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Vectors, mutant viruses and methods for generating mutant viruses

a technology of mutant viruses and vectors, applied in the field of nucleic acid vectors, can solve the problems of inability of a broad spectrum first generation oncolytic virus to replicate in or provide an delay in generating recombinant hsv, and inability to provide effective treatment for all tumour types, etc., to achieve the effect of convenient identification

Inactive Publication Date: 2007-05-17
CRUSADE LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The RL1.dIRES-GFP plasmid streamlines the generation and purification of ICP34.5 null HSV mutants, reducing the time from months to weeks, and enables the production of viruses with enhanced oncolytic activity, suitable for cancer therapy by allowing the expression of specific genes, thereby improving the development of pharmaceutical compositions and vaccines.

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.

Method used

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  • Vectors, mutant viruses and methods for generating mutant viruses
  • Vectors, mutant viruses and methods for generating mutant viruses
  • Vectors, mutant viruses and methods for generating mutant viruses

Examples

Experimental program
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Effect test

example 1

Construction of Plasmid RL1.dIRES-GFP

General Approach

[0255] Plasmid RL1.dIRES-GFP was generated in three stages, illustrated in FIG. 1.

[0256] 1. The DNA sequences containing the CMV IE promoter (pCMV), the NAT gene, the internal ribosome entry site (IRES), the GFP reporter gene and the SV40 polyadenylation sequences were excised from pNAT-IRES-GFP using NsiI and SspI and purified.

[0257] 2. The purified pCMV-NAT-IRES-GFP-PolyA DNA fragment was cloned into RL1.del to form a new plasmid designated RL1.dCMV-NAT-GFP.

[0258] 3. The pCMV-NAT DNA sequences of RL1.dCMV-NAT-GFP were excised using XhoI and the remainder of the plasmid re-ligated to form a novel plasmid designated RL1.dIRES-GFP. This novel plasmid contained a multi-cloning site (all sites shown are unique) upstream of an IRES, the GFP gene and the SV40 polyA sequences all within the HSV-1 RL1 flanking sequences. Recombinant ICP34.5 null HSV-1, expressing a gene of interest in the RL1 locus, can be generated by cloning the ...

example 2

Generation of ICP34.5 Null HSV-1 Expressing a Gene Product of Interest and GFP Using Plasmid RL1.dIRES-GFP

General Approach

[0270] Generation of ICP34.5 null HSV-1 expressing a gene product of interest requires insertion of nucleotide sequence encoding the gene product (polypeptide) of interest, and often a desired promoter, at the MCS of RL1.dIRES.GFP followed by co-transfection of BHK cells with the linearised plasmid, containing the gene of interest, and HSV DNA. Following homologous recombination viral plaques expressing GFP are identified. FIG. 7 illustrates the method steps involved.

[0271] Referring to FIG. 7A plasmid DNA, containing the gene of interest and the desired promoter (X), is digested with restriction endonucleases to release the promoter / gene fragment.

[0272] The promoter / gene fragment is purified and cloned into the multi-cloning site (MCS) of RL1.dIRES.GFP forming a shuttle vector suitable for generating oncolytic HSV-1 (FIG. 7B). This vector contains HSV-1 seq...

example 3

Construction of HSV1716 / CMV-NTR / GFP

General Approach

[0282] HSV1716 / CMV-NTR / GFP was generated by cloning a 1.6 Kbp BamHI fragment from pPS94910, consisting of the E. coli nitroreductase (NTR) gene downstream of the CMV IE promoter (pCMV), into the MCS of the RL1.dIRES-GFP smart cassette, in the forward orientation with respect to the GFP gene in RL1.dIRES-GFP (FIG. 8). The resultant plasmid, named RL1.dCMV-NTR-GFP, was then linearised and recombinant virus generated and purified as described above. The plasmid pPS949 (referred to as ‘pxLNC-ntr’ in Ref 10) containing the NTR gene downstream of the CMV IE promoter (pCMV-NTR) in a pLNCX (Clontech) backbone, was a kind gift from Professor Lawrence Young, University of Birmingham, UK.

Materials and Methods

[0283] 4×1 μg of pPS949 was digested with 10 units of BamHI (Promega), in a suitable volume of 10× buffer (Promega) and nuclease free water (Promega), at 37° C. for 16 hrs. The reaction mixture was electrophoresed in a 1% agarose gel...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/86C12N7/00A61K35/13A61K35/76A61K39/00C12N15/113C12N15/869
CPCA01K2267/0331A61K35/13A61K48/00A61K2039/5256C12N7/00C12N15/1135C12N15/86C12N2710/16643C12N2840/203A61P35/00A61P35/02A61P35/04A61P43/00
Inventor BROWN, SUSANNE MOIRACONNER, JOEDUNN, PAUL
Owner CRUSADE LAB
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