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) cell line through a regulatable expression cassette which induces no toxic effects in the cells, thus making the generation and production of the vector easier and efficient. As the deleted gene is highly specific of adenovirus, no complementation of the gene in transduced cells is to be expected, which increases the safety and suitability of the protease gene deleted vectors for gene transfer purposes.

[0017] When additionally deleted for E1 region of adenoviral genome, the vectors of the invention are blocked for replication, but are capable of a single round of replication if deleted only for the protease gene. The latter feature permits an enhanced expression of the transgene in transduced cells, which may be of importance in some applications, for example to achieve localized enhanced expressions of transgenes (in situ tumor therapy) or efficient vaccinations without boosting.

[0018] 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 gene or DNA under control of a suitable promoter, which may be a regulatable (e.g., inducible) promoter, inserted in place of E1 region in a shuttle vector. In another embodiment, the exogenous gene or expressible exogenous DNA is put into a separate expression cassette, under control of a suitable promoter. 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 ensures generation of a large number of high purity recombinant adenovirus vectors and allows generation of adenovirus-based expression libraries with diversity exceeding 106 clones.

[0020] It is a further object of the present invention to provide a method for producing novel cell lines capable of complementing in trans an adenovirus mutant deleted for the protease gene, which cell lines contain DNA expressing the adenovirus protease.

[0027] According to one aspect of the present invention, novel cell lines have been generated which are capable of expressing the Ad2 protease gene from a dicistronic expression cassette, under control of a tetracycline inducible promoter. The protease is expressed in these cells together with the green fluorescent protein (GFP), the latter used to facilitate cell cloning and expression monitoring. The novel cell lines have been prepared by transfecting derivatives of 293 cells with pieces of DNA encoding the Ad2 protease and GFP, selecting cells harboring these pieces (cells expressing the GFP) and amplifying them. The novel cell lines, stably expressing the Ad2 protease, produce amounts of protease equal to or greater than those reached after comparable infections by adenovirus. The biological activity of the novel cell lines has been demonstrated by their ability to fully support the reproduction of Ad2ts1 mutant, a temperature-sensitive mutant expressing a functionally defective protease and to restore normal yields of replication of two novel adenovirus mutants in which the protease gene has been deleted.

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 replication deficiency (and the return to a phenotypic state of multiplication) through complementation in trans in some cells which produce proteins capable of replacing proteins encoded by the deleted regions of the viral genome.

However, at present the construction of AdVs remains a cumbersome and lengthy process that is not readily amenable to the generation of large collection of clones.

Although useful, this approach still suffer from the intrinsic limitation that, in a library of several thousand of clones, an even larger number of parental viruses would have to be screened against, a process which is fairly time consuming.

Furthermore, recombinant AdV are sometimes at growth disadvantage relative to the parental virus and these clones might be more difficult to isolate in a library, unless recombinant viruses are positively selected for growth.

Thus far, a positive selection system compatible with the generation of very large number of AdV clones has not yet been developed.

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