Vectors, mutant viruses and methods for generating mutant viruses

Abstract

A nucleic acid vector comprising first and second nucleotide sequences corresponding to nucleotide sequences flanking an insertion site in the genome of a selected herpes simplex virus strain; and a cassette located between said first and second nucleotide sequences comprising nucleic acid encoding: (a) one or a plurality of insertion sites and / or a nucleotide sequence of interest; and (b) a ribosome binding site or a regulatory nucleotide sequence; and (c) a marker is disclosed. Herpes simplex viruses generated using said vector, methods for their generation and herpes simplex viruses having a genome comprising heterologous nucleic acid are also disclosed.

Description

FIELD OF THE INVENTION [0001] The present invention relates to nucleic acid vectors for delivery of a nucleic acid cassette to an insertion site in a selected viral genome, to methods of generating mutant virus using said vectors and to the mutant viruses generated, and particularly, although not exclusively, to mutant herpes simplex viruses and nucleic acid vectors for use in generating mutant herpes simplex viruses. BACKGROUND TO THE INVENTION [0002] Existing procedure for generating herpes simplex virus (HSV) mutants requires generation of a unique plasmid by cloning an entire expression cassette consisting of a promoter, gene of interest and polyadenylation sequences into a plasmid separately constructed to contain the relevant flanking sequences and then co-transfecting BHK cells with the resultant plasmid and HSV-1 DNA. Homologous recombination drives the formation of recombinant HSV-1 expressing the gene of interest, which is identified by Southern blotting. The recombinant v...

AI Technical Summary

Benefits of technology

[0011] The inventors have provided a generic plasmid vector designated RL1.dIRES-GFP. RL1.dIRES-GFP provides a platform for generating a plurality of ‘shuttle vectors’ which can exploit the process of homologous recombination to transfer a nucleotide sequence of interest (downstream of a selected promoter) into the disabling RL1 locus of HSV-1, generating easily identifiable, oncolytic, ICP34.5 null HSV-1 mutants expressing the products of the nucleotide sequence of interest, e.g. an RNA transcript or a polypeptide, and GFP. RL1.dIRES-GFP thus provides for ease of generation and purification of ICP34.5 null HSV.

[0012] RL1.dIRES-GFP is a useful vector for making second-generation oncolytic viruses having enhanced cytotoxic potential and which may express the product(s) of selected gene(s) to enhance the oncolytic and / or therapeutic effect of the administered virus.

[0016] RL1.dIRES-GFP is promoterless, thus enabling a promoter of choice to be incorporated in the homologously recombined shuttle vector for controlling expression of the nucleotide sequence of interest from the inserted cassette.

[0018] By using the plasmid RL1.dIRES-GFP to generate a shuttle vector, designated RL1.dCMV-NTR-GFP, containing the E. coli nitroreductase gene downstream of a CMV IE promoter, both inserted at the MCS, the inventors have further provided a novel second generation oncolytic mutant HSV. The genome of this mutant HSV comprises the heterologous (i.e. non-HSV) E. coli nitroreductase protein coding sequence inserted at one or each ICP34.5 locus, disrupting the ICP34.5 protein coding sequence such that the ICP34.5 gene is non-functional and cannot express a functional ICP34.5 gene product. The generated HSV is capable of expressing the E. coli nitroreductase gene product under control of the inserted promoter. This virus thus has the oncolytic activity of HSV strain 1716 and can be used in gene directed enzyme-prodrug therapy and has shown significantly enhanced tumour cell killing in vitro and in vivo when used with the prodrug CB1954. The mutant virus is designated HSV1716 / CMV-NTR / GFP.

Problems solved by technology

This process takes several months.

Most tumours exhibit individual characteristics and the ability of a broad spectrum first generation oncolytic virus to replicate in or provide an effective treatment for all tumour types is not guaranteed.

The prior art provides technically challenging, procedurally slow and inefficient materials and methods for generating recombinant HSV.

In particular the prior art does not provide methods of, and materials for, generating recombinant HSV which are easy to detect, may be designed to be specific null mutants and which may express a selected gene of interest.

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