Rna transcription vector and uses thereof

Abstract

The present invention in general relates to an improved RNA transcription vector, which is very suitable for the production of mRNA for in vivo therapeutic purposes. The improvements in the vector in particular reside in the presence of a transcription enhancer and a nuclear retention element.

What is claimed is: 
     
         1 - 16 . (canceled) 
     
     
         17 . An RNA production vector comprising:
 a transcribable nucleic acid sequence;   a 5′ translation enhancer (TE) sequence comprising a tandem repeat of a 9-nucleotide segment of the mouse Gtx homeodomain protein, interspaced by 9-nucleotide fragments of the human beta globin 5′ UTR; and   a 3′ nuclear retention sequence (ENE) comprising the poly-adenylated non-translated RNA (PAN) region of the Kaposi's sarcoma associated Herpes virus (KSHV).   
     
     
         18 . The RNA production vector according to  claim 17 , wherein said TE sequence is a 10× tandem repeat of a wild type 9-nucleotide sequence from a Gtx leader sequence (CCGGCGGGT) (SEQ ID NO: 6) linked by a 9-nucleotide sequence derived from a 5′UTR of the human beta globin (TTCTGACAT) (SEQ ID NO: 7). 
     
     
         19 . The RNA production vector according to  claim 17 , wherein said ENE is a 79-nucleotide sequence of the poly-adenylated non-translated RNA (PAN) region of the Kaposi's sarcoma associated Herpes virus (KSHV), comprising a stem-loop structure with an asymmetric internal U-rich loop. 
     
     
         20 . The RNA production vector according to  claim 17 , wherein said transcribable nucleic acid sequence is selected from the group consisting of mRNA encoding CD40L, mRNA encoding CD70, mRNA encoding caTLR4, and antigen/disease specific mRNA. 
     
     
         21 . A method of increasing stability and/or translation efficiency of in vitro transcribed RNA, the method comprising:
 (i) providing an RNA production vector according to  claim 17 , wherein said transcribable nucleic acid sequence is a transcribable DNA sequence that corresponds to said RNA to be transcribed; and   (ii) transcribing in vitro said transcribable DNA sequence.   
     
     
         22 . An RNA molecule comprising:
 a transcribable nucleic acid sequence;   a 5′ translation enhancer (TE) sequence comprising a tandem repeat of a 9-nucleotide segment of the mouse Gtx homeodomain protein, interspaced by 9-nucleotide fragments of the human beta globin 5′ UTR; and   a 3′ nuclear retention sequence (ENE) comprising the poly-adenylated non-translated RNA (PAN) region of the Kaposi's sarcoma associated Herpes virus (KSHV).   
     
     
         23 . The RNA molecule according to  claim 22 , wherein said transcribable nucleic acid sequence is selected from the group consisting of mRNA encoding CD40L, mRNA encoding CD70, mRNA encoding caTLR4, and antigen/disease specific mRNA. 
     
     
         24 . A composition comprising one or more RNA molecules according to  claim 22 . 
     
     
         25 . A method for introducing the RNA molecule according to  claim 22  into a host cell, the method comprising transfecting the RNA molecule into the host cell. 
     
     
         26 . A method for introducing the composition according to  claim 24  into a host cell, the method comprising transfecting the composition into the host cell. 
     
     
         27 . A method for treating a disorder in a subject in need thereof, the method comprising administering to the subject the RNA molecule according to  claim 22 . 
     
     
         28 . The method according to  claim 27 , wherein said disorder is selected from the group consisting of cancer, allergy, and infectious disease. 
     
     
         29 . The method according to  claim 28 , wherein the infectious disease is selected from the group consisting of bacterial infections, viral infections, and fungal infections. 
     
     
         30 . A method for treating a disorder in a subject in need thereof, the method comprising administering to the subject the composition according to  claim 24 . 
     
     
         31 . The method according to  claim 30 , wherein said disorder is selected from the group consisting of cancer, allergy, and infectious disease. 
     
     
         32 . The method according to  claim 31 , wherein the infectious disease is selected from the group consisting of bacterial infections, viral infections, and fungal infections. 
     
     
         33 . A kit comprising one or more RNA molecules according to  claim 22 . 
     
     
         34 . A kit comprising the composition according to  claim 24 .