An oncolytic adenoviral vector carrying the tyrosinase promoter for glioma gene therapy.

Targeting gene expression to cancer cells remains a challenge for the development of gene and viral therapy for gliomas. Recent studies have highlighted transcriptional targeting as one of the possible solutions to overcome this limitation. In this context, melanoma associated antigens (MAAs) are usually over-expressed in brain tumors in comparison to normal brain tissue. For this reason, we investigated the use of the tyrosinase promoter as a transcriptional element to target oncolytic therapy for gliomas. Tyrosinase mRNA expression was evaluated by qRT-PCR in normal human brain tissue as well as in human glioma specimens. We found that this gene was significantly over-expressed in glioma cell lines and in primary glioma samples. Tyrosinase expression correlated with the grade of the tumor (p-value range: 0.05-0.001). Furthermore, transfection of several cell cultures with human and mouse tyrosinase promoters driving a luciferase reporter gene confirmed the activity of this promoter in mouse and human cells. To evaluate whether tyrosinase-activated conditionally replicative adenoviruses (CRAds) could induce toxicity in glioma cells, two vectors (Ad h/m and Ad24TYR) were tested in a mouse glioma model. C57BL/6 mice underwent intracranial injection of tumor cell line GL261. Survival was used to evaluate efficacy of the tested vectors. Mice receiving 1 x 10(9) MOI of Ad h/m and Ad24TYR following intracranial tumor implants had a median survival of 46+/-3 days (p<0.05); in contrast, those treated with medium had a median survival of 31+/-2 days. These results suggest that injection of tyrosinase CRAds leads to prolongation of survival in mice with experimental brain tumors. The tyrosinase promoter stands as a proof of principle of the potential use of MAA over-expression patterns for targeting novel anti-glioma therapies.

Comparative evaluation of survivin, midkine and CXCR4 promoters for transcriptional targeting of glioma gene therapy.

OBJECTIVE: Transcriptional targeting is a key strategy to enhance therapeutic efficacy of gene therapy applications. In the context of oncolytic virotherapy, transcriptional promoter elements are used from genes that are over expressed in a variety of malignant cancers. In the present study, we examined the feasibility of transcriptional targeting to glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor-specific promoters. METHODS: To evaluate the expression level of several glioma related genes, we performed quantitative RT-PCR analyses on samples obtained from cell lines and patients. To determine specific level of gene expression mediated by selective promoter elements, we measured luciferase expression in glioma samples transduced with replication deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a conditionally replicative adenoviral vector, CRAd-5/3, and examined the cytopathic effect in vitro. RESULTS: The survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a surviving-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain. A novel vector, CRAd-survivin5/3, with E1a under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro. CONCLUSIONS: Our data demonstrate that the survivin promoter element is very active in glioma samples and has low activity in normal human brain and liver. A novel oncolytic virus, CRAd-survivin-5/3, was effective against a panel of glioma cell lines in vitro. Our results suggest that employing the survivin promoter element in the context of CRAd-5/3 may present a new opportunity for the development of glioma specific oncolytic vectors.

Association between genital tract cytomegalovirus infection and bacterial vaginosis.

Women with sexually transmitted diseases (STDs) and bacterial vaginosis (BV) have increased rates of cytomegalovirus (CMV) seroprevalence and CMV seroconversion. To characterize the association between genital tract CMV infection and BV, vaginal wash specimens from 52 women attending an STD clinic were analyzed. Significantly more women with BV shed CMV in the lower genital tract than did women without BV. In addition, most of the women who were shedding CMV were infected with >1 virus strain. These results suggest that local CMV replication and infection with multiple CMV strains is facilitated by the presence of BV.

Congenital cytomegalovirus infection: association between virus burden in infancy and hearing loss.

OBJECTIVE: To determine the relationship between the virus burden in infancy and hearing loss in congenital CMV infection. STUDY DESIGN: A cohort of 76 infants with congenital cytomegalovirus (CMV) infection identified by means of newborn virologic screening was monitored for outcome. The amount of infectious CMV was analyzed in urine specimens obtained during early infancy. Peripheral blood (PB) samples obtained during early infancy were available from 75 children and CMV DNA was quantitated with a real-time quantitative polymerase chain reaction. RESULTS: Infants with clinical abnormalities at birth (symptomatic congenital CMV infection) had higher amounts of CMV in urine (P = .005) and CMV DNA in PB (P = .001) than infants with no symptoms. Eight children with and 4 children without symptoms had hearing loss. Among children without symptoms, those with hearing loss had a significantly greater amount of CMV in urine (P = .03) and PB virus burden (P = .02) during infancy than those with normal hearing. Infants with < 5 x 10(3) pfu/mL of urine CMV and infants with < 1 x 10(4) copies/mL of viral DNA in PB were at a lower risk for hearing loss. CONCLUSION: In children with asymptomatic congenital CMV infection, hearing loss was associated with increased amounts of urine CMV and PB CMV DNA during early infancy.

Predictors of hearing loss in children with symptomatic congenital cytomegalovirus infection.

OBJECTIVE: Congenital cytomegalovirus (CMV) infection is a major cause of sensorineural hearing loss (SNHL) and neurologic impairment in children. Although the majority of children with symptomatic congenital CMV infection develop hearing loss, many symptomatic infants have normal hearing. The purpose of this study was to identify indicators present in the newborn period that have predictive value for the development of hearing loss in children with symptomatic congenital CMV infection. METHODS: Of the 190 children who had symptomatic congenital CMV infection and were born between 1966 and 1997 and enrolled in a follow-up study, hearing outcome was known for 180 children. Follow-up data were analyzed using univariate and multivariate logistic regression analyses to determine the specific demographic, newborn clinical, and laboratory findings predictive of hearing loss. The amount of infectious CMV in urine was quantified in a subset of 21 children who were born between 1994 and 1998. RESULTS: The presence of intrauterine growth retardation, petechiae, hepatosplenomegaly, hepatitis, thrombocytopenia, and intracerebral calcifications was associated with the development of hearing loss on univariate analysis. The presence of microcephaly and other neurologic abnormalities was not predictive of hearing loss. Logistic regression analysis revealed that only petechiae and intrauterine growth retardation independently predicted hearing loss. None of the demographic and other newborn findings predicted progressive hearing loss. The children who developed hearing loss had higher urine CMV titers during infancy than those with normal hearing. CONCLUSION: In children with symptomatic congenital CMV infection, evidence of disseminated infection with or without the presence of neurologic involvement at birth was predictive of the development of hearing loss. However, it was not possible to identify factors that are independently predictive of the development of progressive hearing loss.