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-vacicine-compositions of above for causing death of cancer cells that form part of a tunoour and virus infected Denguue, Measles and other diseased cells; the derivative comprising replicating as well as non-replicating dervivaties 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 describved above.

What is claimed is: 
     
         1 . A non-replicating derivative of an attenuated negative stranded RNA virus belonging to family paramyxoviridae, wherein the derivative comprises artificially designed transcriptional units coding for non-viral genes inserted in the same. 
     
     
         2 . The non-replicating derivative of attenuated negative stranded RNA virus of  claim 1 , wherein the derivative comprises of two or more non-viral genes. 
     
     
         3 . The non-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 non-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 MV genome like replicon RNA coding for non-MV genes along with a subset of 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 non-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, and wherein the non-replicating derivative is a Virosome. 
     
     
         6 . The non-replicating derivative of the attenuated negative stranded RNA virus of  claim 5 , wherein the Virosome, comprises one or more of: (a) non-MV genes, or (b) non-MV genes and a subset of MV genes, or (c) a subset of MV genes. 
     
     
         7 . The non-replicating derivative of the attenuated negative stranded RNA of  claim 5 , wherein the Virosomes are capable of one of more of following features:
 a. displaying antigens from other pathogenic organisms including viruses like Dengue,   b. delivering and inducing the expression of genes coding for antigens from other pathogenic organisms including viruses like Dengue,   c. inducing immunity against different non-measles virus antigens,   d. delivering non-measles virus genes into human/animal cells for modulating the expression of cellular genes,   e. capable of serving as vaccines against different diseases,   f. serving as therapeutic agents for treatment of different diseases.   
     
     
         8 . The non-replicating derivative of the attenuated negative stranded RNA virus of  claim 6 , wherein the Virosome comprises any one of:
 a. a GFP virosome comprising genome coding for green fluorescent protein and produced from the plasmid pMTX-P1T (SEQ ID NO: 1)—by inserting the cDNA encoding green fluorescence protein (GFP),   b. a Virosome comprising a genome that codes for prM and E proteins of dengue virus and produced from the plasmid pMTX-P1T-D2G comprising SEQ ID NO: 21,   c. a Virosome comprising a genome that codes for the prM and E proteins of Dengue Virus along with the N, P and L proteins of the Measles Virus, produced from a plasmid derived from pMTX-P1T-High (SEQ ID NO: 7) by inserting the cDNA coding for prM and E proteins,   d. a Virosome comprising a genome that codes for a truncated prM and E proteins of Dengue virus, and produced by deleting the region corresponding to SEQ ID NO: 22 from SEQ ID NO: 21,   e. a Virosome comprising a genome that codes for a truncated prM and E proteins of Dengue virus along with N, P and L proteins of the Measles Virus, and produced from a plasmid derived from pMTX-P1T-High by inserting a cDNA corresponding to prM truncated by deleting SEQ ID NO: 22 and E proteins,   f. a Virosome, comprising a genome that codes the H and F proteins of the Measles Virus and is derived from pMV (SEQ ID NO: 9 or SEQ ID NO: 28) by deleting the sequences corresponding to the N, P, M and L genes of MV, and   g. a Virosome, comprising a genome for one or more of therapeutically useful genes and a sub-set of MV genes derived from pMTX-P1T-NP-RE1-FH-RE2-RE3 (SEQ ID NO: 8) or by replacing one or more of the MV protein coding regions from pMV (SEQ ID NO: 9 or SEQ ID NO: 28) with other therapeutically useful genes.   
     
     
         9 . The non-replicating derivative of the attenuated negative stranded RNA virus of  claim 8 , wherein the Virosome of sub-claim g. of  claim 8  comprises a Virosome comprising one or more of genes comprising dominant negative mutant of Cyclin G1, cytocidal genes, Cytosine deaminase gene, human granulocyte macrophage colony stimulating factor gene (GMCSF) gene, soluble PD-1 blockade gene, PD1 blocking antibody gene and gene for prostate specific acid phosphatase (PAP). 
     
     
         10 . A composition comprising:
 a. the non-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.   
     
     
         11 . The composition of  claim 10 , wherein the virosome is one or more selected from the group comprising a genome that codes for either (a) exclusively non-measles genes or (b) a combination of non-MV and a subset of MV genes or (c) exclusively a subset of MV genes. 
     
     
         12 . The composition of  claim 10 , wherein the composition is a vaccine. 
     
     
         13 . The composition of  claim 10 , wherein the vaccine comprises one or more of:
 a. a Virosome comprising a genome that codes for prM and E proteins of dengue virus,   b. a Virosome comprising a genome that codes for the prM and E proteins of Dengue Virus along with the N, P and L proteins of the Measles Virus,   c. a Virosome comprising a genome that codes for a truncated-prM and E proteins of Dengue virus,   d. a Virosome comprising a genome that codes for a truncated-prM and E proteins of Dengue virus along with N, P and L proteins of the Measles Virus, and   e. a Virosome, comprising a genome that codes the H and F proteins of the Measles Virus.   
     
     
         14 . The composition of  claim 13 , wherein the vaccine comprising virosomes of  claim 13  sub-claims b., c., d. and e corresponds to one or more of serotype 1, serotype 2, serotype 3 or serotype 4 of Dengue Virus. 
     
     
         15 . The composition of  claim 13 , wherein virosomes of  claim 13  sub-claims b., c., d. and e. are used as vaccine for prevention of Dengue. 
     
     
         16 . The composition of  claim 10 , wherein the composition is a non-vaccine medication. 
     
     
         17 . The composition of  claim 16 , wherein the non-vaccine medication comprises a Virosome, comprising a genome that codes for one or more of therapeutically useful genes and a sub-set of MV genes. 
     
     
         18 . 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:
 the Virosomes comprising artificially designed Measles Virus genome like replicon RNAs that comprise exclusively of non-Measles Virus genes or a combination of non-Measles Virus and a subset of MV genes.