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Abstract: Objective: The purpose of this study was to investigate the effect of preexisting neutralizing antibody (NAbs) in naive mice and the effect of induced NAbs in mice immunized with either an RGD or nonmodified Ad5 vector on the transduction efficiency of adenoviral vectors. Methods: BALB/c mice were immunized with Ad5LucRGD, with the unmodified Ad5Luc1, or with Opti-MEM intraperitoneally (ip) from one to three times. Sera were collected on day 27 and serially diluted to block Ad5Luc1 or Ad5LucRGD prior to infection of SKOV3.ip1 human ovarian carcinoma cells with these same vectors. Forty-eight hours post Ad infection, a luciferase assay was performed to determine the titer of NAbs. Results: Luciferase assay data showed that the gene transfer efficacy of Ad5LucRGD was 1.56-fold higher than Ad5Luc1 in the presence of serum from naive mice. In the presence of serum from Ad5Luc1-challenged mice, the transduction efficiency of Ad5LucRGD was 3.27-fold higher (single challenge) and 4.2-fold higher (triple challenge) than Ad5Luc1. In the presence of serum from Ad5LucRGD-challenged mice, the transduction efficiency of Ad5LucRGD was 2.24-fold higher (single challenge) and 2.53-fold higher (triple challenge) than Ad5Luc1. Conclusion: The RGD-modified human Ad vectors appear to be less recognizable than unmodified Ad to preexisting NAbs in mouse models. RGD-modified Ad vectors also appear to elicit a relatively lower level of NAbs that may also contribute to the higher gene transduction efficiency of these modified vectors. Therefore, RGD-modified Ad vectors may be reagents of clinical utility in the context of preformed anti-Ad immunity and in the setting of repetitive dosing. [Copyright &y& Elsevier]

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. [Copyright &y& Elsevier]

Adenovirus serotype 5 (Ad5) displays unparalleled gene transfer efficacy to cells with high coxsackie-adenovirus receptor (CAR) expression. Unfortunately, cells isolated from clinical human cancers, both ovarian and other types, express highly variable and often low levels of CAR. Fortunately, native Ad5 tropism can be modified to circumvent CAR deficiency and to enhance infectivity. Ad5/3luc1 incorporates the serotype 3 fiber knob and binds to a receptor distinct from CAR, while the fiber of Ad5lucRGD is modified with an RGD-4C motif, allowing CAR-independent binding to integrins. We studied the liver tropism and blood clearance of these viruses after intravenous (i.v.) injection, and biodistribution after intraperitoneal (i.p.) injection to tumor-bearing mice. To estimate efficacy, we assessed gene transfer to purified human primary ovarian cancer cells, and in a mouse model of ovarian cancer. Ad5/3luc1 achieved improved gene transfer over Ad5lucRGD, and both infectivity-enhanced viruses were superior to the isogenic control with an unmodified Ad5 capsid. In the presence of malignant ascites, gene transfer was improved with both Ad5/3luc1 and Ad5lucRGD. Thus, retargeting to the Ad3 receptor enhances gene transfer to clinically relevant ovarian cancer substrates, while the mouse toxicity and biodistribution profile of both fiber-modified Ad vectors is comparable to Ad5. [Copyright &y& Elsevier]