Cancer Terminator Viruses (CTV): A Better Solution for Viral-Based Therapy of Cancer

Abstract

In principle, viral gene therapy holds significant potential for the therapy of solid cancers. However, this promise has not been fully realized and systemic administration of viruses has not proven as successful as envisioned in the clinical arena. Our research is focused on developing the next generation of efficacious viruses to specifically treat both primary cancers and a major cause of cancer lethality, metastatic tumors (that have spread from a primary site of origin to other areas in the body and are responsible for an estimated 90% of cancer deaths). We have generated a chimeric tropism-modified type 5 and 3 adenovirus that selectively replicates in cancer cells and simultaneously produces a secreted anti-cancer toxic cytokine, melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24), referred to as a Cancer Terminator Virus (CTV) (Ad.5/3-CTV). In preclinical animal models, injection into a primary tumor causes selective cell death and therapeutic activity is also observed in non-injected distant tumors, i.e., “bystander anti-tumor activity”. To enhance the impact and therapeutic utility of the CTV, we have pioneered an elegant approach in which viruses are encapsulated in microbubbles allowing “stealth delivery” to tumor cells that when treated with focused ultrasound causes viral release killing tumor cells through viral replication, and producing and secreting MDA-7/IL-24, which stimulates the immune system to attack distant cancers, inhibits tumor angiogenesis and directly promotes apoptosis in distant cancer cells. This strategy is called UTMD (ultrasound-targeted microbubble-destruction). This novel CTV and UTMD approach hold significant promise for the effective therapy of primary and disseminated tumors. This article is protected by copyright. All rights reserved

1. Introduction to viral gene therapy and what attributes would constitute an ideal therapeutic virus

Gene therapy is a process of delivering genetic materials (genes, gene products, oligonucleotides, etc.) into particular cells to gain a therapeutic result by providing a functional copy of a deficient gene or switching off an undesirable function of a specific gene (Stone, 2010; Katare & Aeri, 2010). The main focus of gene therapy initially was inherited genetic disorders; however, gene therapy is now amenable to a diverse array of diseases, including some autosomal dominant or recessive disorders, and X-linked recessive disorders (Stone, 2010). Additionally, gene therapy is pertinent to different forms of cancer, neurodegenerative disorders, inflammatory conditions, vascular disorders and other acquired diseases (Stone, 2010; Strachan, 1999). Gene therapy can be performed ex vivo, or through in vivo approaches. In the ex vivo approach, targeted cells are first isolated from the patient, grown and maintained in culture in vitro, the gene of interest is manipulated by insertion of a new or corrected gene and then implanted back in the host (Herrero et al., 2012; Suhonen et al., 2006). For the in vivo approach, the genetic material is delivered through an appropriate vector into the target tissue, either a viral or non-viral vector (Suhonen et al., 2006).

Diverse Vector Systems for Gene Therapy

Success of gene therapy relies largely on the effectiveness of gene transfer. An ideal vector should have certain characteristics including a high safety profile, an ability to protect the genetic material from degradation and an efficient release mechanism for the genetic material into the target cell (Bhatia et al., 2013).

Viral vector

When a virus is transduced into the body, it first binds to target cells and releases the payload of genetic material into the cells. Several in vitro and in vivo studies have established the gene transfer efficacy of recombinant viruses. To date, numerous viruses have been proposed for preclinical studies including adeno-associated viruses (AAVs), adenoviruses, herpes viruses, lenti-viruses, and others (described below and summarized in Table 1).

Table 1.

Advantages and disadvantages of different gene therapy vectors

Vector	Advantages	Disadvantages
Viral vectors
Adeno-associated Viruses	Low immunogenicity Stable transgene expression	Small transgene insert capacity (~4.8 kb) Complex process of vector production
Adenovirus	Biologically safe Large transgene insert capacity (~38 kb)	High immunogenicity Short term gene expression
Herpes simplex virus	Large transgene insert capacity (~150 kb)	Immunogenicity
Lentivirus	Infect both dividing and nondividing cells Long term stable expression of transgene Low immunogenicity	Insertional mutagenesis
Measles Virus	Better safety profile	Wild type virus is immunosuppressive Many adults are immune
Retrovirus	Infect only dividing cells Stable transgene expression	Risk of insertion Small transgene insert capacity Immunogenicity, low efficiency in vivo
Vaccinia virus	Large insert capacity Better safety profile	High immunogenicity Replication in skin lesion
Vesicular Stomatitis virus	Selective replication competent in tumor cells	High immunogenicity Animal pathogen-safety concern
Non viral vectors
Plasmid/Naked DNA	Easy to engineer Low immunogenicity	Rapid clearance, low transfection efficiency
Cationic liposomes	Large gene carrying capacity Better accumulation of nanoparticle in tumors	Low transfection efficacy and inflammatory toxicity

Adeno-associated virus (AAV)

AAVs are considered fairly safe because of their inherent characteristics including low immunogenicity, long-term gene expression and broad host range (Thomas et al., 2003). This virus integrates stably into the host DNA in a specific site (region) on chromosome 19, which allows its long-term expression in vivo. The key limitation of this vector is inadequate transgene carrying capacity (only up to ~5 kb) and the complex process required for vector production (Lai et al., 2002; Kay et al., 2000). A recent clinical trial using AAV8 vector (exhibited improved liver tropism and enhanced transduction efficiency in animal models) to deliver the hemophilia B transgene (Naldini, 2015). The results are promising, which showed long-term expression of factor IX in most patients with prompt administration of immunosuppressive corticosteroid treatment.

Adenoviruses (Ads)

Adenoviruses are double-stranded DNA viruses that have very high transduction efficiencies, are able to deliver large DNA particles in replicating or quiescent cells and which can be produced commercially in large quantities (Kotterman et al., 2015; Mathis et al., 2005). However, since these viruses do not integrate into the host genome the expression of the delivered transgene is relatively short. Additionally, immunological responses that are either acute or natural can be a limiting step for systemic delivery. However, this vector is considered a suitable vehicle for targeted delivery to the liver, lungs or for localized cancer gene therapy.

Herpes simplex virus (HSV)

HSV is a recent addition to the virus-based gene therapy genre. HSV is a double-stranded DNA virus that is neurotropic in nature, which makes it a potential vehicle for gene delivery to the nervous system and CNS tumors. HSV is capable of carrying very large payloads of transgenes (~150 kb), including a pro-drug-activating gene thymidine kinase that enhances tumor lysis upon intravenous administration of ganciclovir. Additionally, HSV is used to carry immunomodulatory transgenes that enhance the antitumor immune response (talimogene laherparepvec- T-Vec, trade named Imlygic, formerly called OncoVex^GM-CSF) (Hu et al., 2006) and antiangiogenic genes to suppress tumor vasculature (Liu et al., 2006). Currently, modified oncolytic herpes simplex viruses such as TVEC and others, are being tested either as a monotherapy, or in combination with surgery, chemotherapy and radiation therapy predominantly in patients with high-grade glioma.

Lentivirus vector

Lentiviruses are retroviruses that infect nonhuman primates, bovine, equine and humans (Norton & Miller, 2016; Kafri, 2004; Wong et al., 2006). An exclusive feature of lentiviral vectors is their ability to efficiently infect and integrate into both dividing and nondividing cells, which is the rationale for these to be used as gene delivery vectors. Other advantages of this virus include, its ability to carry relatively large transgenes (~8 kb), long-term and stable transgene expression, and low immunogenicity. Because of its strong tropism to neural stem cells this virus has been used efficiently in several animal models of neurological diseases including Alzheimer, Parkinson, and Huntington’s disease and also in spinal injury and other motor neuron diseases (Kafri, 2004; Wong et al., 2006).

Besides the viruses described above, several other viruses including reovirus, measles viruses, poxviruses, simian virus 40 recombinant (SV40r), retroviruses, and vesicular stomatitis viruses are also being evaluated in several preclinical studies.

Non-Viral vector

Nonviral systems for gene delivery include physical and chemical systems such as cationic liposomes and polymers (chemical), or electroporation, gene gun, ultrasound approaches, particle bombardment, and magnetofection (physical). The gene transduction efficiency is poorer than viral delivery systems, however, some of the advantages include low cost production, reduced immunogenicity and no limitation in the size of the transgenic DNA (Tong et al., 2009; Marrero et al., 2014; Shirley et al., 2013; Hirai et al., 1997).

2. MDA-7/IL-24: novel multifunctional cancer therapeutic cytokine

mda-7/IL-24 was cloned in our laboratory using a subtraction hybridization method in human melanoma cells undergoing terminal differentiation after treatment with interferon-beta and mezerein, a PKC-activator (Fisher et al., 1985; Huang et al., 2001). MDA-7/IL-24 contains an IL-10 signature sequence from amino acids 101 to 121, which classifies it as a member of the IL-10 gene family of cytokines (Huang et al., 2001). The mda-7/IL-24 gene consists of 7 exons and 6 introns on chromosome 1q. The mda-7/IL-24 cDNA encodes a 206-amino acid protein with a predicted molecular weight of ~23.8 kDa (Huang et al., 2001; Persaud et al., 2016; Whitaker et al., 2012). Further genetic sequence analysis found a signal peptide (49-aa long) and a cleavage site, which allows the molecule to be cleaved and secreted (Huang et al., 2001; Whitaker et al., 2012; Caudell et al., 2002; Menezes et al., 2014). Structural analysis further revealed the presence of several glycosylation sites and disulfide bonds in MDA-7/IL-24 indicating the possibility of dimerization (Wang et al., 2002; Sauane et al., 2003).

Immune cells, such as myeloid cells and lymphoid cells and monocytes can produce MDA-7/IL-24 in response to treatment with lipopolysaccharides or specific cytokines (Buzas et al., 2011). In response to cytokine-stimulation epithelial cells can secret MDA-7/IL-24, which is established by several in vitro and in vivo studies (Persaud et al., 2016; Whitaker et al., 2012; Buzas et al., 2011). Non-lymphoid cells can also produce MDA-7/IL-24 in response to cytokines secreted by immune cells (Persaud et al., 2016). Additionally, IL-1 can stimulate MDA-7/IL-24 expression in keratinocytes and human colon cells (Andoh et al., 2009). A basal expression of MDA-7/IL-24 at the physiological level is found in melanocytes and the expression gradually decreases as the melanocyte begin to transform to metastatic melanoma (Jiang et al., 1995; Ekmekcioglu et al., 2001; Ellerhorst et al., 2002).

MDA-7/IL-24, when expressed at supraphysiological levels, using an adenovirus-mediated delivery system (Ad.mda-7/IL-24), induces apoptosis and growth suppression in a wide array of human cancer cells, without exerting any considerable adverse effects on their normal counterparts (Menezes et al., 2014; Mhashilkar et al., 2001; Emdad et al., 2009; Fisher, 2005; Fisher et al., 2003; Fisher et al., 2007; Gopalan et al., 2008; Levedeva et al., 2005; Chada et al., 2004; Dash et al., 2010a; Gupta et al., 2006). Mechanistically, MDA-7/IL-24 induces cancer-selective apoptosis through multiple pathways by regulating endoplasmic reticulum (ER) stress markers [such as Bip/GRP78, PKR-like endoplasmic reticulum kinase (PERK), growth arrest and DNA damage inducible genes (GADD)-34, GADD-153/CHOP, PP2A, and XBP-1]; mitochondrial markers (ceramide, BCL-2, BCL-xL, Bax, Bak, Mcl-1, reactive oxygen species, CD95 etc.) (Whitaker et al., 2012; Caudell et al., 2002; Sauane et al., 2003; Dash et al., 2014; Dash et al., 2010a; Gopalan et al., 2008; Gupta et al., 2006; Lebedeva et al., 2007; Lebedeva et al., 2003a; Lebedeva et al., 2002; Lebedeva et al., 2005; Lebedeva et al., 2003b; Menezes et al., 2014; Sarkar et al., 2002; Sauane et al., 2010). In melanoma and ovarian cancer, MDA-7/IL-24 activates an extrinsic apoptosis pathway by regulating Fas–Fas ligands, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and their respective death receptor signaling pathways (Gopalan et al., 2005; Ekmekcioglu et al., 2008). Besides apoptosis induction, MDA-7/IL-24 is also reported to induce autophagy and blocking the autophagic process suppressed cytotoxicity suggesting a toxic version of autophagy (Park et al., 2008; Yacoub et al., 2008b). Additionally, a switch from autophagic to apoptotic processes following mda-7/IL-24 infection of tumor cells has also been described (Bhutia et al., 2011). The tumor inhibitory properties of MDA-7/IL-24 not only involve apoptosis and toxic autophagy, but also involve anti-angiogenesis, and inhibition of invasion, migration and metastasis (Lebedeva et al., 2007; Ramesh et al., 2004; Huo et al., 2013; Ramesh et al., 2003; Nishikawa et al., 2004; Saeki et al., 2002; Wang et al., 2010). MDA-7/IL-24 binds to its cognate receptors IL-20R1/IL-20R2 or IL-22R1/IL-20R2 or IL-20R1/IL-22R1 receptor complexes and induces expression of endogenous MDA-7/IL-24 by stabilizing mda-7/IL-24 mRNA (Dumoutier et al., 2001; Sauane et al., 2003; Wang et al., 2002; Sauane et al., 2008). As a secreted cytokine, MDA-7/IL-24, facilitates immunostimulatory, chemo- and radiosensitizing, antiangiogenic and “bystander” antitumor activities, which allow this cytokine to eradicate primary tumors as well as the distant tumors resembling metastasis (Chada et al., 2004; Dash et al., 2014; Dash et al., 2010a; Emdad et al., 2009; Fisher, 2005; Fisher et al., 2003; Fisher et al., 2007; Gopalan et al., 2008; Gupta et al., 2006; Lebedeva et al., 2007; Menezes et al., 2014; Mhashilkar et al., 2001; Persaud et al., 2016; Sarkar et al., 2007; Sauane et al., 2003; Su et al., 2005a; Su et al., 2003; Whitaker et al., 2012; Yacoub et al., 2008a; Yacoub et al., 2003; Hamed et al., 2013a).

A recent study from our group discovered a novel molecular mechanism of MDA-7/IL-24-mediated cell death in neuroblastoma. Specifically, we found that mda-7/IL-24 induces ATM-mediated activation of H2AX and AIF resulting in caspase-independent apoptosis that appears unique to neuroblastoma cells (Bhoopathi et al., 2016). Another recent study from our group demonstrates that overexpression of mda-7/IL-24, either via Ad. or as a recombinant protein, downregulates miR-221 and upregulates p27 and PUMA, resulting in cancer cell death (Pradhan et al., 2017). This study also identified beclin-1 as a new transcriptional target of miR-221 and showed that mda-7/IL-24 regulates autophagy through miR-221/beclin-1 feedback (Pradhan et al., 2017).

Based on its exceptional and compelling success in pre-clinical studies, a Phase I clinical trial was introduced to determine the therapeutic potential of mda-7/IL-24 integrated into an adenoviral vector (Ad.mda-7; INGN 241) in patients with advanced carcinomas and melanomas (Dent et al., 2010a; Dent et al., 2010b; Eager et al., 2008; Inoue et al., 2006; Menezes et al., 2014; Tong et al., 2005). Intratumoral injection of INGN 241(Ad.mda-7) resulted in ~44% clinical response in injected lesions and the therapy was overall well–tolerated with some mild side effects including pain and erythema in the injection site (Tong et al., 2005). The positive results in the preclinical studies and a Phase I/II clinical trial with repeated applications of MDA-7/IL-24 (INGN 241) accentuates the usefulness of this multifunctional cytokine as an efficient anti-cancer agent (Tong et al., 2005; Dent et al., 2010a; Dent et al., 2010b; Eager et al., 2008; Fisher, 2005; Inoue et al., 2006; Menezes et al., 2014).

3. Cancer-selective gene expression: targeting viral replication uniquely in cancer

The purpose of cancer gene therapy is to deliver therapeutic genes in a cancer-specific manner, which will result in targeted expression only or preferentially in cancer cells while sparing normal cells, thereby exerting minimal toxic side effects (Hood et al., 2002; Godbey & Atala, 2003; Bhatia et al., 2013). Targeted gene delivery can be accomplished through various approaches including attachment of ligands to gene delivery complexes, which will home in on cancer-specific receptors or transmembrane proteins (Hood et al., 2002; Godbey & Atala, 2003). Additionally, promoter-based gene targeting is frequently used as a method for targeted delivery of therapeutic genes (Das et al., 2012; Dash et al., 2011b). Several promoters that are selectively active in cancers as compared to their normal counterparts have been identified and are being used for guiding reporter gene expression and selective gene delivery specifically in cancers. These include the survivin promoter (Bao et al., 2002; Lu et al., 2005), the human telomerase reverse transcriptase (hTERT) promoter (Wirth et al., 2005; Kishimoto et al., 2006), the Astrocyte elevated gene (AEG)-1 promoter (Bhatnagar et al., 2014; Emdad et al., 2013; Kang et al., 2005), and a truncated version of Cysteine-rich protein 61 (tCCN1) gene promoter (Dash et al., 2010c; Sarkar et al., 2015). Survivin is the smallest member of the inhibitor of apoptosis (IAP) family and its selective-overexpression is found in a variety of cancer cells (Zhu et al., 2005; Chen et al., 2006). Several investigators have confirmed the ability of the human survivin promoter to specifically target different types of cancers (Zhu et al., 2005; Chen et al., 2006). Recently, the survivin promoter was used in oncolytic viruses to target therapeutic agents in a cancer-specific manner (Li et al., 2006; Van Houdt et al., 2006; Zhu et al., 2006; Ulasov et al., 2007). The hTERT promoter is robustly active in human cancer cells, but not in normal differentiated human cells (Wirth et al., 2005).

Gene promoters, which exhibit enhanced activity uniquely in a wide array of cancer cells, represent the holy-grail of gene therapists. One such promoter is the minimal active promoter of a specific rodent transformation-associated gene, i.e., the Progression Elevated Gene-3 (PEG-3) promoter. The PEG-3 gene was first identified as a gene in rat embryonic fibroblast cells upregulated upon malignant transformation by diverse oncogenes (Su et al., 1997). Rodent PEG-3 is a C-terminal truncated mutant form of the GADD-34 gene, and functions as a dominant-negative inhibitor of the apoptosis inducing function of GADD-34 (Hollander et al., 2003; Su et al., 1997; Su et al., 2005a). Ectopic expression of PEG-3, in human cancer cells, by a replication-incompetent adenovirus (Ad.PEG-3), induces genomic instability, and increases invasion, angiogenesis, and metastasis (Emdad et al., 2005). Since no human counterpart of this gene exists there is a negligible chance for the rodent PEG-3 promoter to recombine and chromosomally insert into the human genome by homologous recombination. Robust activity of the PEG-3 promoter was observed in a number of human cancer cell lines including breast, prostate, glioma and others with minimal activity in their normal counterparts (Su et al., 2005b). AP-1 and PEA-3, two transcription factors, often upregulated in cancer cells are important for the cancer-selective activity of PEG-3 promoter. Adenovirus expressing GFP or luciferase under the control of the PEG-Prom was used to infect a series of normal and cancer cells, which documented enhanced GFP, or luciferase expression in cancer cells, with negligible expression in normal cells (Figure 1). Further research using the reporter genes under transcriptional control of the PEG-3 promoter documented successful detection of micrometastatic disease in in vivo models of melanoma and breast cancer by both bioluminescent and radionuclide-based molecular-genetic imaging approaches (Bhang et al., 2011).

Figure 1.

PEG-prom drives expression of GFP selectively in cancer cells, but not in normal cells. Upper panel, Schematic representation of utility of PEG promoter to deliver reporter genes (green fluorescence protein (GFP) or luciferase (luc)) using replication incompetent Ads. Lower panel, PEG-Prom drives the expression of GFP only in cancer cells but not in normal cells. A. Normal primary human prostate epithelial cells (HuPEC) and human prostate carcinoma cells (Du-145, PC-3 and LNCaP); B. Immortal normal prostate epithelial cells (P69) and progressed tumorigenic P69-derived cells (M2182) and progressed P69-derived metastatic cells (M12); C. Normal primary human breast epithelial cells (HMEC) and human breast carcinoma cells (MCF7, T47D, MDA-MB-157, MDA-MB-231 and MDA-MB-453). The indicated cells were infected with either Ad.CMV-GFP or Ad.PEG-GFP at a moi of 100 pfu per cell, and GFP expression was analyzed by an immunofluorescence microscope at 2 day postinfection. (Adapted from Su et al, PNAS; 2005)

4. It takes more than viral replication alone to treat cancer, the Cancer Terminator Virus (CTV)

Gene therapy usually involves delivery of a cancer-selective apoptosis-inducing gene or suicide gene into tumor cells with replication incompetent Ads frequently used to transfer these genes. However, multiple administrations of Ads are predominantly required to achieve a significant anticancer response, which conversely stimulates immune responses resulting in viral neutralization and clearance. Considering these disadvantages, conditionally replication competent Ads (CRCAs) are currently being evaluated because of their enhanced efficacy in killing cancer cells through replication and cytolysis (Heise & Kirn, 2000; Kirn & McCormick, 1996). In this context, we hypothesized that inducing cancer cell lysis (through viral replication) combined with apoptosis-induction with a secreted cytokine would foster a potent cancer killing effect when combined into a single therapeutic reagent. Additionally, using a cancer-specific promoter that could precisely induce viral replication only in cancer cells, but not in normal cells, would restrict virus replication and transgene release only in cancer cells. Using this strategy, we created a first-generation CTV in a type 5 serotype adenoviral background, in which viral replication is controlled by a minimal active region of the PEG-3 promoter and as discussed above, to enhance the therapeutic potential of these CTVs, we further modified these Ads to produce mda-7/IL-24 (Ad.PEG-E1A-mda-7 or Ad.CTV) under the control of a cytomegalovirus (CMV) promoter (Sarkar et al., 2006; Sarkar et al., 2008; Sarkar et al., 2005a; Sarkar et al., 2005b).

CTV construction

To generate CTVs, two shuttle vectors, pE1.2 and pE3.1, were used (Sarkar et al., 2005a) with the PEG-Prom driving the E1A gene (PEG-Prom E1A) and the CMV promoter controlling the therapeutic gene (mda-7/IL-24) were inserted into the multiple cloning sites of the vectors pE1.2 and pE3.1, respectively (Figure 2). These transgene cassettes were inserted into the Ad backbone in a four-fragment ligation exploiting the presence of unique restriction enzyme sites in the shuttle vectors and in the Ad plasmid. The cosmid DNA was amplified by standard large-scale preparation using a CsCl gradient, digested with PacI restriction enzyme, and transfected into human embryonic kidney-293 cells for in vivo recombination. The Ad was purified and viral titer was determined by measuring absorbance at 260 nm and using BD AdenoX rapid titer kit (BD Biosciences) (Sarkar et al., 2005a).

Figure 2.

Constructing a tropism-modified adenovirus (Ad.5/3) carrying the mda-7/IL-24 gene. The genome of Ad.5/3.mda-7 was generated by homologous recombination between the linearized plasmid pShCMV.mda-7 and 8st81-digested genomic DNA of Ad.5/3-Luc, and kanamycin selection resulted in the pAd.5/3-mda-7 genome, where the CMV promoter in place of the early viral El region drives mda-7/IL-24 expression. This plasmid was digested with Pad to release viral ITRs and transfected in A549 cells to rescue the Ad.5/3-mda-7. (Das et al., Adv. Cancer Res., 2012)

We evaluated the therapeutic efficacy of CTV (Ad.PEG-E1A-mda-7) in various cancer models including breast, prostate, pancreatic, renal, melanoma, glioma and neuroblastoma (Sarkar et al., 2005a; Bhoopathi et al., 2016; Sarkar et al., 2008; Hamed et al., 2013a; Greco et al., 2010; Sarkar et al., 2014; Azab et al., 2014; Hamed et al., 2013b). Initial studies were performed using the breast cancer model. Ad.CTV induced viral replication and generated significant MDA-7/IL-24 protein production only in breast cancer cell lines, but not in normal immortal mammary epithelial cells. Supporting the in vitro observations, in vivo assays established that Ad.CTV was more efficacious in eradicating not only the primary tumor but also distant tumors as compared to replication incompetent Ad.null (empty vector) and Ad.mda-7, and replication competent Ads lacking the apoptosis-inducing mda-7/IL-24 transgene (Ad.PEG-E1A) (Sarkar et al., 2005a). The positive results of CTV in breast cancer provided an impetus for testing the CTVs in other human cancer models to evaluate potential for treating primary tumors as well as distant metastases. Next, we explored the efficacy of CTV in therapy-resistant prostate cancer. Similar to the breast cancer study, tumors were established in both flanks of the mice and therapeutic Ads were injected only into the left flank tumors. This experiment demonstrated that intratumoral injection of Ad.CTV could completely eradicate primary and distant tumors (comparable to a metastasis) in a therapy-resistance prostate cancer model (Greco et al., 2010; Sarkar et al., 2015).

The efficacy of Ad.CTV was also determined in human melanoma (Sarkar et al., 2008), using a series of melanoma cells and in normal immortal human melanocytes. The functionality of all Ads, was confirmed in a series of in vitro and in vivo studies. As was found in breast and prostate cancer, injecting Ad.CTV completely eliminated not only the primary-treated tumors but also distant non-treated tumors in nude mice, offering a potent antitumor response in this immune compromised animal model (Sarkar et al., 2008).

Serotype 5 Ads use Coxsackie-Adenovirus Receptors (CARs) on the cell surface as the primary means of infecting cell (Glasgow et al., 2004). The efficiency of infection with a type 5 Ad depends on the level of CAR expression (Volk et al., 2003; Glasgow et al., 2004). Dash et al. demonstrated that Ad.5-CTV is capable of efficiently infecting high-CAR cells (such as DU-145) resulting in expression of high levels of mda-7/IL-24, whereas infection is restricted and expression of MDA-7/IL-24 protein is minimal in low-CAR cells, such as PC-3 (Dash et al., 2011a; Dash et al., 2010b). To forestall this problem in infectivity of low-CAR cells, we employed “tropism modification” techniques in which virus capsid proteins that normally associate with CAR are modified, allowing both CAR-dependent and CAR-independent infectivity of tumor cells (Tsuruta et al., 2007; Park et al., 2011; Hamed et al., 2010; Dash et al., 2010b; Dash et al., 2011b; Azab et al., 2012; Azab et al., 2014). Studies using various tumor cell types have shown that inclusion of type 3 Ad sequences within the Ad type 5 virus knob (Ad.5/3 recombinant virus) fosters viral infectivity in tumor cells displaying reduced or no CAR expression (Figure 2) (Azab et al., 2012; Azab et al., 2014; Dash et al., 2010b; Hamed et al., 2010; Park et al., 2011). Additionally, Ad.5/3 also retains high infectivity in CAR-expressing tumor cells showing equal efficacy when compared with wild-type Ad.5, thereby providing an expanded range of utility for Ad.5/3, in both low- and high-CAR-expressing tumor cells (Dash et al., 2010b; Eulitt et al., 2010). This second-generation CTV in a tropism-modified Ad.5/3 virus, Ad.5/3-CTV has shown profound primary and ‘bystander’ antitumor activity in additional tumor models including prostate cancer, glioblastoma multiforme, pancreatic cancer, renal cancer and neuroblastoma (Azab et al., 2014; Bhoopathi et al., 2016; Hamed et al., 2013a; Hamed et al., 2013b; Sarkar et al., 2014).

Cancer is a complex and extremely heterogeneous disease and a single-arm treatment is not always sufficient to cure this deadly disease. In a quest to further enhance the therapeutic benefit of Ad.5/3-CTV, we employed combinatorial therapeutic approach with the CTV. Ad.5/3-CTV in combination with a ROS inducing agent (perillyl alcohol; POH) exerts a significant antitumor ‘bystander’ effect in vivo in therapy-resistant pancreatic cancer (Sarkar et al., 2014). In a study with human renal cancer, Hamed et al. demonstrated that histone deacetylase inhibitors enhanced MDA-7/IL-24-mediated toxicity (delivered with Ad.5/3-CTV) and tumor-specific adenoviral delivery (Hamed et al., 2013a).

5. A path to systemic delivery of viruses: Ultrasound-Targeted Microbubble-Destruction (UTMD)

An effective cancer therapy relies on the specific delivery of the therapeutic to diseased tissue. Ultrasound (US) contrast agents (microbubbles) have recently come to light as a potential agent for effective delivery of molecules to target tissues (Howard et al., 2006; Larina et al., 2005; Lawrie et al., 2000; Ng & Liu, 2002). Microbubbles consist of a gas core stabilized by a surrounding shell made from materials such as phospholipids, biocompatible polymers, or proteins. Microbubbles can be injected via the intravenous route and it can survive in the bloodstream for several minutes (Howard et al., 2006; Goldberg et al., 1994). The ideal diameter for microbubbles is between 2.5- and 4-μm which prevents entrapment within the pulmonary capillary bed (ranging from 5- to 8-μm in diameter), however, large enough to protect the viral vectors from the surrounding microenvironment. Ultrasound-targeted microbubble-destruction (UTMD) allows focal release of trapped-materials and creates small shock waves that enhance cellular permeability (Pitt et al., 2004). The protective properties of microbubbles protects the encapsulated agent from rapid degradation by the immune system. This feature allows repeated intravenous administration, which is particularly important in cancer therapy to reach inaccessible tumors because the microbubbles may also limit the amount of inflammatory response of the delivered agents. Targeted gene delivery using microbubbles was first evaluated by Howard et al. in both in vitro and in vivo systems (Howard et al., 2006). Systemic delivery of complement pretreated Ad.GFP microbubbles resulted in ultrasound-guided transduction in the targeted tissue only, with no uptake in liver, lung or heart.

Greco et al. confirmed the ability of the ultrasound contrast agent to deliver viruses efficiently to specific target sites in vivo using prostate tumor xenografts (Greco et al., 2010). The xenografts were established in both flanks of athymic nude mice by injecting DU-145, an aggressive human prostate cancer cell line. The tumor-bearing nude mice were then intravenously injected with contrast agent that was reconstituted with Ad.GFP. A portable SonoSite Micro-Maxx US (ultrasound) platform (SonoSite, Inc., Bothell, WA) equipped with a L25 linear array transducer set at 0.7 Mechanical Index, 1.8 MPa for 10 min, was used to sonoporate the tumor implanted only on the right side. GFP expression was checked by Western blotting in lysates from different organs and tumors from both sites (left and right flanks). Interestingly, GFP expression was seen only in the tumors that were exposed to ultrasound (Greco et al., 2010), confirming its target specificity. This study was further expanded and the feasibility of systemic delivery of Ad.CTV by microbubbles was evaluated. Tumors were established in both flanks with therapy-resistant prostate cancer cells and Ad.CTV treated with complement was injected via tail vein with 100 μL of Targestar-P contrast agent (Greco et al., 2010). The tumors were sonoporated only on the left side and the ultrasound-guided delivery completely eradicated not only the-targeted tumor, but also the one established in the opposite flank confirming a “bystander” antitumor effect (Greco et al., 2010). In a recent study, we also confirmed the efficacy of the UTMD approach (Figure 3) in a spontaneous model of prostate cancer, the Hi-Myc mouse (Dash et al., 2011a).

Figure 3.

Delivery of Cancer Terminator Viruses (CTVs) systemically following complexing with microbubbles (MB) coupled with ultrasound-targeted MB destruction, the UTMD approach. Complexes of CTVs with MBs are delivered intravenously (Box A, Ad incorporated in the lipid shell of MBs), which are released at the primary tumor site by the application of ultrasound (Box B, sonoporation of MBs in the tumor with an ultrasound probe). After intracellular entry, the CTVs replicate selectively in tumor cells, resulting in robust production of mda-7/IL-24 that when translated into MDA-7/IL-24 protein cause ER stress and “unfolded protein stress response” and cancer cell death. MDA-7/IL-24 is subsequently released into the circulatory system and due to virtue of its “bystander activity” (Box C, binding of MDA-7/IL-24 with IL-20R1/IL-20R2 or IL-20R1/IL-22R1 and promoting intracellular signaling leading to autocrine production of MDA-7/IL-24), which would be anticipated to induce tumor-specific apoptosis of primary and distant tumors, antiangiogenic effects in primary and distant tumor vasculatures, and immune modulatory effects targeting tumors for immune destruction. It is believed to be the “summation” of these multifaceted antitumor properties of MDA-7/IL-24 that promotes selective destruction of both primary and distant tumors. (Adapted from Das et al., Adv. Cancer Res., 2012).

6. Summary and Future Perspectives

Although significant advances have been made in early detection and treatment of localized cancers, metastatic cancer has become a veritable death sentence because no single or combinatorial treatment approach to date has demonstrated potential in decreasing morbidity or establishing a cure. Most cancer-related death is attributed to disseminated tumor growth or invasion into normal tissue thereby impairing vital organ(s) function. Several challenges exist that compromise the effectiveness of a therapeutic to function efficiently including nonspecific delivery, failure to reach the metastatic site, destruction by the immune system, and nonspecific trapping in the liver or other organ sites not harboring metastases. In this regard, our CTV approach is exciting because it provides cancer-specific replication based on expression of the PEG-Prom of the tCCN1-Prom and selective local and systemic killing of cancer cells by means of the cancer-specific apoptosis- and toxic autophagy-inducing cytokine mda-7/IL-24 (Das et al., 2012; Menezes et al., 2014; Sarkar et al., 2015). Our laboratory and others are finding solutions to efficiently deliver viruses, including the CTV, using targeted microbubbles by the UTMD approach. This would not only shield Ad vectors from neutralizing antibodies following intravenous administration, but also avoid its trapping in the liver and other non-specific tissues, thus maximizing the biological outcome. It is expected that, with these targeted approaches, we will be able to eliminate both primary and distant metastatic tumors, with minimal toxicity to normal organs, leading to a potential “cure” in patients with diverse solid cancers.