carrying the
GRP78 promoter. Results demonstrated improved imaging and decreased tumor size upon GCV administration, as a result of ligand-directed accumulation of the processed drug within the tumor [
59]. A schematic representation of these theranostic AAVP vectors is provided in .
Well-known, biologically active peptides are also a source of targeting ligands that do not require a priori screening. For example, octreotide (OCT), a synthetic analog of somatostatin, was exposed on AAVP for the delivery of tumor necrosis factor (TNF) to somatostatin receptor type-2 (SSTR-2) expressing pancreatic neuroendocrine tumors. Mid- and long-term systemic injection of OCT-AAVP-TNF in transgenic mice with pancreatic insulinomas induced tumor response, as determined by the decreased insulin secretion, total choline levels, and tumor sizes [
60]. Transduction in AAVP systems remains a challenge due to physiological limitations in viral delivery and processing. In particular, both the ECM and the proteasome system pose a barrier to AAVP efficacy. Hence, ECM depletion by specific enzymes (collagenase and hyaluronidase) and proteasome inhibition by peptide aldehyde inhibitors (
N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal (MG132) and
N-acetyl-L-leucyl-L-leucyl-leucyl-L-norleucinal (LLnL)) were shown to improve both viral uptake and transgene expression [
61,
62].
As a peculiar yet promising application in phage-derived therapeutics, RNA nanotechnology offers the possibility of manipulating the DNA packaging unit of bacteriophage Φ29, to make chimeric RNAs that form dimers via the right and left-hand loop interactions. Within the bacteriophage, packaging RNA (pRNA) dimers serve as building blocks for bottom-up assembly of pRNA hexamers that compact its DNA in the procapsid. pRNA dimers are 25 nm in size and sequence replacement of their helical domain does not alter dimer formations. Thus, redesign of monomers to carry siRNAs for gene downregulation and aptamers/ribozymes for cell targeting is possible and is shown to produce dimers that mediate gene silencing in different cell types [
66]. As an example, specific dimers of pRNA-folate and pRNA-siRNA survivin or metallothionein II were shown to degrade the corresponding mRNAs in ovarian cancer cells much more efficiently than siRNAs alone [
67]. Furthermore, changing these assemblies to build modules of up to 12 monomers with different geometries was demonstrated to lead to stable RNA nanoparticles that can also be systemically administered [
64]. Recently it was also shown that the 3-way core junction of Φ29 pRNA could be functionalized to carry two siRNAs and a targeting RNA aptamer. Specifically, Her2+ breast cancer cells were targeted with an RNA nanoparticle carrying two siRNAs for
MED1 (a well-known transcriptional co-activator of Her2) and an RNA aptamer targeting Her2. pRNA-Her2apt-siMED1 effectively silenced
MED1 in breast cancer cells, blocked tumor growth, and completely eradicated lung metastases [
65] (b). These results clearly point towards future application of these RNA-based nanoparticles in the clinic.