Interleukins, growth factors, and transcription factors are key targets for gene therapy in osteoarthritis: A scoping review.

Objective: Osteoarthritis (OA) is the most common degenerative joint disease, characterized by a progressive loss of cartilage associated with synovitis and subchondral bone remodeling. There is however no treatment to cure or delay the progression of OA. The objective of this manuscript was to provide a scoping review of the preclinical and clinical studies reporting the effect of gene therapies for OA. Method: This review followed the JBI methodology and was reported in accordance with the PRISMA-ScR checklist. All research studies that explore in vitro, in vivo, or ex vivo gene therapies that follow a viral or non-viral gene therapy approach were considered. Only studies published in English were included in this review. There were no limitations to their date of publication, country of origin, or setting. Relevant publications were searched in Medline ALL (Ovid), Embase (Elsevier), and Scopus (Elsevier) in March 2023. Study selection and data charting were performed by two independent reviewers. Results: We found a total of 29 different targets for OA gene therapy, including studies examining interleukins, growth factors and receptors, transcription factors and other key targets. Most articles were on preclinical in vitro studies (32 articles) or in vivo animal models (39 articles), while four articles were on clinical trials related to the development of TissueGene-C (TG-C). Conclusion: In the absence of any DMOAD, gene therapy could be a highly promising treatment for OA, even though further development is required to bring more targets to the clinical stage.

Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models.

OBJECTIVE: Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long-term expression of therapeutic proteins within the joint. This study was undertaken to investigate the use of a helper-dependent adenovirus (HDAd)-mediated intraarticular gene therapy approach for long-term expression of interleukin-1 receptor antagonist (IL-1Ra) as sustained symptomatic and disease-modifying therapy for OA. METHODS: In mouse models of OA, efficacy of HDAd-IL-1Ra was evaluated by histologic analysis, micro-computed tomography (micro-CT), and hot plate analysis. In a horse OA model, safety and efficacy of HDAd-IL-1Ra were evaluated by blood chemistry, analyses of synovial fluid, synovial membrane, and cartilage, and gross pathology and lameness assessments. RESULTS: In skeletally immature mice, HDAd-IL-1Ra prevented development of cartilage damage, osteophytes, and synovitis. In skeletally immature and mature mice, treatment with HDAd-interleukin-1 receptor antagonist post-OA induction resulted in improved-albeit not significantly-cartilage status assessed histologically and significantly increased cartilage volume, cartilage surface, and bone surface covered by cartilage as assessed by micro-CT. Fewer osteophytes were observed in HDAd-IL-1Ra-treated skeletally immature mice. Synovitis was not affected in skeletally immature or mature mice. HDAd-IL-1Ra protected against disease-induced thermal hyperalgesia in skeletally mature mice. In the horse OA model, HDAd-IL-1Ra therapy significantly improved lameness parameters, indicating efficient symptomatic treatment. Moreover, macroscopically and histologically assessed cartilage and synovial membrane parameters were significantly improved, suggesting disease-modifying efficacy. CONCLUSION: These data from OA models in small and large animals demonstrated safe symptomatic and disease-modifying treatment with an HDAd-expressing IL-1Ra. Furthermore, this study establishes HDAd as a vector for joint gene therapy.

Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity.

In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI) to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate-and eventually the long-lasting adaptive immunity against cancer.

Oncolytic Adenovirus Expressing Monoclonal Antibody Trastuzumab for Treatment of HER2-Positive Cancer.

Monoclonal anti-HER2 antibody trastuzumab has significantly improved the survival of patients with HER2-overexpressing tumors. Nevertheless, systemic antibody therapy is expensive, limited in efficacy due to physical tumor barriers, and carries the risk of severe side effects such as cardiomyopathy. Oncolytic viruses mediate cancer-selective transgene expression, kill infected cancer cells while mounting antitumor immune responses, and have recently demonstrated promising efficacy in combination treatments. Here, we armed an oncolytic adenovirus with full-length trastuzumab to achieve effective in situ antibody production coupled with progressive oncolytic cancer cell killing. We constructed an infectivity-enhanced serotype 5 oncolytic adenovirus, Ad5/3-Δ24-tras, coding for human trastuzumab antibody heavy- and light-chain genes, connected by an internal ribosome entry site. Infected cancer cells were able to assemble full-length functional antibody, as confirmed by Western blot, ELISA, and antibody-dependent cell-mediated cytotoxicity assay. Importantly, oncolysis was required for release of the antibody into tumors, providing additional spatial selectivity. Ad5/3-Δ24-tras showed potent in vitro cytotoxicity and enhanced antitumor efficacy over oncolytic control virus Ad5/3-Δ24 or commercial trastuzumab in HER2-positive cancer models in vivo (both P < 0.05). Furthermore, Ad5/3-Δ24-tras resulted in significantly higher tumor-to-systemic antibody concentrations (P < 0.001) over conventional delivery. Immunological analyses revealed dendritic cell activation and natural killer cell accumulation in tumor-draining lymph nodes. Thus, Ad5/3-Δ24-tras is an attractive anticancer approach combining oncolytic immunotherapy with local trastuzumab production, resulting in improved in vivo efficacy and immune cell activation in HER2-positive cancer. Moreover, the finding that tumor cells can produce functional antibody as directed by oncolytic virus could lead to many valuable antitumor approaches. Mol Cancer Ther; 15(9); 2259-69. ©2016 AACR.

Incomplete but infectious vaccinia virions are produced in the absence of oncolysis in feline SCCF1 cells.

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.

Oncolytic adenovirus and doxorubicin-based chemotherapy results in synergistic antitumor activity against soft-tissue sarcoma.

Despite originating from several different tissues, soft-tissue sarcomas (STS) are often grouped together as they share mesenchymal origin and treatment guidelines. Also, with some exceptions, a common denominator is that when the tumor cannot be cured with surgery, the efficacy of current therapies is poor and new treatment modalities are thus needed. We have studied the combination of a capsid-modified oncolytic adenovirus CGTG-102 (Ad5/3-D24-GMCSF) with doxorubicin, with or without ifosfamide, the preferred first-line chemotherapeutic options for most types of STS. We show that CGTG-102 and doxorubicin plus ifosfamide together are able to increase cell killing of Syrian hamster STS cells over single agents, as well as upregulate immunogenic cell death markers. When tested in vivo against established STS tumors in fully immunocompetent Syrian hamsters, the combination was highly effective. CGTG-102 and doxorubicin (without ifosfamide) resulted in synergistic antitumor efficacy against human STS xenografts in comparison with single agent treatments. Doxorubicin increased adenoviral replication in human and hamster STS cells, potentially contributing to the observed therapeutic synergy. In conclusion, the preclinical data generated here support clinical translation of the combination of CGTG-102 and doxorubicin, or doxorubicin plus ifosfamide, for the treatment of STS, and provide clues on the mechanisms of synergy.

Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles.

We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification.

Proteoglycan 4 expression protects against the development of osteoarthritis.

Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

Capsid-modified adenoviral vectors for improved muscle-directed gene therapy.

Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy.

Nitric-oxide supplementation for treatment of long-term complications in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is required for the synthesis and channeling of L-arginine to nitric oxide synthase (NOS) for nitric oxide (NO) production. Congenital ASL deficiency causes argininosuccinic aciduria (ASA), the second most common urea-cycle disorder, and leads to deficiency of both ureagenesis and NO production. Subjects with ASA have been reported to develop long-term complications such as hypertension and neurocognitive deficits despite early initiation of therapy and the absence of documented hyperammonemia. In order to distinguish the relative contributions of the hepatic urea-cycle defect from those of the NO deficiency to the phenotype, we performed liver-directed gene therapy in a mouse model of ASA. Whereas the gene therapy corrected the ureagenesis defect, the systemic hypertension in mice could be corrected by treatment with an exogenous NO source. In an ASA subject with severe hypertension refractory to antihypertensive medications, monotherapy with NO supplements resulted in the long-term control of hypertension and a decrease in cardiac hypertrophy. In addition, the NO therapy was associated with an improvement in some neuropsychological parameters pertaining to verbal memory and nonverbal problem solving. Our data show that ASA, in addition to being a classical urea-cycle disorder, is also a model of congenital human NO deficiency and that ASA subjects could potentially benefit from NO supplementation. Hence, NO supplementation should be investigated for the long-term treatment of this condition.

Cytokine-conditioned dendritic cells induce humoral tolerance to protein therapy in mice.

A major obstacle in the genetic therapy of inherited metabolic disease is host immune responses to the therapeutic protein. This is best exemplified by inhibitor formation in the protein therapy for hemophilia A. An approach to overcoming this is induction of immunological tolerance to the therapeutic protein. Tolerogenic dendritic cells (DCtols) have been reported to induce tolerance. In addition, cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-ß(1) are known to induce tolerance. To model protein therapy, we used ovalbumin (OVA) as antigen in BALB/c mice and their transgenic derivative, DO11.10 mice. In this study we show that adoptive transfer of antigen-pulsed dendritic cells (DCs) treated with a combination of IL-10 and TGF-ß(1) can suppress the antibody response in mice. Adoptive transfer of cytokine-conditioned DCs in preimmunized mice results in reduction of antibody response in the mice. Furthermore, the effect is antigen specific, as the recipient mice were able to mount a potent antibody response to the control antigen. Last, we show that TGF-ß(1) and IL-10-conditioned DCs are able to inhibit anti-FVIII antibody responses in FVIII knockout (KO) mice. Analysis of the contribution of IL-10 and TGF-ß(1) to the DCtol phenotype shows that IL-10 treatment of DCs is sufficient for inducing OVA-specific tolerance in BALB/c mice, but we observed a requirement for treatment with both human TGF-ß(1) and human IL-10 to significantly inhibit anti-FVIII antibody responses in FVIII KO mice. This paper demonstrates that autologous cell therapy for antigen-targeted immune suppression may be developed to facilitate long-term therapy.

Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors.

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development.

Oncolytic vaccinia virus for the treatment of cancer.

INTRODUCTION: Gene therapy offers promising approaches for the development of anticancer agents with new modes of action. Among gene therapy vectors, vaccinia virus has emerged as an attractive agent especially when used as an oncolytic virus. AREAS COVERED: This review describes the use of vaccinia virus in cancer therapy as a gene therapy vector, as an oncolytic virus and in the generation of oncolysates. The main achievements of each field are summarized with a special emphasis on vaccinia as an oncolytic vector and its combination therapies. The virus that has advanced furthest in clinical trials, GM-CSF expressing JX-594, is described in detail and its preclinical and clinical data are reviewed. EXPERT OPINION: Vaccinia virus has great potential in cancer gene therapy, especially when used as an oncolytic virus. In particular, JX-594 has shown promising preclinical and clinical data, and a multi-continental randomized Phase III trial in hepatocellular carcinoma is expected to start soon.

Mitochondrial myopathy induces a starvation-like response.

Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, fibroblast growth factor 21 (Fgf21). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.

Treatment of cancer patients with a serotype 5/3 chimeric oncolytic adenovirus expressing GMCSF.

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.

Oncolytic adenovirus coding for granulocyte macrophage colony-stimulating factor induces antitumoral immunity in cancer patients.

Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell-killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus-mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing.

Oncolytic adenovirus ICOVIR-7 in patients with advanced and refractory solid tumors.

PURPOSE: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7. EXPERIMENTAL DESIGN: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 x 10(10) to 1 x 10(12) viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies. RESULTS: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication. CONCLUSIONS: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing.

Targeted chemotherapy for head and neck cancer with a chimeric oncolytic adenovirus coding for bifunctional suicide protein FCU1.

PURPOSE: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations. EXPERIMENTAL DESIGN: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Delta24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. RESULTS: We examined the efficacy of Ad5/3-Delta24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Delta24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination. CONCLUSIONS: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system.

Antiangiogenic arming of an oncolytic vaccinia virus enhances antitumor efficacy in renal cell cancer models.

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.