Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

The poor prognosis of patients with malignant gliomas necessitates the development of novel therapies. Virotherapy, using genetically engineered adenovectors that selectively replicate in and kill neoplastic cells, represents one such strategy. In this study, we examined several oncolytic vectors with modified transcriptional and transductional control of viral replication. First, we incorporated the survivin promoter (S) to drive E1A gene expression. We then modified the adenovirus serotype 5 (Ad5) fiber protein via genetic knob switching or incorporation of peptide ligands to target the following glioma-associated receptors: the Ad3 attachment protein, or CD46, alpha(v) beta(3)/alpha(v)beta(5) integrins, or heparan sulfate proteoglycans. The three conditionally replicative adenoviruses, CRAd-S-5/3, CRAd-S-RGD, and CRAd-S-pk7, were then examined in vitro with respect to transduction efficiency and tissue specificity. The most promising virus was then tested in vivo for evidence of tumor growth inhibition. CRAd-S-pk7 provided the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft tumor growth by more than 300% (p < 0.001). Sixty-seven percent of treated mice with intracranial tumors were long-term survivors (>110 days; p < 0.005). Analysis of tumor tissue indicated increased adenoviral infectivity, decreased mitotic activity, and enhanced tumor apoptosis. These findings demonstrate the effectiveness of CRAd-S-pk7 and provide the rationale for further development of this novel oncolytic virus for glioma gene therapy.

IgA deficiency and common variable immunodeficiency are heritable disorders that can occur within the same family. Both immunodeficiencies are characterized by arrests in B-cell differentiation that vary in the extent of the immunoglobulin isotypes involved. A high frequency of major histocompatibility complex supratypes associated with a null allele of the gene encoding the C4A isotype of complement component C4 has been observed in IgA-deficient individuals. In search of a genetic linkage between the two immunodeficiencies, we examined the major histocompatibility complex (MHC) class HI genes encoding complement components C2, C4A, and C4B and steroid 21-hydroxylase in addition to the HLA serotypes in individuals with either common variable immunodeficiency or IgA deficiency. Twelve of 19 patients with common variable immunodeficiency (63%, P < 0.001) and 9 of 16 patients with IgA deficiency (56%, P < 0.01) had rare C2 alleles and/or C4A and 21-hydroxylase A deletions, whereas these gene features were seen in only 5 of 34 healthy individuals (15%) in the control group. Nine of 11 patients with C4A deletion had an HLA haplotype consistent with the MHC

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a ''tumor on'' and ''liver off'' profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.

Together these data indicate that the survivin promoter is a promising tumor-specific promoter for transcriptional targeting of adenovirus-based vectors and CRAds for malignant gliomas. The strategy of using survivin-CRAds may thus translate into an experimental therapeutic approach that can be used in human clinical trials.

To improve the efficacy and selectivity of virotherapy for malignant glioma, we designed a strategy to amplify adenoviral replication in conjunction with radiotherapy using a radioinducible promoter. First, we compared the radiationinducible activity of FLT-1, vascular endothelial growth factor, DR5, Cox2, and survivin. We then examined the capacity of the optimal promoter to modulate transgene expression followed by E1A activity in vitro and in vivo in a glioma stem cell model. In the presence of radiation, survivin mRNA activity increased 10-fold. Luciferase transgene expression was dose dependent and optimal at 2 Gy. A novel oncolytic adenovirus, CRAd-Survivin-pk7, showed significant toxicity and replication against a panel of passaged and primary CD133

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 survivin-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.

Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P¼0.0087), 16.7% (P¼0.1) and 20% (P¼0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a 'tumor-on' and 'liver-off' status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.