Method for modulating gene expression by modifying the cpg content

Abstract

The invention relates to nucleic acid modifications for a directed expression modulation by the targeted insertion or removal of CpG dinucleotides. The invention also relates to modified nucleic acids and expression vectors.

1 . Method for the targeted modulation of the gene expression, comprising the steps:
 (i) Provision of a target nucleic acid sequence to be expressed,   (ii) Modification of the target nucleic acid sequence, in which the number of CpG dinucleotides present in the target nucleic acid sequence is raised using the degeneracy of the genetic code to increase the gene expression, or is lowered to reduce the gene expression,   (iii) Cloning of the thereby modified target nucleic acid sequence with a modified number of the CpG dinucleotides in a suitable expression vector in operative coupling with a suitable transcription control sequence,   (iv) Expression of the modified target nucleic acid sequence in a suitable expression system.   
     
     
         2 . Method according to  claim 1 , in which in step (ii) the modification of the target nucleic acid sequence is carried out so that, in addition to increasing or reducing the number of CpG dinucleotides, one or more additional modifications is/are carried out at the nucleic acid level. 
     
     
         3 . (canceled) 
     
     
         4 . Method according to  claim 1 , in which the modification of the target nucleic acid sequence by increasing or reducing the number of CpG dinucleotides is carried out having regard to a codon choice optimised for the expression system. 
     
     
         5 . (canceled) 
     
     
         6 . Method according to  claim 1 , in which the gene expression is raised. 
     
     
         7 . Method according to  claim 1 , in which the gene expression is reduced. 
     
     
         8 . Method according to  claim 1 , in which the target nucleic acid sequence to be expressed is heterologous to the expression system. 
     
     
         9 . Method according to  claim 1 , in which a eukaryotic or prokaryotic expression system is used as the expression system. 
     
     
         10 .- 12 . (canceled) 
     
     
         13 . Method according to  claim 1 , in which the modified target nucleic acid sequence and the transcription control sequence are not associated with CpG islands. 
     
     
         14 . (canceled) 
     
     
         15 . Method according to  claim 1 , in which the number of CpG dinucleotides is increased or reduced by at least 10%, preferably at least 50%, more preferably at least 100%. 
     
     
         16 . Method according to  claim 1 , in which all CpG dinucleotides are removed using the degeneracy of the genetic code. 
     
     
         17 .- 20 . (canceled) 
     
     
         21 . Method according to claim  17 , in which the target nucleic acid sequence codes for a functional RNA. 
     
     
         22 . (canceled) 
     
     
         23 . Modified nucleic acid with a region capable of transcription that can be expressed in an expression system, and which is derived from a wild-type sequence, in which the region capable of transcription is modified so that it is codon-optimised in relation to the employed expression system, and so that the number of CpG dinucleotides is increased compared to the codon-optimised sequence derived from the wild-type sequence, by using the degeneracy of the genetic code. 
     
     
         24 . Nucleic acid according to  claim 23 , in which the number of CpG dinucleotides is increased compared to the wild-type sequence by at least 10%, preferably at least 25%, more preferably at least 50%, particularly preferably at least 100%, more particularly preferably at least 200%, especially by a factor of 5 and most especially by a factor of 10 or more. 
     
     
         25 .- 26 . (canceled) 
     
     
         27 . Vector comprising a nucleic acid according to  claim 23  in operative coupling with a suitable transcription control sequence. 
     
     
         28 .- 30 . (canceled) 
     
     
         31 . Vector according to  claim 27 , in which the promoter is an inducible promoter. 
     
     
         32 . (canceled) 
     
     
         33 . Vector according to  claim 27 , in which the promoter is not associated with a CpG island. 
     
     
         34 .- 35 . (canceled) 
     
     
         36 . Vector according to  claim 27 , with the nucleic acid sequence shown in SEQ 10 NO. 25. 
     
     
         37 . Cell containing a nucleic acid or a vector according to  claim 23 . 
     
     
         38 . Expression system comprising
 a) a modified nucleic acid sequence with a region capable of transcription, which is derived from a wild-type sequence, wherein the modified nucleic acid sequence has an increased or reduced number of CpG dinucleotides compared to the wild-type sequence, in operative coupling with a transcription control sequence, and   b) an expression environment selected from a cell and a cell-free expression environment wherein a) can be expressed, in which the expression system in the case of expression of a modified nucleic acid sequence with an increased number of CpG dinucleotides exhibits an increased expression, and in the case of expression of a modified nucleic acid sequence with a reduced number of CpG dinucleotides exhibits a reduced expression.   
     
     
         39 . (canceled) 
     
     
         40 . Use of a nucleic acid and/or a vector and/or a cell and/or an expression system according to  claim 23  for the production of a medicament for a diagnostic and/or therapeutic treatment. 
     
     
         41 - 42 . (canceled)