Method for the repair of mutated rna from genetically defective dna and for the specific destruction of tumor cells by rna trans-splicing, and a method for the detection of naturally trans-spliced cellular rna

Abstract

Methods and products for the repair of genetically defective DNA in the region of a mutated exon, for the specific destruction of tumor cells, and for the identification of naturally trans-spliced RNA. The methods inter alia are based on the utilization of cellular RNA splicing components.

What is claimed is: 
     
         1 . A method for selective destruction of tumor cells in a cell population, comprising introducing a DNA into the cell population, which DNA encodes an artificially-shortened cell death pre-mRNA, said cell death pre-mRNA being extended selectively at a N terminus via trans-splicing by means of RNA components occurring in the tumor cells, including an AUG codon as start of translation, and naturally-occurring splicing components, thus obtaining a complete cell death mRNA and encoding a complete cell death protein selectively destroying the tumor cells on a direct or indirect route. 
     
     
         2 . The method according to  claim 1 , wherein said DNA has a nucleotide sequence comprising a replication origin, an RNA polymerase-II promoter, and a signal sequence at a 3′ end for pre-mRNA polyadenylation. 
     
     
         3 . The method according to  claim 1 , wherein the shortened cell death pre-mRNA derived from said DNA comprises an anterior 5′ outron and an exon, the exon having DNA in a distal portion thereof having a nucleotide sequence as cDNA which encodes from amino acid 2 on and up to a last amino acid of the cell death protein. 
     
     
         4 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA a 5′ end of an exon has a frame shift sequence 0, 1, or 2 nucleotides. 
     
     
         5 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA an exon downstream of a frame shift nucleotide sequence and upstream of a nucleotide sequence from amino acid 2 of the cell death protein on, includes a nucleotide sequence of a protease recognition region encoding a peptide sequence which can be cleaved by naturally-occurring cellular proteases. 
     
     
         6 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA an exon has an exonic splice enhancer (ESE) sequence in a region of a protease recognition region and/or in a region of a cell death protein region encoding from amino acid 2 on, with no additional or other amino acids being coded for by said ESE sequence(s). 
     
     
         7 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA an outron in a distal region towards an exon has a 3′ splice site including a branch A site, a polypyrimidine base stretch, and an AG dinucleotide at a border to the exon. 
     
     
         8 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA a branch A site of a 3′splice site has an 8-mer sequence U-A/G-C/UU-A/G-A-C/U-A/G and a polypyrimidine base stretch has the sequence of 15 to 18-mer (U-C). 
     
     
         9 . The method according to  claim 1 , wherein the shortened cell death pre-mRNA in an outron upstream of a 3′ splice site thereof includes an antisense sequence of at least 18 nucleotides which undergo specific antisense pairing to a particular tumor pre-mRNA. 
     
     
         10 . The method according to  claim 1 , wherein an antisense sequence in a 5′ outron of the incomplete cell death pre-mRNA pairs to the specific tumor cell pre-mRNA in its polypyrimidine base sequence of a 3′ splice site upstream of a second coding exon of said RNA. 
     
     
         11 . The method according to  claim 1 , wherein in the shortened cell death pre-mRNA a 5′ outron has a translation start AUG which is not in a reading frame of an exon for the cell death protein from amino acid 2 on, and which initiates a short nonsense protein. 
     
     
         12 . The method according to  claim 1 , wherein the protein selectively destroying the tumor cells is a Herpes Simplex Virus-Thymidine Kinase (HSV-TK) protein.