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A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus.

ABSTRACT: BACKGROUND: Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, in vitro ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, in vitro ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field. METHODS AND RESULTS: A modified in vitro ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of E1a, deletion of the E1B55k gene, and HR-free cloning of the recombined E1Delta55k gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1Delta55k CRAd was 1.5-2% of Ad without E1Delta55k deletion. CONCLUSION: A 3-step cloning approach can generate a double-targeted, E1B55k-deleted CRAd using a straight-forward, modified in vitro ligation procedure.

SUBMITTER: Haviv YS

PROVIDER: S-EPMC2647529 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus.

Haviv Yosef S YS

Virology journal 20090207

<h4>Background</h4>Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously, in vitro ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, in vitro ligation has only rarely been used to generate CRAds and may be a complex proced ...[more]

PMID: 19200390

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Project description:In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity.We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5'-untranslated region (5'-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA.Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity.Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

Dataset Information

A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus.

Publications

A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus.

Similar Datasets

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