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Autosomal recessive congenital ichthyosis (ARCI) is a diverse group of cornification diseases associated with severe clinical complications and decreased quality of life. Germline mutations in the TGM1 gene, which encodes the enzyme TGM1, are the predominant cause of ARCI. These TGM1 mutations trigger the abnormal epidermal differentiation and impaired cutaneous barrier function observed in patients with ARCI. Unfortunately, current ARCI therapies focus solely on symptomatic relief. Thus, there is a significant unmet need for therapeutic strategies aimed at correcting the TGM1 deficiency underlying ARCI. In this study, we investigated the ability of KB105, a gene therapy vector encoding full-length human TGM1, to deliver functional human TGM1 to keratinocytes. In vitro, KB105 efficiently infected TGM1-deficient human keratinocytes, produced TGM1 protein, and rescued transglutaminase enzyme function. In vivo studies demonstrated that both single and repeated topical KB105 administration induced TGM1 protein expression in the target epidermal layer without triggering fibrosis, necrosis, or acute inflammation. Toxicity and biodistribution assessments on repeat dosing indicated that KB105 was well-tolerated and restricted to the dose site. Overall, our results demonstrate that rescuing TGM1 deficiency in patients with ARCI through topical KB105 application represents a promising strategy for safely and noninvasively treating this debilitating disease.

We have conducted a phase 1 study of intravenous vvDD, a Western Reserve strain oncolytic vaccinia virus, on 11 patients with standard treatment-refractory advanced colorectal or other solid cancers. The primary endpoints were maximum tolerated dose and associated toxicity while secondary endpoints were pharmacokinetics, pharmacodynamics, immune responses, and antitumor activity. No dose-limiting toxicities and treatment related severe adverse events were observed. The most common adverse events were grades 1/2 flu-like symptoms. Virus genomes were detectable in the blood 15–30 minutes after virus administration in a dose-dependent manner. There was evidence of a prolonged virus replication in tumor tissues in two patients, but no evidence of virus replication in non-tumor tissues, except a healed injury site and an oral thrush. Over 100-fold of anti-viral antibodies were induced in patients' sera. A strong induction of inflammatory and Th1, but not Th2 cytokines, suggested a potent Th1-mediated immunity against the virus and possibly the cancer. One patient showed a mixed response on PET-CT with resolution of some liver metastases, and another patient with cutaneous melanoma demonstrated clinical regression of some lesions. Given the confirmed safety, further trials evaluating intravenous vvDD in combination with therapeutic transgenes, immune checkpoint blockade or complement inhibitors, are warranted.

Genetically engineered tumor-selective vaccinia virus (VV) has been demonstrated to be a highly effective oncolytic agent, but immune clearance may limit its therapeutic potential. As previously demonstrated, immunosuppression can lead to significant enhancement of viral recovery and therapeutic effect, but the magnitude of complement-mediated viral inactivation has not been fully elucidated and warrants further investigation. Using fluorescent microscopy and quantitative plaque assays, we have determined complement's key role in viral clearance and its multi-faceted means to pathogen destruction. Complement can lead to direct viral destruction and inhibition of viral uptake into cells, even in the absence of anti-vaccinia antibodies. Our data demonstrate C5 to be integral to the clearance pathway, and its inhibition by Staphylococcal superantigen-like protein (SSL7) leads to a 90-fold and 150-fold enhancement of VV infectivity in both the presence and absence of anti-VV antibodies, respectively. This study suggests that complement inhibition may reduce vaccinia viral neutralization and may be critical to future in vivo work.

Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV).

Pseudomyxoma peritonei (PMP) is characterized by intraperitoneal dissemination of mucinous ascites. This malignancy frequently recurs despite aggressive locoregional therapies, demonstrates chemo-insensitivity and lacks targeted therapies. This review addresses some intriguing questions in PMP; what role does mucin play in this malignancy?; what genetic alterations and dysregulated signaling pathways lead to a putative goblet cell-lineage differentiation or mucin overexpression?; are targeted therapies against known transcriptional pathways for mucin production a novel therapeutic strategy in this malignancy?

Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP.The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays.Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count.Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

Pre-existing antipoxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic double-deleted vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B-cell knockout mice, we confirmed that the neutralizing antibodies had a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (interleukin (IL)-10(hi)/IL-12(low)) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results showed the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy.

We describe the clinical, pathologic and molecular characteristics of a xenograft model of metastatic mucinous appendiceal adenocarcinoma. Tumours from patients with mucinous appendiceal neoplasms were implanted in nude mice and observed for evidence of intraperitoneal tumour growth. Morphologic and immunohistochemical features, temporal growth characteristics relative to controls, and loss of heterozygosity (LOH) at multiple chromosomal alleles were assessed in a successfully engrafted tumour. Two of seventeen implanted tumours successfully engrafted and only one mucinous adenocarcinoma propagated throughout the course of the study. The successful xenograft is morphologically similar to the original tumour, produces abundant extracellular mucin and exhibits non-invasive growth on peritoneal surfaces. The temporal growth characteristics of the xenograft tumour relative to controls reveal that tumour burden can be followed indirectly by measuring the weight or abdominal girth of engrafted animals. The cytokeratin, mucin core protein, CDX2, Ki-67 and p53 expression patterns are identical in the xenograft and resected tumour and are consistent with the expected pattern of protein expression for mucinous adenocarcinoma of the appendix. LOH was found in 1 of 10 informative chromosomal loci (chromosome 10p23) in xenograft tumour cells. Although we were unable to engraft a low-grade appendiceal mucinous neoplasm, the engrafted adenocarcinoma will be useful for future evaluation of novel therapeutic strategies directed at mucinous appendiceal adenocarcinoma and evaluation of strategies for treating widespread, bulky, mucinous peritoneal surface neoplasms. Xenograft tumour enrichment can facilitate molecular studies of appendiceal epithelial neoplasia.

We have explored a unique combination therapy for metastatic colorectal cancer. This strategy combines a potent and new oncolytic poxvirus expressing a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or TNFSF10) and oxaliplatin (Ox) chemotherapy. We hypothesized that TRAIL expression would increase the efficacy of the oncolytic poxvirus, and that the therapeutic efficacy would be further enhanced by combination with chemotherapy. The cytotoxicity to cancer cells by Ox, oncolytic vaccinia virus (VV) and trail gene-armed VV alone or in combination was tested in vitro. The trail gene armed oncolytic VV-expressed high levels of TRAIL in infected cancer cells and had greater potency as a cytotoxic agent compared with the parent VV. Ox alone exerted concentration-dependent cytotoxicity. In vitro, the combination of the two agents applied at suboptimal concentrations for individual therapy displayed synergy in inducing cancer cells into enhanced levels of apoptosis/necrosis. Western blot analyses were consistent with the notion that TRAIL induced cancer cell death mainly through apoptosis, whereas Ox and vJS6 induced cell death more through non-apoptotic death pathways. In two aggressive colorectal carcinomatosis models derived from human HCT116 and murine MC38 cells, the combination therapy displayed synergistic or additive antitumor activity and prolonged the survival of the tumor-bearing mice compared with either Ox chemotherapy or vvTRAIL-mediated oncolytic gene therapy alone. This combination strategy may provide a new avenue to treating peritoneal carcinomatosis and other types of metastases of colorectal cancer.

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

Complement is a central part of both the innate and adaptive immune response and its activation has traditionally been considered part of the immunosurveillance response against cancer. Its pro-inflammatory role and its contribution to the development of many illnesses associated with inflammatory states implicate complement in carcinogenesis. We evaluated the role of three protein inhibitors of complement—cobra venom factor, humanized cobra venom factor, and recombinant staphylococcus aureus superantigen-like protein 7—in the setting of a transplantable murine colon cancer model. Outcomes were evaluated by monitoring tumor growth, and flow cytometry, ELISPOT, and quantitative real-time PCR were used to determine the impact of complement inhibition on the host immune response. Complement inhibitors were effective at depleting complement component C3 in tumor bearing mice and this was temporally correlated with a decreased rate of tumor growth during the establishment of tumors. Treatment with cobra venom factor resulted in increased CD8+ T cells as a percentage of tumor-infiltrating cells as well as a reduced immunosuppressive environment evidenced by decreased myeloid derived suppressor cells in splenocytes of treated mice. Complement inhibition resulted in increased expression of the chemoattractive cytokines CCL5, CXCL10, and CXCL11. Complement depletion represents a promising mode of immunotherapy in cancer by its ability to impair tumor growth by increasing the host’s effective immune response to tumor and diminishing the immunosuppressive effect created by the tumor microenvironment and ultimately could be utilized as a component of combination immunotherapy.

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

Background The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. Methods To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. Results In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (PhCMV*-1) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original PhCMV*-1 in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses. Copyright © 2004 John Wiley & Sons, Ltd.

We examined whether a herpes simplex virus vector that expresses human proenkephalin could be used to attenuate nociception in a model of bone cancer pain in mice. Osteolytic sarcoma cells were implanted into the medullary space of the right femur, followed by a subcutaneous inoculation of a replication-defective herpes simplex virus vector expressing human proenkephalin (vector SHPE) or a lacZ-expressing control vector (vector SHZ). SHPE-inoculated mice demonstrated a significant, naltrexone-reversible decrease in pain-related behavior assessed during open-field motor activity. These results suggest that gene transfer with an enkephalin-expressing vector may be used to treat pain resulting from cancer in bone.

Oncolytic viral therapy utilizes a tumor-selective replicating virus which preferentially infects and destroys cancer cells and triggers antitumor immunity. The Western Reserve strain of vaccinia virus (VV) is the most virulent strain of VV in animal models and has been engineered for tumor selectivity through two targeted gene deletions (vvDD). We performed the first-in-human phase 1, intratumoral dose escalation clinical trial of vvDD in 16 patients with advanced solid tumors. In addition to safety, we evaluated signs of vvDD replication and spread to distant tumors, pharmacokinetics and pharmacodynamics, clinical and immune responses to vvDD. Dose escalation proceeded without dose-limiting toxicities to a maximum feasible dose of 3 × 10(9) pfu. vvDD replication in tumors was reproducible. vvDD genomes and/or infectious particles were recovered from injected (n = 5 patients) and noninjected (n = 2 patients) tumors. At the two highest doses, vvDD genomes were detected acutely in blood in all patients while delayed re-emergence of vvDD genomes in blood was detected in two patients. Fifteen of 16 patients exhibited late symptoms, consistent with ongoing vvDD replication. In summary, intratumoral injection of the oncolytic vaccinia vvDD was well-tolerated in patients and resulted in selective infection of injected and noninjected tumors and antitumor activity.

Previous studies from our group have demonstrated an upregulation in nerve growth factor (NGF) RNA and protein in the cortex 24 h following traumatic brain injury (TBI) in a rat model. This increase in NGF is suppressed if rats are subjected to 4 h of whole-body hypothermia following TBI. In the present study we used in situ hybridization to extend our initial RNA gel-blot (Northern) hybridization findings by demonstrating that NGF RNA is increased in the cortex following TBI and that hypothermia diminishes this response. Further, by combining in situ hybridization with immunocytochemistry for glial fibrillary acidic protein we demonstrate that astrocytes are the major cellular source for the upregulation in NGF and that this upregulation can be observed in the hippocampus as early as 3 h posttrauma. The predominantly astrocytic origin suggests that the NGF upregulation is not related primarily to cholinotrophic activities. We hypothesize that its function is to stimulate upregulation of antioxidant enzymes, as part of an injury-induced cascade, and that supplementation of NGF or antioxidants may be warranted in hypothermic therapies for head injury.

Paclitaxel-induced peripheral neuropathy (PIPN) is devastating constellation of symptoms that arise as a complication in patients receiving paclitaxel chemotherapy for cancer, most notably breast cancer. It presents as a glove and stocking neuropathy characterized by numbness, tingling, and occasionally burning pain in the feet and hands. Approximately 230,000 women will be diagnosed with breast cancer this year and over half, or about 120,000 women, will develop peripheral neuropathy as a consequence of paclitaxel therapy; this neuropathy will last for 5 years or longer in a quarter of these women. The development of PIPN may result in dose reduction of the paclitaxel, a switch to less efficacious agent, or even cessation of all chemotherapy treatment. Currently there are no approved therapies for the treatment of PIPN.PGN-503 is a new investigation drug designed to prevent and/or treat PIPN. It is a herpes simplex virus type 1 based vector expressing human neurotrophin-3. Following a skin injection, PGN-503 is taken up by sensory nerves, transported back to the cell body, and directs the expression of neurotrophin-3 (NT3). NT3 is a well-studied neurotrophic factor necessary for the growth and maintenance of sensory neurons, and thus represents an ideal candidate for the treatment of primary sensory neuropathies. In a series of preclinical studies, we examined the efficacy of PGN-503 in a mouse model of PIPN.Paclitaxel, dosed at 30 mg/kg/day for 6 days over two weeks, causes a peripheral neuropathy in BALB/c mice characterized by a long-lasting decrease in evoked sensory nerve action potentials (SNAPs) and conduction velocities (SNCVs). Mice, pretreated with a single injection of PGN-503 into the plantar surface of the hind paws 3 days prior to paclitaxel dosing, were completely protected against this decrease in SNAPs and SNCVs for the length of the study (approximately 4 months). This protection was dose-dependent on the total plaque forming units of PGN-503 applied to the paws. In a separate set of animals, we determined that a single application of PGN-503 was completely protective against the development of neuropathy following a second round of paclitaxel dosing beginning at 47 days post vector injection and partially protective against a third round beginning at 99 days post vector injection. Importantly, we have also demonstrated that PGN-503, applied one week after paclitaxel dosing, is capable of reversing the peripheral neuropathy. A Phase I/II trial of PGN-503 in preventing the development of PIPN in adjuvant breast cancer treatment is currently planned to begin next year.

Pseudomyxoma peritonei (PMP) is characterized by intraperitoneal dissemination of mucinous ascites. This malignancy frequently recurs despite aggressive locoregional therapies, demonstrates chemo-insensitivity and lacks targeted therapies. This review addresses some intriguing questions in PMP; what role does mucin play in this malignancy?; what genetic alterations and dysregulated signaling pathways lead to a putative goblet cell-lineage differentiation or mucin overexpression?; are targeted therapies against known transcriptional pathways for mucin production a novel therapeutic strategy in this malignancy?

Tumor necrosis factor superfamily members, including Fas ligand and TRAIL, have been studied extensively for cancer therapy, including as components of gene therapy. We examined the use of FasL expression to achieve tumor-selective replication of an oncolytic poxvirus (vFasL), and explored its biology and therapeutic efficacy for FasR- and FasR+ cancers. Infection of FasR+ normal and MC38 cancer cells by vFasL led to abortive viral replication owing to acute apoptosis and subsequently showed both reduced pathogenicity in non-tumor-bearing mice and reduced efficacy in FasR+ tumor-bearing mice. Infection of FasR- B16 cancer cells by vFasL led to efficient viral replication, followed by late induction of FasR and subsequent apoptosis. Treatment with vFasL as compared with its parental virus (vJS6) led to increased tumor regression and prolonged survival of mice with FasR- cancer (B16) but not with FasR+ cancer (MC38). The delayed induction of FasR by viral infection in FasR- cells provides for potential increased efficacy beyond the limit of the direct oncolytic effect. FasR induction provides one mechanism for tumor-selective replication of oncolytic poxviruses in FasR- cancers with enhanced safety. The overall result is both a safer and more effective oncolytic virus for FasR- cancer.

Tumor vaccines can induce robust immune responses targeting tumor antigens in the clinic, but antitumor effects have been disappointing. One reason for this is ineffective tumor infiltration of the cytotoxic T lymphocytes (CTLs) produced. Oncolytic viruses are capable of selectively replicating within tumor tissue and can induce a strong immune response. We therefore sought to determine whether these therapies could be rationally combined such that modulation of the tumor microenvironment by the viral therapy could help direct beneficial CTLs induced by the vaccine. As such, we examined the effects of expressing chemokines from oncolytic vaccinia virus, including CCL5 (RANTES), whose receptors are expressed on CTLs induced by different vaccines, including type-1-polarized dendritic cells (DC1). vvCCL5, an oncolytic vaccinia virus expressing CCL5, induced chemotaxis of lymphocyte populations in vitro and in vivo, and displayed improved safety in vivo. Interestingly, enhanced therapeutic benefits with vvCCL5 in vivo correlated with increased persistence of the viral agent exclusively within the tumor. When tumor-bearing mice were both vaccinated with DC1 and treated with vvCCL5 a further significant enhancement in tumor response was achieved which correlated with increased levels of tumor infiltrating lymphocytes. This approach therefore represents a novel means of combining biological therapies for cancer treatment.

Top of pageAbstract Background: Recombinant vaccinia virus is currently being explored as a potential replicating oncolytic virus for cancer virotherapy due to its exceptional ability to replicate in tumor cells. Vaccinia tropism is not limited to tumor tissues, but also other tissues including ovarian follicles, possibly due to the presence of angiogenic factors such as vascular endothelial growth factor (VEGF). We therefore investigated the utility of vaccinia virus as an oncolytic therapy in a murine model of ovarian cancer. Methods: A murine ovarian surface epithelial cell (MOSEC) line was employed for this model. Intraperitoneal inoculation of the MOSEC cell line leads to a highly malignant neoplasm containing both carcinomatous and sarcomatous components as well as production of hemorrhagic ascitic fluid. These mice were treated with a thymidine kinase (TK) and vaccinia growth factor (VGF) double-deleted virus with the cytokine deaminase (CD) gene inserted into the TK locus. This recombinant virus (vvddCD) was found to be equivalent to the native WR strain in tumor tropism with minimal systemic growth. Results: Following intraperitoneal (i.p.) inoculation with 7.5x106 cells, mice were treated with 109 pfu of the vvddCD strain of vaccinia virus i.p. on the same day (concurrent treatment). In vivo studies appeared to show complete inhibition of tumor growth as compared to the control group (median mouse weight with ascites at 88 days: 40.7+/-1.49 (control [n=10]) vs 27.43+/-1.12 (virus [n=10])) (p

Traumatic brain injury (TBI) induces astrocytic and microglial activation and proliferation and augmented production of the cytokine interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF). The increase in NGF temporally follows the increase in IL-1 beta, suggesting that the IL-1 beta up-regulation after trauma directly induces the increase in NGF. We examined the effect of IL-1 receptor antagonist protein (IL-1ra) on microglial proliferation and NGF production in rat cortex, following two different models of TBI. Rabbit fibroblasts infected with a retroviral vector containing the human IL-1ra gene were implanted into the wound cavity immediately following a cortical stab wound or 6 hours after a weight drop-induced trauma. Both microglial proliferation and NGF up-regulation were decreased significantly in animals receiving IL-1ra-expressing cells compared with animals receiving naive (untransfected) fibroblasts. These data demonstrate that the increase in NGF after central nervous system trauma is directly mediated through IL-1 beta and that blocking IL-1 beta following brain injury leads to suppression of an NGF-mediated reparative response. Such blockade of inflammation, however, may prove to be of significant therapeutic benefit in human brain injury and other inflammatory states.

Promising phase II clinical results have been reported recently for several oncolytic viral therapeutics, including strains based on vaccinia virus. One reason for this has been an increased appreciation of the critical therapeutic importance of the immune response raised by these viruses. However, the most commonly used approaches to enhance these immunotherapeutic effects in oncolytic viruses, typically though expression of cytokine transgenes, often also result in a reduction in oncolytic activity and premature clearance of the virotherapy from the tumor. Approaches that enhance the immunotherapeutic effects while maintaining oncolytic activity would therefore be beneficial. Here, it is demonstrated that the expression of the chemokine CCL19 (ELC) from an oncolytic vaccinia virus (vvCCL19) results in increased antitumor effects in syngeneic mouse tumor models. This corresponded with increased t cell and dendritic cell infiltration into the tumor. However, vvCCL19 persisted in the tumor at equivalent levels to a control virus without CCL19, demonstrating that oncolytic activity was not curtailed. Instead, vvCCL19 was cleared rapidly and selectively from normal tissues and organs, indicating a potentially increased safety profile. The therapeutic activity of vvCCL19 could be further significantly increased through combination with adoptive transfer of therapeutic immune cells expressing CCR7, the receptor for CCL19. This approach therefore represents a means to increase the safety and therapeutic benefit of oncolytic viruses, used alone or in combination with immune cell therapies.

Introduction: Pseudomyxoma peritonei (PMP) is characterized by compressive organ dysfunction from progressive intra-peritoneal accumulation of mucinous ascites. MUC2 is the primary secreted mucin in PMP and the MAPK signaling pathway is involved in MUC2 promoter regulation. We hypothesized that targeted inhibition of the MAPK pathway using small molecule drugs would be a novel, effective and safe therapeutic strategy in this malignancy by reducing mucinous tumor burden. Methods: The therapeutic effect of RDEA119 (BAY 86-9766), a specific MEK 1/2 inhibitor, was assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo, by serial parametric measurements, MUC2 transcripts via real time RT-PCR and MUC2 protein expression via immunofluorescence assays. Results: MUC2 mRNA accounted for approximately 98% of the overall message encoding for secreted gel-forming mucins, with a relatively small contribution from other known gel-forming mucins (MUC5AC, MUC5B, MUC6 and MUC19) in LS174T cells and in the murine xenograft models. RDEA119 significantly inhibited basal MUC2 mRNA levels (DMEM: 0.02 ± 0.0007 vs. RDEA119 5x10−6M: 0.007 ± 0.0003; 48 hr; p 43 days; p=0.02). In the intra-peritoneal PMP murine xenograft model treated with chronic oral RDEA119, MUC2 protein analysis by immunofluorescence demonstrated non-significant gross reduction in MUC2 protein volume (PBS-PO: 41695993 ± 13154727 µm2 vs. RDEA-PO: 35447861 ± 6079224 µm2; p>0.05) and tumor cell count (PBS-PO: 79889 ± 25654 cells vs. RDEA-PO: 52093 ± 15433 cells; p>0.05), suggesting a dual inhibitory effect of RDEA119 on mucin production and tumor cell proliferation. Conclusions: MEK-ERK pathway inhibitor RDEA119 demonstrated effective inhibition of MUC2 production in vitro and in vivo and improved survival in a subcutaneous murine xenograft model of PMP. Reduced mucinous tumor burden by targeted inhibition of the MAPK pathway may be a novel and effective strategy to reduce tumor-specific symptoms, improve overall survival and reduce the need for morbid cytoreductive surgical procedures in patients with recurrent PMP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5253. doi:1538-7445.AM2012-5253

Introduction. Oncolytic viruses have therapeutic potential in cancer by selectively replicating within malignant cells and/or other cells in the tumor tissues, causing cell death. Vaccinia virus (VACV) with deletion of viral genes encoding for thymidine kinase and vaccinia growth factor and armed with a chemokine gene encoding CCL5 (vvDD-CCL5) has been previously shown to be safe and efficacious in a murine colon cancer model (Li J. et al., Mol Ther., 19: 650-7; 2011). Here we explored combination therapy with the use of this armed oncolytic virus and immunotherapy in a murine malignant mesothelioma model in syngeneic BALB/c mice. Methods. The viral replication and oncolysis was examined in murine AB12 mesothelioma cancer cells in vitro, and the combination of oncolytic virus, IL-2 and anti-CD25 and anti-CTLA4 antibodies, was investigated in vivo to treat a murine model of peritoneal malignant mesothelioma (PMM) derived from AB12 cells in BALB/c mice. Results. In vitro, vvCCL5 and its parental virus vvDD replicate well in AB12 cancer cells and lead to efficient cytotoxicity in these cancer cells. In vivo biodistribution analysis indicated that both viruses displayed highly tumor selective replication; however, vvDD-CCL5 replicated to a greater extent and displayed increased persistence in the tumor tissue. As a result, vvDD-CCL5 was more efficient than vvDD, working in a viral dose-dependent manner and leading to increased survival of tumor-bearing mice. Combination therapy of virus with antibodies against CD25 and CTLA4 and IL-2 was investigated. Clearly, this has been the best strategy to treat AB12 PMM, achieving the best long-term survival of mice. We are currently investigating its cellular and molecular mechanisms by this combination leading to tumor regression and long-term survival of mice. Conclusions: The chemokine gene-armed oncolytic virus (vvDD-CCL5) is effective in a PMM tumor model. The combination of this virus with an immunologic regimen of Treg depletion and IL-2 treatment has been demonstrated to be highly efficacious for PMM. Further understanding of its cellular and molecular mechanisms of this treatment strategy will facilitate the optimization of combined therapeutic regimens for PMM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1544. doi:1538-7445.AM2012-1544

The cholinergic deficit in Alzheimer’s disease (AD) brain has given rise to numerous pharmacological strategies designed to enhance cholinergic metabolism and efficacy and improve cognition. Although the cholinergic system is important to cognition we know that correlations between cortical cholinergic function and level of cognition are relatively weak (DeKosky and Scheff, 1990). Synapse loss is more robustly linked to decline in cognition (DeKosky and Scheff, 1990; Terry et al., 1991; DeKosky et al., 1992). Ultrastructural assessment of cortical biopsy samples from AD patients showed a strong correlation with cognition (DeKosky and Scheff, 1990). Confocal immunohistochemical examination of postmortem brain confirmed this correlation between synapse loss and (antemortem) measurements of cognition (Terry et al., 1991). Thus, therapeutic strategies must consider both cholinergic status and the degree of synaptic loss in AD brain.

Background Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene. Findings In Rhox5 -silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo . Conclusions Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.

The pre-existing anti-poxvirus immunity in cancer patients presents a hurdle to successful poxvirus-mediated oncolytic virotherapy. To overcome this hurdle, we have explored strategies of immunosuppression (IS) or/and immune evasion (IE) for successful delivery of an oncolytic vaccinia virus to tumor models in mice pre-vaccinated with a poxvirus. These include transient IS using a cocktail of three immunosuppressive drugs (IS drugs) (tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate) that have been used in organ transplant, carrier cells as a means of IE, or the combined strategy. The three drug cocktail, which targeted multiple components of innate and adaptive immunity, displayed no enhancing effects on viral infection in tumor tissue in the pre-immunized host. Using B cell knockout mice, we confirmed that B cells and the neutralizing antibodies, the later of which were not targeted by the drug cocktail, played a significant role in preventing poxvirus infection. Cancer cells as carrier cells alone were not effective to deliver the virus to tumor. Instead, the application of both IS drugs and carrier cells worked synergistically to promote the delivery to, viral infection, replication and spread inside the tumor mass and thus enhanced the efficacy of oncolytic virotherapy in an ovarian carcinomatosis model. The administraton of these IS drugs led to facilitated development of tumor-associated macrophages (TAMs) into immunosuppressive M2 phenotype (IL-10hi/IL-12low) in the tumor microenvironment. These results revealed a novel mechanism for induction of the immunosuppressive tumor microenvironment by the IS drugs, and demonstrated in principle that it is feasible to treat pre-vaccinated cancer patients with an oncolytic poxvirus when combined with both IS and IE strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1498.

Top of pageAbstract We have previously studied vaccinia as an oncolytic virus and demonstrated enhanced tumor selectivity by deleting viral genes encoding thymidine kinase and vaccinia growth factor. Vaccinia virus encodes at least three anti-apoptosis genes, spi-1, spi-2 and F1L. Spi-2 is a homoloque of cowpox virus CrmA gene, whose product inhibits ICE and other caspase activity. Spi-1 is thought to inhibit the mitochondrial pathway of apoptosis by binding cathespin G. F1L is a newly characterized gene encoding a protein inhibiting apoptosis thorough stablization of the mitochondrial membrane. Cancer cells are well known to possess the ability to evade apoptosis. Therefore, the viral anti-apoptosis genes may be redundant for the virus to replicate in cancer cells, but essential for virus to replicate in normal cells. We have constructed mutant viruses deleted for either F1L alone (vF1L), for spi-1 and spi-2 (vSP), or for all three viral genes (vSPF). Infection of several tumor cell lines including murine colon adenocarcinoma (MC38), human colon adenocarcinoma (HT29) and human epithelial ovarian cancer cell (A2780) with vF1L, vSP, vSPF and a wild type vF13, yielded nearly equivalent viral recovery. The mechanism of cell death in infected cancer and normal cells was investigated by annexin V and PI staining utilizing flow cytometry. In the human colon carcinoma cells infected with WT virus, 11.5% were annexin V positive, while 30.4% were annexin V positive in vSPF-infected cells. However, both vF13L+ and vSPF induced more cell death and more apoptosis in human normal fibroblasts. The virus vF13L+ induced 34.5% and vSPF induced 58% of annexin V postive cells. These results suggest that normal cells are more sensitive to virus-induced apoptosis than cancer cells, and the cells-infected with vSPF are more prone to die via apoptosis. The biodistribution of these viruses were studied in nude mice bearing subcutaneous MC38 tumor. These mice were injected i.p. with either a mutant virus or WT at 107 plaque-forming units (pfu). The results demonstrated that vSPF reduced dramatically viral yields in brain and liver, as well as other normal tissues, while retaining similar yields in tumor. The deletion of F1L alone (vF1L) displayed significant reduction of virus in the liver. The triple deletion virus vSPF also displayed reduced pathogenicity. Naive nude mice receiving 108 pfu of vSPF i.p. lived more than 100 days without illness, whereas those receiving WT virus had median survivals of 7 days and died of viral pathogenicity. The efficiency for tumor regression is currently under study.

Top of pageAbstract We have previously demonstrated the enhanced tumor selectivity of a replicating oncolytic vaccinia virus based on the double deletion of viral genes encoding thymidine kinase and vaccinia growth factor. In this study, we explore the effects of deleting viral anti-apoptosis genes (spi-1 and spi-2) on the proliferation of the virus in normal cells and transformed cells and the effects on the toxicity and oncolytic activity in murine tumor models. Tumor cells have accrued genetic defects in the apoptosis pathways, thus becoming resistant to apoptosis, one of the hallmarks of cancer (Hanahan and Weinberg, Cell, 2000). This may allow the cancer cells to have a survival advantage over the normal cells once infected with a vaccinia lacking the anti-apoptosis genes. Indeed, the deletant virus preferentially replicates in transformed cells or p53-deleted cells as compared to their normal counterparts, as demonstrated in three paired cell types. In vivo, the deletant virus retains full ability to replicate in tumor tissues, while replication in normal tissues is decreased as compared to the wild type virus after systemic delivery. Mice treated with the deleted virus survive much longer compared to those mice treated with the wild type virus. Finally, the deletant virus and the wild type virus have similar antitumor effects as demonstrated in a subcutaneous tumor model in nude mice. The effects of the deletions on the apoptosis of the infected tumor cells and normal cells are being analyzed. In summary, these results demonstrate that this mutant vaccinia replicates as well as the wild type virus in transformed cells or cancerous cells but is significantly attenuated in normal cells. In vivo, the deletant virus exhibits significantly less toxicity but similar anti-tumor efficacy as compared to the wild type virus. We conclude that the spi-2/spi-1 deleted vaccinia is a promising vector for tumor-directed gene therapy, given its enhanced safety profile and tumor selectivity, and high oncolytic effects after systemic delivery.

SummaryVaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.

Controlled cortical impact (CCI) is a contemporary model of experimental cerebral contusion. We examined the cerebrovascular and neuropathologic effects of a severe CCI in rats. The utility of magnetic resonance imaging (MRI) for the assessment of contusion volume after severe CCI was also established. Severe CCI (3.0 mm depth, 4 m/sec velocity) to the left (L) parietal cortex was produced in anesthetized (isoflurane/N2O/O2), intubated, and mechanically ventilated male Sprague-Dawley rats (n = 58). Physiologic parameters were controlled. The time course of alterations in edema [L-R% brain water (% BW) in 3-mm coronal sections through injured and contralateral hemispheres, wet-dry weight] was evaluated at 2 h, 24 h, 48 h, and 7 days posttrauma. Local cerebral blood flow (ICBF, measured in 8 structures in each hemisphere by autoradiography) was evaluated at 2 h, 24 h, and 7 days. Contusion volume (measured by histology and image analysis) was assessed at 14 days and measured in 6 rats by both MRI and histology. The survival rate after severe CCI was 96.2%. The L-R difference in % BW increased to 1.69 +/- 0.18% at 2 h, 3.00 +/- 0.08% at 24 h, 2.69 +/- 0.09% at 48 h, and 0.94 +/- 0.21% at 7 days. These values all differed from the control (p0.05). The % BW was greater at 24 h and 48 h than at 2 h and 7 days (p0.05). Marked reductions in ICBF were limited to structures in the injured hemisphere and were observed in the parietal cortex (2 and 24 h), subcortical white matter (2 and 24 h), and hippocampus (2 h), (p0.05) vs control rats. In the contusion core, ICBF was 19.4 +/- 8.8 mL 100 g-1 min-1 at 24 h (p = 0.011 vs normal). Necrosis was seen in large portions of the parietal cortex and subcortical white matter, and portions of the hippocampus and thalamus. Contusion volume was 47.8 +/- 9.2 mm3, which represented 14.4 +/- 2.1% of the traumatized hemisphere. Estimates of contusion volume by MRI and histology were closely correlated (r = 0.941, p0.017). Severe CCI in rats is accompanied by contusion, reproducible edema, and marked hypoperfusion, involving over 14% of the injured hemisphere, and can be produced with minimal mortality. T2-weighted MRI successfully and noninvasively identifies contusion volume in this model.

Nerve growth factor β subunit (β-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk − K mutant background) and replication-defective (ICP4 − ;tk − S mutant background) vectors were engineered to contain the murine β-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of β-NGF-specific transcripts and β-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of β-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active β-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.