Dna molecules producing custom designed replicating and non-replicating negative stranded rna viruses and uses there of

Abstract

This invention comprises: compositions comprising a derivative, plasmids, a reagent kit and methods of making these compositions a derivative, vaccine- and non-vaccine-compositions of above for causing death of cancer cells that form part of a tumour and virus infected Dengue, Measles and other diseased cells; the derivative comprising replicating as well as non-replicating derivatives of an attenuated negative stranded RNA virus belonging to family paramyxoviridae, including Measles Virus, comprising a single additional transcriptional unit carrying either only one or two or more non-viral genes, and the non-replicating derivatives being free from contaminating replicating Measles Virus (b) a Measles Virus packaging cell line for making above compositions, expressing the M, F and H proteins of MV stably. And (c) a reagent kit for producing the Measles Virus derivatives described above.

What is claimed is: 
     
         1 . A replicating derivative of an attenuated negative stranded RNA virus belonging to family paramyxoviridae, wherein the derivative comprises a single artificially designed additional transcriptional unit coding for two or more non-viral genes inserted in the same. 
     
     
         2 . The replicating derivative of attenuated negative stranded RNA virus of  claim 1 , wherein the derivative comprises of three or more or four or more non-viral genes. 
     
     
         3 . The replicating derivative of an attenuated negative stranded RNA virus of  claim 2 , wherein the virus is a Measles Virus or any other virus with equivalent attenuation, equivalent safety for a human being and with requirements for rescuing the any other virus from cDNA, the requirements comprising use of viral N, P and L proteins expressed from three distinct helper plasmids and use of a plasmid coding a viral anti-genomic RNA modified by insertion of a single additional transcriptional unit. 
     
     
         4 . The replicating derivative of an attenuated negative stranded RNA virus of  claim 3 , wherein the process of making the same comprises use of (a) a two plasmid system and comprising (i) one cloning plasmid comprising the entire anti-genome of the measles virus with or without an additional transcriptional unit (ATU) coding for non-MV genes, wherein MV stands for “Measles Virus”, (ii) one helper plasmid coding for and expressing N, P, L proteins respectively, and (b) a cell line supporting measles virus replication; the cell line may or may not be modified to express one or more of M or F or H proteins of MV stably,
 but not requiring the help of exogenous vaccinia virus or exogenous T7 RNA polymerase. 
 
     
     
         5 . The replicating derivative of an attenuated negative stranded RNA virus of  claim 3 , wherein the attenuated virus is a Measles Virus; the term “Measles Virus” being abbreviated as MV hereafter. 
     
     
         6 . The replicating derivative of an attenuated negative stranded RNA virus of  claim 4 , wherein the cloning plasmid coding for the anti-genome of replicating Measles Virus derivatives comprises a single additional transcriptional unit (ATU) that comprises two or more non-viral genes inserted in the same. 
     
     
         7 . The replicating derivative of an attenuated negative stranded RNA virus of  claim 6 , wherein the cloning plasmid comprises any one selected from the group consisting of pMV-GP of SEQ ID NO: 16, pMV-GC of SEQ ID NO: 17, pMV-GsPP of SEQ ID NO: 23, pMV-GsPC of SEQ ID NO: 25, pMV-GsPDP of SEQ ID NO: 24, and pMV-GsPDC of SEQ ID NO: 26. 
     
     
         8 . A composition comprising:
 a. the replicating derivative of an attenuated negative stranded RNA virus as claimed in  claim 1 , wherein the virus is a Measles Virus, the term “Measles Virus” being abbreviated as “MV” hereafter, or any other virus with equivalent attenuation, equivalent safety for a human being and with requirements for rescuing the any other virus from cDNA, the requirements comprising use of viral N, P and L proteins expressed from three distinct helper plasmids and use of a plasmid coding a viral anti-genomic RNA modified by insertion of a single additional transcriptional unit, and   b. pharmaceutically acceptable excipients,
 wherein the replicating derivatives comprise two or more non-viral genes inserted in the same. 
   
     
     
         9 . The composition of  claim 8 , wherein the replicating derivative of an attenuated virus comprises derivatives that are capable to induce the death of cancer cells but do not adversely affect non-cancerous cells. 
     
     
         10 . The composition of  claim 9 , wherein the cancer cells comprise breast cancer cells, lung cancer cells or prostate cancer cells and the non-cancerous cells are human non-cancerous cells or Vero cell line. 
     
     
         11 . The composition of  claim 10 , wherein the cancer cells comprise at least one of T47D, A-549 or PC-3 and human non-cancerous cells comprising at least one of human normal dermal fibroblasts or mesenchymal stem cells. 
     
     
         12 . The composition of  claim 9 , wherein the replicating derivative is one or more selected from the group consisting of rMV-GP, wherein an additional transcriptional unit (ATU) comprising SEQ ID NO: 10 is inserted upstream of N protein coding region, rMV-GC, wherein an ATU comprising SEQ ID NO: 11 is inserted upstream of N protein coding region, rMV-GsPP, wherein an ATU comprising SEQ ID NO: 12 is inserted upstream of N protein coding region, rMV-GsPC, wherein an ATU comprising SEQ ID NO: 13 is inserted upstream of N protein coding region, rMV-GsPDP, wherein an ATU comprising SEQ ID NO: 14 is inserted upstream of N protein coding region or rMV-GsPDC, wherein an ATU comprising SEQ ID NO: 15 is inserted upstream of N protein coding region. 
     
     
         13 . A composition of plasmids comprising:
 a. the plasmid coding for an anti-genome of replicating Measles virus derivatives, comprising a single additional transcriptional unit (ATU),   b. a helper plasmid coding for and expressing N, P, and L proteins of the Measles Virus, and   c. pharmaceutically acceptable excipients,
 wherein the ATU comprises two or more non-viral genes inserted in the same. 
   
     
     
         14 . The composition of  claim 13 , wherein:
 a. the plasmid coding for the anti-genome of replicating Measles Virus derivatives, the term “Measles Virus” being abbreviated as “MV” hereafter, comprising a single additional transcriptional unit (ATU) coding for 2 to 4 non-Measles Virus genes comprise one or more of pMV-GP of SEQ ID NO: 16, pMV-GC of SEQ ID NO: 17, pMV-GsPP of SEQ ID NO: 23, pMV-GsPC of SEQ ID NO: 25, pMV-GsPDP of SEQ ID NO: 24, pMV-GsPDC of SEQ ID NO: 26;   b. the helper plasmid comprises a plasmid of SEQ ID NO: 18.   
     
     
         15 . A method of reducing a number of cancer cells, wherein the cancer cells are part of a tumour, the method comprising the steps of administering:
 a. an oncolytic virus, selected from the group consisting of rMV-GP, wherein an additional transcriptional unit (ATU) comprising SEQ ID NO: 10 is inserted upstream of N protein coding region, rMV-GC wherein an ATU comprising SEQ ID NO: 11 is inserted upstream of N protein coding region, rMV-GsPP wherein an ATU comprising SEQ ID NO: 12 is inserted upstream of N protein coding region, rMV-GsPC wherein an ATU comprising SEQ ID NO: 13 is inserted upstream of N protein coding region, rMV-GsPDP wherein an ATU comprising SEQ ID NO: 14 is inserted upstream of N protein coding region or rMV-GsPDC wherein an ATU comprising SEQ ID NO: 15 is inserted upstream of N protein coding region, or   b. a combination of a helper plasmid of SEQ ID NO: 18 and one or more of the plasmids selected from the group consisting of pMV-GP of sequence ID #16, pMV-GC of SEQ ID NO: 17, pMV-GsPP of SEQ ID NO: 23, pMV-GsPC of SEQ ID NO: 25, pMV-GsPDP of SEQ ID NO: 24, pMV-GsPDC of SEQ ID NO: 26.