Actin-resistant DNAse I Expression From Oncolytic Adenovirus Enadenotucirev Enhances Its Intratumoral Spread and Reduces Tumor Growth.

Spread of oncolytic viruses through tumor tissue is essential to effective virotherapy. Interstitial matrix is thought to be a significant barrier to virus particle convection between "islands" of tumor cells. One way to address this is to encode matrix-degrading enzymes within oncolytic viruses, for secretion from infected cells. To test the hypothesis that extracellular DNA provides an important barrier, we assessed the ability of DNase to promote virus spread. Nonreplicating Ad5 vectors expressing actin-resistant DNase (aDNAse I), proteinase K (PK), hyaluronidase (rhPH20), and chondroitinase ABC (CABC) were injected into established DLD human colorectal adenocarcinoma xenografts, transcomplemented with a replicating Ad5 virus. Each enzyme improved oncolysis by the replicating adenovirus, with no evidence of tumor cells being shed into the bloodstream. aDNAse I and rhPH20 hyaluronidase were then cloned into conditionally-replicating group B adenovirus, Enadenotucirev (EnAd). EnAd encoding each enzyme showed significantly better antitumor efficacy than the parental virus, with the aDNAse I-expressing virus showing improved spread. Both DNase and hyaluronidase activity was still measurable 32 days postinfection. This is the first time that extracellular DNA has been implicated as a barrier for interstitial virus spread, and suggests that oncolytic viruses expressing aDNAse I may be promising candidates for clinical translation.

Oncolytic viruses: finally delivering.

Oncolytic viruses can be found at the confluence of virology, genetic engineering and pharmacology where versatile platforms for molecularly targeted anticancer agents can be designed and optimised. Oncolytic viruses offer several important advantages over traditional approaches, including the following. (1) Amplification of the active agent (infectious virus particles) within the tumour. This avoids unnecessary exposure to normal tissues experienced during delivery of traditional stoichiometric chemotherapy and maximises the therapeutic index. (2) The active cell-killing mechanisms, often independent of programmed death mechanisms, should decrease the emergence of acquired drug resistance. (3) Lytic death of cancer cells provides a pro-inflammatory microenvironment and the potential for induction of an anticancer vaccine response. (4) Tumour-selective expression and secretion of encoded anticancer biologics, providing a new realm of potent and cost-effective-targeted therapeutics.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. "Stealthing" of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

Quantification of siRNA using competitive qPCR.

We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1-50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2'-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.

The role of cancer metabolism in defining the success of oncolytic viro-immunotherapy.

Oncolytic viruses infect, replicate in, and kill cancer cells selectively without harming normal cells. The rapidly expanding clinical development of oncolytic virotherapy is an exciting interdisciplinary field that provides insights into virology, oncology, and immunotherapy. Recent years have seen greater focus on rational design of cancer-selective viruses together with strategies to exploit their immunostimulatory capabilities, ultimately to develop powerful oncolytic cancer vaccines. However, despite great interest in the field, many important experiments are still conducted under optimum conditions in vitro, with many nutrients present in excess and with cellular stress kept to a minimum. Whilst this provides a convenient platform for cell culture, it bears little relation to the typical conditions found within a tumour in vivo, where cells are often subject to a range of metabolic and environmental stresses. Viral infection and cancer will both lead to production of metabolites that are also not present in media in vitro. Understanding how oncolytic viruses interact with cells exposed to more representative metabolic conditions in vitro represents an under-explored area of study that could provide valuable insight into the intelligent design of superior oncolytic viruses and help bridge the gap between bench and bedside. This review summarises the major metabolic pathways altered in cancer cells, during viral infection and highlights possible targets for future studies.

P-selectin dependent targeting to inflamed endothelium of recombinant P-selectin glycoprotein ligand-1 immunoglobulin chimera-coated poly[N-(2-hydroxypropyl) methacrylamide]-DNA polyplexes in vivo visualised by intravital microscopy.

BACKGROUND: Developing vectors that target specifically to disease sites after systemic injection is an important goal in gene therapy research. METHODS: We prepared fluorescent DNA polyplexes (< or =150 nm in diameter) comprising plasmid DNA condensed with poly(L-lysine) and coated with a multivalent reactive copolymer based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). These polyplexes were then surface modified with a recombinant P-selectin glycoprotein ligand-1 immunoglobulin chimera (rPSGL-Ig) previously investigated as a selectin antagonist in clinical studies. RESULTS: Five minutes after jugular vein injection of these polyplexes, fluorescence accumulation in inflamed cremasteric venules of C57BL6 mice was more than eight-fold higher than that observed after injection of Fc-blocked control polyplexes. Fluorescence above background was not observed in P-selectin deficient mice, confirming the specificity for P-selectin in this model. CONCLUSIONS: These data provide encouragement for the further development of rPSGL-Ig-coated polyplexes as potential nonviral vectors for targeted gene therapy in inflammatory conditions, such as ischaemia reperfusion injury, unstable atherosclerotic plaques and myocarditis. This approach may also be transferable to the use of other targeting ligands whose cognate partner is specifically upregulated on the vascular endothelium in individual pathological situations.

In vitro evaluation of a 'stealth' adenoviral vector for targeted gene delivery to adult mammalian neurones.

BACKGROUND: Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells. METHODS: In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat. RESULTS: sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with tagged viral DNA within transfected DRGN, showing that sAd5 did not uncoat and viral DNA did not traffic to the nucleus. Noncoated Ad5 transduced non-neuronal DRG cells more efficiently than DRGN, whereas A9(CAR) cells were more significantly transduced than any other cell type. Retargeting of the sAd5.gfp with either fibroblast growth factor-2 or nerve growth factor (NGF) enhanced internalisation by DRGN into endocytic vesicles allowing uncoating and thus GFP expression. Retargeting with NGF resulted in significantly higher numbers of DRGN expressing GFP than non-neuronal DRG cells. CONCLUSIONS: These findings indicate that DRGN pinocytose atropic genetic particles at higher levels than non-neuronal DRG cells and the environment of pinocytic vesicles is not conducive to sAd5 uncoating and capsid dismantling, requiring reformulation of sAd5 with either a neurone specific ligand or a self-dismantling coat to target sAd5 transgene expression to neurones.

Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples.

BACKGROUND: Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid "on-target off-tumour" toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. METHOD: A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor ß (FRß) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. RESULTS: T cells activated by the CD206- and FRß-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding - the first of its kind - demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. CONCLUSIONS: This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.

Coating of adeno-associated virus with reactive polymers can ablate virus tropism, enable retargeting and provide resistance to neutralising antisera.

BACKGROUND: Copolymers based on poly-[N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8. METHODS: Three strategies for modifying transductional targeting of AAV were employed. The first involved direct reaction of AAV5 or AAV8 with amino-reactive HPMA copolymer. The second approach used carbodiimide (EDC) chemistry to increase the number of surface amino groups on the AAV5 capsid, thereby improving coating efficiency. In the third approach, the AAV5 genome was isolated from capsid proteins and delivered in a synthetic polyplex consisting of polyethylenimine (PEI) and HPMA. RESULTS: Efficient covalent attachment of HPMA copolymer to AAV5 could only be achieved following modification of the virus with EDC. Coating inhibited sialic acid dependent infection and provided a platform for retargeting via new ligands, including basic fibroblast growth factor. Retargeted infection was shown to be partially resistant to neutralising antisera. Delivery of AAV5 genomes using PEI and HPMA was efficient and provided absolute control of tropism and protection from antisera. In contrast AAV8 could be reacted directly with HPMA copolymer and allowed specific retargeting via the epidermal growth factor receptor, but gave no protection against neutralising antisera. CONCLUSIONS: Reactive HPMA polymers can be used to ablate the natural tropism of both AAV8 and EDC-modified AAV5 and enable receptor-specific infection by incorporation of targeting ligands. These data show transductional targeting strategies can be used to improve the versatility of AAV vectors.

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons.

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague-Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.

Solid Tumor Immunotherapy with T Cell Engager-Armed Oncolytic Viruses.

Oncolytic viruses (OVs) are novel anticancer agents that combine direct cancer cell killing with the stimulation of antitumor immunity. In addition, OVs can be engineered to deliver biological therapeutics directly to tumors, offering unique opportunities to design multimodal anticancer strategies. Here, a case for arming OVs with bispecific T cell engagers (BiTEs) is put forward. BiTEs redirect the cytotoxicity of polyclonal T cells to target cells of choice, and have demonstrated efficacy against a number of hematological cancers. However, the success of BiTEs in the treatment of solid tumors appears more limited, at least in part due to: (i) poor delivery kinetics and penetration into tumors, and (ii) on-target off-tumor activity, leading to dose-limiting toxicities. Linking the production of BiTEs to OV replication provides an exciting means to restrict production to the tumor site, widen their therapeutic window, and synergize with direct oncolysis. This review summarizes progress thus far in the preclinical development of BiTE-armed OVs, and explores the possibility of cotargeting cancer cells and nontransformed stromal cells.

Expression of human CD46 and trans-complementation by murine adenovirus 1 fails to allow productive infection by a group B oncolytic adenovirus in murine cancer cells.

BACKGROUND: Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS: Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS: Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION: Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.

An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.

: Effective immunotherapy of stromal-rich tumors requires simultaneous targeting of cancer cells and immunosuppressive elements of the microenvironment. Here, we modified the oncolytic group B adenovirus enadenotucirev to express a stroma-targeted bispecific T-cell engager (BiTE). This BiTE bound fibroblast activation protein on cancer-associated fibroblasts (CAF) and CD3ε on T cells, leading to potent T-cell activation and fibroblast death. Treatment of fresh clinical biopsies, including malignant ascites and solid prostate cancer tissue, with FAP-BiTE-encoding virus induced activation of tumor-infiltrating PD1+ T cells to kill CAFs. In ascites, this led to depletion of CAF-associated immunosuppressive factors, upregulation of proinflammatory cytokines, and increased gene expression of markers of antigen presentation, T-cell function, and trafficking. M2-like ascites macrophages exhibited a proinflammatory repolarization, indicating spectrum-wide alteration of the tumor microenvironment. With this approach, we have actively killed both cancer cells and tumor fibroblasts, reversing CAF-mediated immunosuppression and yielding a potent single-agent therapeutic that is ready for clinical assessment. SIGNIFICANCE: An engineered oncolytic adenovirus that encodes a bispecific antibody combines direct virolysis with endogenous T-cell activation to attack stromal fibroblasts, providing a multimodal treatment strategy within a single therapeutic agent.

Passive tumour targeting of polymer-coated adenovirus for cancer gene therapy.

Adenovirus provides many opportunities as a vector for delivery of cytotoxic genes to tumours. Polymer coating of adenovirus is known to increase its plasma circulation kinetics, affording the possibility of active and passive targeting to tumours. Here we show that polymer-coating adenovirus (pc-virus) abrogates its normal infectivity in vitro and also in liver following intravenous injection. The coated virus accumulates within solid subcutaneous AB22 mesothelioma tumours 40-times more than unmodified virus, and mediates higher levels of transgene expression within tumours. This is the first demonstration of passive tumour targeting of polymer-coated adenoviruses administered by intravenous injection, and also the first time pc-virus has been shown to be infectious following passive targeting to tumours in vivo. This technology provides an interesting option for delivery of therapeutic viruses to disseminated tumour masses by intravenous injection.

Bacteriophage biopanning in human tumour biopsies to identify cancer-specific targeting ligands.

Intravenous targeting of anticancer agents should improve both efficacy and therapeutic index. However, rational design of targeting constructs requires detailed definition of receptor targets and must take account of polarised tissue architecture that may restrict access to chosen receptors from the bloodstream. Bacteriophage biopanning provides a solution to this problem, identifying targeting sequences by functional selection rather than design, although reiterative panning in polarized human tumours has not previously been attempted. Here, we report an ex vivo, intra-arterial method for biopanning in freshly-resected human tumours, enabling reiterative selection of oligopeptide sequences capable of intravascular targeting to human colorectal tumours. Significant consensus was observed after two rounds of panning in tumours from different patients, and lead sequences demonstrated tumour targeting in samples from unrelated patients. This novel approach may be applicable to a wide range of settings, thus enabling iteration of consensus targeting sequences for tumour imaging and selective delivery of anticancer agents.

Making Oncolytic Virotherapy a Clinical Reality: The European Contribution.

Oncolytic viruses (OVs) are quickly moving toward the forefront of modern medicines. The reward for the decades of research invested into developing viral platforms that selectively replicate in and lyse tumor cells while sparking anticancer adaptive immunity is presenting in the form of durable therapeutic responses. While this has certainly been a concerted global effort, in this review for the 25th anniversary of the European Society of Gene and Cell Therapy, we focus on the contributions made by European researchers. Research centers across Europe have held central roles in advancing OVs, from the earliest reports of coincidental viral infections leading to antitumor efficacy, to advanced mechanistic studies, and now through Phase I-III trials to imminent regulatory approvals. While challenges still remain, with limitations in preclinical animal models, antiviral immune clearance, and manufacture restrictions enforced by poor viral yields in certain cases, the field has come a very long way in recent years. Thoughtful mechanistic integration of OVs with standard of care strategies and other newly approved therapies should provide potent novel approaches. Combination with immunotherapeutic regimes holds significant promise, and the ability to arm the viral platform with therapeutic proteins for localized expression at the tumor site provides an opportunity for creating highly effective synergistic treatments and brings a new age of targeted cancer therapeutics.

Group B adenovirus enadenotucirev infects polarised colorectal cancer cells efficiently from the basolateral surface expected to be encountered during intravenous delivery to treat disseminated cancer.

Enadenotucirev (EnAd) is a group B oncolytic adenovirus developed for systemic delivery and currently undergoing clinical evaluation for advanced cancer therapy. For differentiated carcinomas, systemic delivery would likely expose virus particles to the basolateral surface of cancer cells rather than the apical surface encountered during natural infection. Here, we compare the ability of EnAd and adenovirus type-5 (Ad5) to infect polarised colorectal carcinoma cells from the apical or basolateral surfaces. Whereas Ad5 infection was more efficient via the apical than basolateral surface, EnAd readily infected cells from either surface. Progeny particles from EnAd were released preferentially via the apical surface for all cell lines and routes of infection. These data further support the utility of group B adenoviruses for systemic delivery and suggest that progeny virus are more likely to be released into the tumour rather than back through the basolateral surface into the blood stream.

Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

Combining Oncolytic Adenovirus with Radiation-A Paradigm for the Future of Radiosensitization.

Oncolytic viruses and radiotherapy represent two diverse areas of cancer therapy, utilizing quite different treatment modalities and with non-overlapping cytotoxicity profiles. It is, therefore, an intriguing possibility to consider that oncolytic ("cancer-killing") viruses may act as cancer-selective radiosensitizers, enhancing the therapeutic consequences of radiation treatment on tumors while exerting minimal effects on normal tissue. There is a solid mechanistic basis for this potential synergy, with many viruses having developed strategies to inhibit cellular DNA repair pathways in order to protect themselves, during genome replication, from unwanted interference by cell processes that are normally triggered by DNA damage. Exploiting these abilities to inhibit cellular DNA repair following damage by therapeutic irradiation may well augment the anticancer potency of the approach. In this review, we focus on oncolytic adenovirus, the most widely developed and best understood oncolytic virus, and explore its various mechanisms for modulating cellular DNA repair pathways. The most obvious effects of the various adenovirus serotypes are to interfere with activity of the MRE11-Rad50-Nbs1 complex, temporally one of the first sensors of double-stranded DNA damage, and inhibition of DNA ligase IV, a central repair enzyme for healing double-stranded breaks by non-homologous end joining (NHEJ). There have been several preclinical and clinical studies of this approach and we assess the current state of progress. In addition, oncolytic viruses provide the option to promote a localized proinflammatory response, both by mediating immunogenic death of cancer cells by oncosis and also by encoding and expressing proinflammatory biologics within the tumor microenvironment. Both of these approaches provide exciting potential to augment the known immunological consequences of radiotherapy, aiming to develop systems capable of creating a systemic anticancer immune response following localized tumor treatment.