[hTERT promoter-based conditionally replicative adenoviruses are useful for lung cancer and other solid cancer treatment].

Treatment of advanced SCLC remains one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and it also has an extremely poor prognosis. As a result, new therapeutic approaches are needed. Telomere maintenance to the regulation of replicative life span strongly implies that alterations in telomere biology play an important role during malignant transformation. Cancers that exhibit high levels of telomerase activity, such as all of the small-cell lung cancers were examined in a previous study. In this study, we turned the expression of hTERT by tumors to a therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the hTERT promoter. This virus achieved good levels of viral replication in SCLC cells and induced a substantial anti-cancer effect in vitro and in vivo. As a further enhancement the cancer cell killing effect was improved with a tropism modification of the virus to express the knob domain of Ad3, and this improved infectivity for cancer cells. Conversely, the hTERT promoter has low activity in normal tissues, and the CRAd caused no damage to normal lung fibroblast cells. Since the telomerase activity is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that the use of hTERT promoter-based CRAds may be a potentially effective strategy for cancer treatment.

A mosaic fiber adenovirus serotype 5 vector containing reovirus sigma 1 and adenovirus serotype 3 knob fibers increases transduction in an ovarian cancer ex vivo system via a coxsackie and adenovirus receptor-independent pathway.

PURPOSE: Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success due to insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). Evidence that adenovirus serotype receptors other than CAR may be of use was presented in previous studies that showed that the Ad3 receptor is expressed at high levels in ovarian cancer cells. We hypothesized that combined use of unique chimeric fibers in the context of novel mosaic adenovirus vectors would enhance infectivity via non-CAR pathways in ovarian cancer cells. EXPERIMENTAL DESIGN: We constructed and characterized Ad5 vectors that use Ad3 knob and reovirus fibers to generate a mosaic fiber virion. Serotype 3 Dearing reovirus uses a fiber-like sigma 1 protein to infect cells expressing sialic acid and junction adhesion molecule 1. We therefore constructed a mosaic fiber Ad5 vector, designated Ad5/3-sigma 1, encoding two fibers: a sigma 1 chimeric fiber and the chimeric Ad5/3 fiber composed of an Ad3 knob. RESULTS: Functionally, Ad5/3-sigma 1 used sialic acid, junction adhesion molecule 1, and Ad3 receptor for cell transduction and achieved maximum infectivity enhancement in ovarian cancer cells with low CAR expression. Furthermore, Ad5/3-sigma 1 achieved infectivity enhancement in primary tissue slices of human ovarian tumor. CONCLUSIONS: We have developed a new type of Ad5 vector with the novel tropism, possessing fibers from Ad3 and reovirus, which exhibits enhanced infectivity via CAR-independent pathway(s). In addition, the flexible genetic platform of vector allows different combination of fiber variants that can be incorporated within the same particle.

Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses.

Natural and genetically modified oncolytic viruses have been systematically tested as anticancer therapeutics. Among this group, conditionally replicative adenoviruses have been developed for a broad range of tumors with a rapid transition to clinical settings. Unfortunately, clinical trials have shown limited antitumor efficacy partly due to insufficient viral delivery to tumor sites. We investigated the possibility of using mesenchymal progenitor cells (MPC) as virus carriers based on the documented tumor-homing abilities of this cell population. We confirmed preferential tumor homing of MPCs in an animal model of ovarian carcinoma and evaluated the capacity of MPCs to be loaded with oncolytic adenoviruses. We showed that MPCs were efficiently infected with an adenovirus genetically modified for coxsackie and adenovirus receptor-independent infection (Ad5/3), which replicated in the cell carriers. MPCs loaded with Ad5/3 caused total cell killing when cocultured with a cancer cell line. In an animal model of ovarian cancer, MPC-based delivery of the Ad5/3 increased the survival of tumor-bearing mice compared with direct viral injection. Further, tumor imaging confirmed a decrease in tumor burden in animals treated with oncolytic virus delivered by MPC carriers compared with the direct injection of the adenovirus. These data show that MPCs can serve as intermediate carriers for replicative adenoviruses and suggest that the natural homing properties of specific cell types can be used for targeted delivery of these virions.

Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication.

Adenovirus (Ad) serotype 5 (Ad5) continues to be the predominant vector used for cancer gene therapy. However, many tumor types are reported to be relatively refractory to Ad5 infection because of low surface expression of the native Ad5 receptor, CAR. The observation that many tumor cells are CAR deficient has necessitated the development of CAR-independent infection strategies, including the introduction of heterologous ligand sequences into the virus fiber gene and immunological or chemical modifications of the capsid proteins. Alternatively, native Ad5 tropism can be modified by substituting the knob region from other Ad serotypes such as Ad type 3 (Ad3) into the Ad5 knob region. To date, the effect(s) of tropism modification on the replication and oncolytic capacity of these chimeric Ad vectors has not been fully evaluated. To address this issue, Ad5 vectors and isogenically matched chimeric vectors with Ad3 tropism (Ad5/3) were compared in this study. Various parameters of virus infection were compared, including binding, nuclear translocation, E1A transcription, transgene expression, de novo virus production, and oncolysis. Overall, the chimeric Ad5/3 virus was progressively more efficient at each step of the replication cycle compared with its Ad5 counterpart. The higher replication efficiency of the chimeric Ad5/3 vector translated into improved therapeutic efficacy in a murine in vivo tumor rejection model. These findings suggest that in addition to the initial target cell interaction, multiple mechanisms contribute to the enhanced replication of the chimeric Ad5/3 vector. Furthermore, the data demonstrate that alternative Ad serotype receptors can be used to improve infection and subsequent oncolytic replication, which is particularly relevant in gene therapy applications for tumors that are inefficiently infected with Ad5.

Infectivity enhanced, hTERT promoter-based conditionally replicative adenoviruses are useful for SCLC treatment.

Treatment of advanced small-cell lung cancer (SCLC) remains one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and it also has an extremely poor prognosis. As a result, new therapeutic approaches are needed. Telomere maintenance to the regulation of replicative lifespan strongly implies that alterations in telomere biology play an important role during malignant transformation. Cancers that exhibit high levels of telomerase activity, such as all of the SCLC, were examined in a previous study. In this study, we turned the expression of human telomerase reverse transcriptase (hTERT) by tumors to a therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 (early region 1) is controlled by the hTERT promoter. This virus achieved good levels of viral replication in SCLC cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with a tropism modification of the virus to express the knob domain of Ad3 (serotype 3 adenovirus), and this improved infectivity for cancer cells. Conversely, the hTERT promoter has low activity in normal tissues, and the CRAd caused no damage to normal lung fibroblast cells. Since the telomerase activity is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that the use of hTERT promoter-based CRAds may be a potentially effective strategy for cancer treatment.

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system.

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.

High-risk ovarian cancer based on 126-gene expression signature is uniquely characterized by downregulation of antigen presentation pathway.

PURPOSE: High-grade serous ovarian cancers are heterogeneous not only in terms of clinical outcome but also at the molecular level. Our aim was to establish a novel risk classification system based on a gene expression signature for predicting overall survival, leading to suggesting novel therapeutic strategies for high-risk patients. EXPERIMENTAL DESIGN: In this large-scale cross-platform study of six microarray data sets consisting of 1,054 ovarian cancer patients, we developed a gene expression signature for predicting overall survival by applying elastic net and 10-fold cross-validation to a Japanese data set A (n = 260) and evaluated the signature in five other data sets. Subsequently, we investigated differences in the biological characteristics between high- and low-risk ovarian cancer groups. RESULTS: An elastic net analysis identified a 126-gene expression signature for predicting overall survival in patients with ovarian cancer using the Japanese data set A (multivariate analysis, P = 4 × 10(-20)). We validated its predictive ability with five other data sets using multivariate analysis (Tothill's data set, P = 1 × 10(-5); Bonome's data set, P = 0.0033; Dressman's data set, P = 0.0016; TCGA data set, P = 0.0027; Japanese data set B, P = 0.021). Through gene ontology and pathway analyses, we identified a significant reduction in expression of immune-response-related genes, especially on the antigen presentation pathway, in high-risk ovarian cancer patients. CONCLUSIONS: This risk classification based on the 126-gene expression signature is an accurate predictor of clinical outcome in patients with advanced stage high-grade serous ovarian cancer and has the potential to develop new therapeutic strategies for high-grade serous ovarian cancer patients.

Human papillomavirus types 52 and 58 are prevalent in uterine cervical squamous lesions from Japanese women.

Objective. To estimate the prevalence and genotypes of high-risk human papillomavirus (HPV) focusing HPV 16, 18, 52, and 58 in Japan. Methods. Liquid-base cytology specimens were collected from Japanese women (n = 11022), aged 14-98. After classifying cytodiagnosis, specimens were analyzed for HPV DNA by the multiplex polymerase chain reaction method, where 1195 specimens were positive for cervical smear, except adenomatous lesions. Result. HPV genotypes were detected in 9.5% of NILM and 72.2% of ASC-US or more cervical lesions. In positive cervical smears, HPV genotypes were HPV 52 at 26.6%, HPV 16 at 25.2%, HPV 58 at 21.8%, and HPV 18 at 7.1%. Most patients infected with HPV 16 were between 20-29 years old, decreasing with age thereafter. As for HPV 52 and 58, although the detection rate was high in 30- to 39-year-olds, it also was significant in the 50s and 60s age groups. Conclusion. In Japan, as a cause of abnormal cervical cytology, HPV52 and 58 are detected frequently in addition to HPV 16. In older age groups, HPV 52 and 58 detection rates were higher than that observed for HPV 16. After widespread current HPV vaccination, we still must be aware of HPV 52 and 58 infections.

Histological correlation of glandular abnormalities in cervical liquid-based cytology.

Conventional Papanicolaou smear method is still commonly used for cervical cancer screening in Japan, despite the liquid-based cytology (LBC) that has become a global tendency in the world recently. One of the obstacles in the way of popularization of this method seems to be the confusion as to diagnosis upon cervical glandular lesions. We performed comparison study between LBC and conventional Papanicolaou smear about cytological diagnosis using split-sample method in 4522 patients. In 13 cases analyses, which were reported with either AGC or adenocarcinoma by either method, LBC tends to be milder than that by conventional smear, however, the credibility of LBC is considered to be near to that of conventional smear with regard to screening for glandular abnormalities. These results indicate that cervical cancer screening should shift to LBC under the enough experience and appropriate dealing with the cytological diagnosis.

Outcomes of fertility-sparing surgery for stage I epithelial ovarian cancer: a proposal for patient selection.

PURPOSE: The objective of this study was to assess clinical outcomes and fertility in patients treated conservatively for unilateral stage I invasive epithelial ovarian cancer (EOC). PATIENTS AND METHODS: A multi-institutional retrospective investigation was undertaken to identify patients with unilateral stage I EOC treated with fertility-sparing surgery. Favorable histology was defined as grade 1 or grade 2 adenocarcinoma, excluding clear cell histology. RESULTS: A total of 211 patients (stage IA, n = 126; stage IC, n = 85) were identified from 30 institutions. Median duration of follow-up was 78 months. Five-year overall survival and recurrence-free survival were 100% [corrected] and 97.8% for stage IA and favorable histology (n = 108), 100% and 100% for stage IA and clear cell histology (n = 15), 100% and 33.3% for stage IA and grade 3 (n = 3), 96.9% and 92.1% for stage IC and favorable histology (n = 67), 93.3% and 66.0% for stage IC and clear cell histology (n = 15), and 66.7% and 66.7% for stage IC and grade 3 (n = 3). Forty-five (53.6%) of 84 patients who were nulliparous at fertility-sparing surgery and married at the time of investigation gave birth to 56 healthy children. CONCLUSION: Our data confirm that fertility-sparing surgery is a safe treatment for stage IA patients with favorable histology and suggest that stage IA patients with clear cell histology and stage IC patients with favorable histology can be candidates for fertility-sparing surgery followed by adjuvant chemotherapy.

An adenovirus serotype 5 vector with fibers derived from ovine atadenovirus demonstrates CAR-independent tropism and unique biodistribution in mice.

Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.

Reovirus sigma1 fiber incorporated into adenovirus serotype 5 enhances infectivity via a CAR-independent pathway.

Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success because of insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). To enhance infectivity in tissues with low CAR expression, tropism expansion is required via non-CAR pathways. Serotype 3 Dearing reovirus utilizes a fiber-like sigma1 protein to infect cells expressing sialic acid and junction adhesion molecule 1 (JAM1). We hypothesized that replacement of the Ad5 fiber with sigma1 would result in an Ad5 vector with CAR-independent tropism. We therefore constructed a fiber mosaic Ad5 vector, designated as Ad5-sigma1, encoding two fibers: the sigma1 and the wild-type Ad5 fiber. Functionally, Ad5-sigma1 utilized CAR, sialic acid, and JAM1 for cell transduction and achieved maximum infectivity enhancement in cells with or without CAR. Thus, we have developed a new type of Ad5 vector with expanded tropism, possessing fibers from Ad5 and reovirus, that exhibits enhanced infectivity via CAR-independent pathway(s).

In vivo analysis of a genetically modified adenoviral vector targeted to human CD40 using a novel transient transgenic model.

BACKGROUND: Retargeting is necessary to overcome the limitations of adenovirus (Ad)-based gene therapy vectors. To this end, we previously constructed an adenovirus with the fiber knob domain replaced by a fibritin trimerization motif fused to the CD40 ligand (Ad5Luc.FF/CD40L). We demonstrated the utility of this fiber replacement strategy for targeting CD40 (hCD40) on human dendritic cells in vitro. The in vivo targeting capacity of this virus, however, is unknown, and there is a limited repertoire of animal models that present hCD40 at an accessible site. Therefore, a new animal model for evaluating CD40-targeted vectors is required. METHODS: We constructed a recombinant adenovirus that expresses hCD40 under transcriptional control of the flt-1 promoter (AdflthCD40). Expression of hCD40 was validated both in vitro and in transgenic mice expressing the human coxsackie adenovirus receptor (hCAR mice). We then evaluated the targeting efficiency of Ad5Luc.FF/CD40L to hCD40 expressed in the pulmonary vasculature of the hCAR mice. RESULTS: Infection of flt-1-positive cells with AdflthCD40 resulted in abundant hCD40 expression in vitro, which could subsequently be targeted by Ad5Luc.FF/CD40L. In vivo administration of AdflthCD40 to hCAR mice resulted in hCD40 expression in the pulmonary vasculature, which was successfully targeted with systemically administered Ad5Luc.FF/CD40L. CONCLUSIONS: This is the first data showing that genetically modified Ad5Luc.FF/CD40L can successfully target hCD40 in vivo. Our data also establishes the utility of transcriptionally targeted, Ad-mediated transient expression of human target molecules in the pulmonary vasculature of hCAR mice as models for in vivo analysis of targeted gene therapy vectors.

Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors.

Most viruses exploit a variety of host cellular proteins as primary cellular attachment receptors in the context of successful execution of infection. Furthermore, many viral agents have evolved precise mechanisms to subvert host immune recognition to achieve persistence. Herein we present data indicating that adenovirus (Ad) serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. CD80 and CD86 are co-stimulatory molecules that are present on mature dendritic cells and B lymphocytes and are involved in stimulating T-lymphocyte activation. To our knowledge, this is one of the first demonstrations of a virus utilizing immunologic accessory molecules as a primary means of cellular entry. This finding suggests a mechanism whereby viral exploitation of these proteins as receptors may achieve both goals of cellular entry and evading the immune system.

Enhanced gene transfer to mouse dendritic cells using adenoviral vectors coated with a novel adapter molecule.

Adenovirus (Ad)-mediated transduction of dendritic cells (DC) is inefficient because of the lack of the primary Ad receptor, CAR. DC infection with Ad targeted to the CD40 results in increased gene transfer. The current report describes further development of the CD40-targeting approach using an adapter molecule that bridges the fiber of the Ad5 to CD40 on mouse DC. The adapter molecule, CFm40L, consists of CAR fused to mouse CD40 ligand via a trimerization motif. A stable cell line that secretes CFm40L at high levels was generated. Gene transfer to mouse bone marrow-derived DC (mBMDC) using CFm40L-targeted Ad was over 4 orders of magnitude more efficient than that for the untargeted Ad5. Gene transfer was achieved to over 70% of the mBMDC compared to undetectable transduction using untargeted Ad5. In addition to dramatically enhanced gene transfer, the CFm40L-targeted Ad5 induced phenotypical maturation and upregulated IL-12 expression. Most importantly, the CFm40L-targeted Ad5 elicited specific immune response against a model antigen in vivo. The results of this study demonstrate that Ad-mediated gene transfer to DC can be significantly enhanced using nonnative transduction pathways, such the CD40 pathway, which may have important applications in genetic vaccination for cancer and infectious diseases.

Evaluation of tissue-specific promoters in carcinomas of the cervix uteri.

BACKGROUND: Gene therapy is a novel approach for treatment of patients with advanced, recurrent, or metastatic cervical cancer. One effective way to direct transgene expression to specific tissues or tumors is the use of tissue-specific-promoters (TSPs). In the context of adenovirus (Ad)-mediated cancer gene therapy it is rational to choose a TSP which is highly expressed in the tumor but has potentially low activity in non-tumor cells, especially the liver. In this study, we have investigated several promoters which fulfill these criteria. Candidate cervical cancer specific TSPs include promoters of the genes for secretory leukoprotease inhibitor (SLPI), cyclooxygenase-2 (COX-2), Midkine (MK), vascular endothelial growth factor receptor type 1 (flt-1), vascular endothelial growth factor (VEGF), Survivin and the receptor for chemokine SDS-1 (CXCR4). METHODS: To evaluate the specific gene expression of the different promoters in the context of cervical cancer, we constructed a panel of E1-deleted Ads that express luciferase under the control of the promoters of interest. We investigated various established cervical cancer cell lines, as well as purified primary cancer cells and normal control cells from the cervix uteri. RESULTS: In all cell lines tested, promoters for MK, VEGF and CXCR4 showed the highest activity. Both MK and VEGF promoters also resulted in a high activity in primary cervical cancer cells. Interestingly, gene expression profiles correlate with luciferase activity in both cell lines and primary cancer samples. CONCLUSIONS: Our study demonstrates that the promoters for MK and VEGF are active in cervical cancer. We believe that both promoters can be successfully employed as TSPs for gene therapy targeted to cervical cancer.