Mutant viruses

Abstract

An herpes simplex virus wherein the herpes simplex virus genome comprises nucleic acid encoding an antisense to the squamous cell carcinoma related oncogene (asSCCRO); and an herpes simplex virus wherein the herpes simplex virus genome comprises nucleic acid encoding a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide are disclosed together with methods for generation and applications of such viruses.

1 . An herpes simplex virus wherein the herpes simplex virus genome comprises nucleic acid encoding an antisense to the squamous cell carcinoma related oncogene (asSCCRO).  
     
     
         2 . The herpes simplex virus according to  claim 1  wherein said nucleic acid encodes a mammalian asSCCRO.  
     
     
         3 . The herpes simplex virus according to  claim 1  wherein said nucleic acid encodes the human asSCCRO.  
     
     
         4 . The herpes simplex virus according to  claim 1  wherein said nucleic acid encodes a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence complementary to: 
 (i) the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;    (ii) the mRNA transcript of SEQ ID No. 1 or 3; or    (iii) to a fragment of said polynucleotide sequence or mRNA transcript.    
     
     
         5 . The herpes simplex virus according to  claim 4  wherein said nucleic acid encodes a nucleotide sequence complementary to: 
 (i) the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;    (ii) the mRNA transcript of SEQ ID No. 1 or 3; or    (iii) to a fragment of said polynucleotide sequence or mRNA transcript.    
     
     
         6 . The herpes simplex virus according to  claim 4  wherein said degree of sequence identity is at least 70%.  
     
     
         7 . The herpes simplex virus  claim 4  wherein a said fragment comprises at least 20 nucleotides and no more than 900 nucleotides.  
     
     
         8 . The herpes simplex virus according to  claim 1  wherein said nucleic acid hybridises under high stringency conditions to: 
 (i) the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;    (ii) the mRNA transcript of SEQ ID No. 1 or 3; or    (iii) a fragment of said polynucleotide sequence or mRNA transcript.    
     
     
         9 . The herpes simplex virus of  claim 1  wherein said herpes simplex virus genome further comprises a regulatory sequence operably linked to said nucleic acid encoding an antisense to the squamous cell carcinoma related oncogene (as SCCRO), wherein said regulatory sequence has a role in controlling transcription of said asSCCRO.  
     
     
         10 . The herpes simplex virus of  claim 1  wherein said nucleic acid is located in at least one RL1 locus of the herpes simplex virus genome.  
     
     
         11 . The herpes simplex virus of  claim 1  wherein said nucleic acid is located in, or overlaps, at least one of the ICP34.5 protein coding sequences of the herpes simplex virus genome.  
     
     
         12 . The herpes simplex virus of  claim 1  wherein the herpes simplex virus is a mutant of one of HSV-1 strains 17 or F or HSV-2 strain HG52.  
     
     
         13 . The herpes simplex virus of  claim 1  wherein the herpes simplex virus is a mutant of HSV-1 strain 17 mutant 1716.  
     
     
         14 . The herpes simplex virus of  claim 1  which is a gene specific null mutant.  
     
     
         15 . The herpes simplex virus of  claim 1  which is an ICP34.5 null mutant.  
     
     
         16 . The herpes simplex virus of  claim 1  which lacks at least one expressible ICP34.5 gene.  
     
     
         17 . The herpes simplex virus of s claim 1  which lacks only one expressible ICP34.5 gene.  
     
     
         18 . The herpes simplex virus of  claim 1  which is non-neurovirulent.  
     
     
         19 . The herpes simplex virus of  claim 1  wherein said nucleic acid encoding the asSCCRO forms part of a nucleic acid cassette integrated in the genome of said herpes simplex virus, said cassette comprising nucleic acid encoding: 
 (a) said asSCCRO; and nucleic acid encoding:    (b) a ribosome binding site; and    (c) a marker,    wherein the nucleic acid encoding asSCCRO is arranged upstream (5′) of the ribosome binding site and the ribosome binding site is arranged upstream (5′) of the marker.    
     
     
         20 . The herpes simplex virus according to  claim 19  wherein a regulatory nucleotide sequence is located upstream (5′) of the nucleic acid encoding asSCCRO, wherein the regulatory nucleotide sequence has a role in regulating transcription of said nucleic acid encoding the asSCCRO.  
     
     
         21 . The herpes simplex virus as claimed in  claim 19  wherein the cassette disrupts a protein coding sequence resulting in inactivation of the respective gene product.  
     
     
         22 . The herpes simplex virus of  claim 19  wherein a transcription product of the cassette is a bi- or poly-cistronic transcript comprising a first cistron encoding the asSCCRO and a second cistron encoding the marker nucleic acid wherein the ribosome binding site is located between said first and second cistrons.  
     
     
         23 . The herpes simplex virus of  claim 19  wherein the ribosome binding site comprises an internal ribosome entry site (IRES).  
     
     
         24 . The herpes simplex virus of  claim 19  wherein the marker is a defined nucleotide sequence encoding a polypeptide.  
     
     
         25 . The herpes simplex virus as claimed in  claim 24  wherein the marker comprises the Green Fluorescent Protein (GFP) protein coding sequence or the enhanced Green Fluorescent Protein (EGFP) protein coding sequence.  
     
     
         26 . The herpes simplex virus of  claim 19  wherein the marker comprises a defined nucleotide sequence detectable by hybridisation under high stringency conditions with a corresponding labelled nucleic acid probe.  
     
     
         27 . The herpes simplex virus of  claim 19  wherein the cassette further comprises nucleic acid encoding a polyadenylation sequence located downstream (3′) of the nucleic acid encoding the marker.  
     
     
         28 . The herpes simplex virus as claimed in  claim 27  wherein the polyadenylation sequence comprises the Simian Virus 40 (SV40) polyadenylation sequence.  
     
     
         29 .- 32 . (canceled)  
     
     
         33 . A method of lysing or killing tumour cells in vitro or in vivo comprising the step of administering to a patient in need of treatment the herpes simplex virus  claim 1 .  
     
     
         34 . A medicament, pharmaceutical composition or vaccine comprising the herpes simplex virus of  claim 1 .  
     
     
         35 . The medicament, pharmaceutical composition or vaccine as claimed in  claim 34  further comprising a pharmaceutically acceptable carrier, adjuvant or diluent.  
     
     
         36 . An herpes simplex virus, wherein the genome of said virus comprises a nucleic acid sequence encoding an antisense to the squamous cell carcinoma related oncogene (asSCCRO), wherein (1) the nucleic acid sequence is in at least one of the long repeat regions (R L ), or (2) the herpes simplex virus is non-neurovirulent.  
     
     
         37 .- 41 . (canceled)  
     
     
         42 . A method for the treatment of a tumour comprising the step of administering to a patient in need of treatment athe herpes simplex virus of  claim 36 .  
     
     
         43 . (canceled)  
     
     
         44 . The method of  claim 42  wherein said herpes simplex virus is capable of killing tumour cells.  
     
     
         45 . A method of expressing in vitro or in vivo an antisense to the squamous cell carcinoma related oncogene (asSCCRO), said method comprising the step of infecting at least one cell or tissue of interest with the herpes simplex virus of  claim 36 , wherein asSCCRO is operably linked to a transcription regulatory sequence.  
     
     
         46 . (canceled)  
     
     
         47 . HSV1716asSCCRO (ECACC accession number 04051901).  
     
     
         48 . An herpes simplex virus wherein the herpes simplex virus genome comprises nucleic acid encoding a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide.  
     
     
         49 . The herpes simplex virus according to  claim 48  wherein said siRNA is capable of repressing or silencing expression of a mammalian SCCRO.  
     
     
         50 . The herpes simplex virus according to  claim 48  wherein said siRNA is capable of repressing or silencing expression of human SCCRO.  
     
     
         51 . The herpes simplex virus according to  claim 48  wherein said siRNA comprises a nucleic acid of between 10 and 70 nucleotides in length and having the sequence of SEQ ID No.5 or the complement thereof.  
     
     
         52 . The herpes simplex virus according to  claim 48  wherein said siRNA comprises a nucleic acid of between 10 and 70 nucleotides in length and having at least 70% identity to SEQ ID No.5 or the complement thereof.  
     
     
         53 . The herpes simplex virus according to  claim 52  wherein said degree of sequence identity is at least 80%.  
     
     
         54 . The herpes simplex virus of  claim 48  wherein said herpes simplex virus genome fuarther comprises a regulatory sequence operably linked to said siRNA, wherein said regulatory sequence has a role in controlling transcription of said siRNA.  
     
     
         55 . The herpes simplex virus of  claim 48  wherein said nucleic acid is located in at least one RL1 locus of the herpes simplex virus genome.  
     
     
         56 . The herpes simplex virus of  claim 48  wherein the said nucleic acid is located in, or overlaps, at least one of the ICP34.5 protein coding sequences of the herpes simplex virus genome.  
     
     
         57 . The herpes simplex virus of  claim 48  wherein the herpes simplex virus is a mutant of one of HSV-1 strains 17 or F or HSV-2 strain HG52.  
     
     
         58 . The herpes simplex virus of  claim 48  wherein the herpes simplex virus is a mutant of HSV-1 strain 17 mutant 1716.  
     
     
         59 . The herpes simplex virus of  claim 48  which is a gene specific null mutant.  
     
     
         60 . The herpes simplex virus of  claim 48  which is an ICP34.5 null mutant.  
     
     
         61 . The herpes simplex virus of  claim 48  which lacks at least one expressible ICP34.5 gene.  
     
     
         62 . The herpes simplex virus of  claim 48  which lacks only one expressible ICP34.5 gene.  
     
     
         63 . The herpes simplex virus of  claim 48  which is non-neurovirulent.  
     
     
         64 . The herpes simplex virus of  claim 48  wherein said nucleic acid encoding said siRNA forms part of a nucleic acid cassette integrated in the genome of said herpes simplex virus, said cassette comprising nucleic acid encoding: 
 (a) said siRNA; and nucleic acid encoding:    (b) a first regulatory nucleotide sequence; and    (c) a marker,    wherein the nucleic acid encoding said siRNA is arranged upstream (5′) of the first regulatory nucleotide sequence and the first regulatory nucleotide sequence is arranged upstream (5′) of the marker, wherein said first regulatory sequence has a role in controlling transcription of said marker.    
     
     
         65 . The herpes simplex virus according to  claim 64  wherein a second regulatory nucleotide sequence is located upstream (5′) of the nucleic acid encoding said siRNA, wherein the second regulatory nucleotide sequence has a role in regulating transcription of said nucleic acid encoding said siRNA.  
     
     
         66 . The herpes simplex virus as claimed in  claim 64  wherein the cassette disrupts a protein coding sequence resulting in inactivation of the respective gene product.  
     
     
         67 . The herpes simplex virus of  claim 64  wherein the marker is a defined nucleotide sequence encoding a polypeptide.  
     
     
         68 . The herpes simplex virus as claimed in  claim 67  wherein the marker comprises the Green Fluorescent Protein (GFP) protein coding sequence or the enhanced Green Fluorescent Protein (EGFP) protein coding sequence.  
     
     
         69 . The herpes simplex virus of  claim 64  wherein the marker comprises a defined nucleotide sequence detectable by hybridisation under high stringency conditions with a corresponding labelled nucleic acid probe.  
     
     
         70 . The herpes simplex virus of  claim 64  wherein the cassette further comprises nucleic acid encoding a polyadenylation sequence located downstream (3′) of the nucleic acid encoding the marker.  
     
     
         71 . The herpes simplex virus as claimed in  claim 70  wherein the polyadenylation sequence comprises the Simian Virus 40 (SV40) polyadenylation sequence.  
     
     
         72 . The herpes simplex virus of  claim 48  for use in a method of medical treatment.  
     
     
         73 . The herpes simplex virus of  claim 48  for use in the treatment of cancer.  
     
     
         74 . The herpes simplex virus of  claim 48  for use in the oncolytic treatment of a tumour.  
     
     
         75 . (canceled)  
     
     
         76 . A method of lysing or killing tumour cells in vitro or in vivo comprising the step of administering to a patient in need of treatment the herpes simplex virus of  claim 48 .  
     
     
         77 . A medicament, pharmaceutical composition or vaccine comprising the herpes simplex virus of  claim 48 .  
     
     
         78 . The medicament, pharmaceutical composition or vaccine as claimed in  claim 77  further comprising a pharmaceutically acceptable carrier, adjuvant or diluent.  
     
     
         79 . An herpes simplex virus, wherein the genome of said virus comprises a nucleic acid sequence encoding a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide, wherein (a) the siRNA is in at least one of the long repeat regions (R L ), or (b) the herpes simplex virus is non-neurovirulent.  
     
     
         80 .- 85 . (canceled)  
     
     
         86 . A method for the treatment of a tumour comprising the step of administering to a patient in need of treatment a herpes simplex virus, wherein the genome of said virus comprises a nucleic acid sequence encoding a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide and wherein (a) nucleic acid sequence is in at least one of the long repeat regions (R L ), or (b) the herpes simplex virus is non-neurovirulent.  
     
     
         87 . The method of  claim 86  wherein said herpes simplex virus is capable of killing tumour cells.  
     
     
         88 . A method of expressing in vitro or in vivo a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide, said method comprising the step of infecting at least one cell or tissue of interest with a herpes simplex virus, wherein the genome of said virus comprises a nucleic acid sequence encoding said siRNA in at least one of the long repeat regions (R L ), wherein said nucleic acid sequence encoding said siRNA is operably linked to a transcription regulatory sequence.  
     
     
         89 . A method of expressing in vitro or in vivo a short interfering ribonucleic acid (siRNA) molecule that is capable of repressing or silencing expression of squamous cell carcinoma related oncogene (SCCRO) nucleic acid or polypeptide, said method comprising the step of infecting at least one cell or tissue of interest with a non-neurovirulent herpes simplex virus, wherein the genome of said virus comprises a nucleic acid sequence encoding said siRNA, wherein said nucleic acid sequence encoding said siRNA is operably linked to a transcription regulatory sequence.  
     
     
         90 . A medicament, pharmaceutical composition or vaccine comprising the herpes simplex virus of  claim 36 .  
     
     
         91 . The medicament, pharmaceutical composition or vaccine as claimed in  claim 90  further comprising a pharmaceutically acceptable carrier, adjuvant or diluent.  
     
     
         92 . A medicament, pharmaceutical composition or vaccine comprising the herpes simplex virus of  claim 79 .  
     
     
         93 . The medicament, pharmaceutical composition or vaccine as claimed in  claim 92  further comprising a pharmaceutically acceptable carrier, adjuvant or diluent.