Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease

Abstract

Disclosed is a new system for generating recombinant adenovirus vectors and adenovirus-based expression libraries, by positive selection of recombinants deleted for the endogenous protease gene, which gene is expressibly cloned into another region of the adenoviral genome. In a preferred embodiment, the invention allows positive selection of E1-deleted, protease-deleted recombinant adenovirus vectors comprising an exogenous gene or an expressible piece of exogenous DNA, by providing an expression cassette comprising the protease gene and the exogenous DNA inserted in place of E1 region in a shuttle vector. In vivo recombination of the shuttle vector with a protease-deleted adenoviral genome in suitable non-complementing cells generates viable recombinants only when rescuing the protease cloned in E1 region. Non-recombinant viral genomes are not able to grow due to the deletion of the protease gene, ensuring that only recombinant viral plaques are generated. This positive selection can be used for the generation of a large number of high purity recombinant adenovirus vectors and allows generation of adenovirus-based libraries with diversity exceeding 10⁶ clones.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a Divisional of U.S. patent application Ser. No. 09 / 843,949 filed Apr. 30, 2001, the entire content of which is incorporated by reference in this application.FIELD OF THE INVENTION [0002] The present invention relates to a method of generation of adenovirus recombinant vectors and adenovirus-based expression libraries, in particular to a method of generation of adenovirus recombinant vectors and adenovirus-based expression libraries by positive selection of adenovirus recombinant vectors through ectopic expression of the adenovirus protease. BACKGROUND OF THE INVENTION [0003] The term “gene therapy” is usually understood to mean the process in which a gene is introduced into the somatic cells of an individual with the aim of being expressed in the cells, to produce some therapeutic effect. Initially this principle was applied to cases where an additional normal copy of a defective gene was provided to restore the synt...

AI Technical Summary

Benefits of technology

[0016] The present invention provides an adenovirus vector/packaging cell line system in which the vector replication is blocked by deletion of a single gene, not a viral transcriptional region, which deletion does not interfere with any other viral functions. The deleted gene is the gene of the adenovirus protease. The protease encoded by the deleted gene is expressed in a complementing (packaging) c

Problems solved by technology

In particular, the transferred gene (transgene) may code for a protein that is not necessarily missing but that may be of therapeutic benefit and difficult to administer exogenously, for example IL-2 or antitumor cytokines.

When these substances are administered subsequently, they trigger selective destruction of the targeted cells.

The deletion of some parts of the viral genome may render the virus replication-incompetent, i.e., unable to multiply in the infected host cells.

One of critical issues in the development of safe viral vectors is to prevent the generation of replication-competent virus during vector production in a packaging cell line or during the gene therapy treatment.

Even though recombination events are rare for E1-deleted adenovirus vectors, their in vivo replication and the ensuing risks could not be completely prevented, and generation of replication-competent adenovirus was demonstrated during the preparation of viral stocks.

Another danger is the loss of rep

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