Synthetic gagpol genes and their uses

Abstract

The present invention relates to synthetic gag and gagpol genes optimized for high level expression via codon optimization and the uses thereof for the efficient generation of vector particles. The invention further relates to the generation of packaging cells and vaccines based on the synthetic gag and gagpol genes.

1 . - 20 . (canceled) 
     
     
         21 . Nucleic acid sequence encoding the gag and pol polypeptides, whereby the amino acid Ala is encoded by the nucleic acid triplet gcc or get, Arg is encoded by agg or aga, Asn is encoded by aac or aat, Asp is encoded by gac or gat, Cys is encoded by tgc or tgt, Gln is encoded by cag or cat, Glu is encoded by gag or gaa, Gly is encoded by ggc or gga, His is encoded by cac or cat, Ile is encoded by ate or att, Leu is encoded by ctg or ctc, Lys is encoded by aag or aat, Met is encoded by atg, Phe is encoded by ttc or ttt, Pro is encoded by ccc or cct, Ser is encoded by agc or tee, Thr is encoded by acc or aca, Trp is encoded by tgg, Tyr is encoded by tac or tat, Val is encoded by gtg ot gtc and the stop codon is tga or taa. 
     
     
         22 . Nucleic acid sequence according to  claim 21 , whereby the nucleic acid sequence in the region in which the reading frames encoding the gag and pol polypeptides overlap corresponds to the wildtype nucleic acid sequence encoding the gag and pol polypeptides. 
     
     
         23 . Nucleic acid sequence as depicted in SEQ ID NO:1 or 2. 
     
     
         24 . Nucleic acid sequence encoding the gag polypeptide, whereby the amino acid Ala is encoded by the nucleic acid triplet gcc or get, Arg is encoded by agg or aga, Asn is encoded by aac or aat, Asp is encoded by gac or gat, Cys is encoded by tgc or tgt, Gln is encoded by cag or cat, Glu is encoded by gag or gaa, Gly is encoded by ggc or gga, His is encoded by cac or cat, Ile is encoded by ate or att, Leu is encoded by ctg or etc, Lys is encoded by aag or aat, Met is encoded by atg, Phe is encoded by ttc or ttt, Pro is encoded by ccc or cct, Ser is encoded by agc or tee, Thr is encoded by acc or aca, Trp is encoded by tgg, Tyr is encoded by tae or tat, Val is encoded by gtg ot gtc, and wherein said nucleic acid triplets are used only in the 5′ sequence of the gag encoding nucleic acid, in particular from nucleotide 1 to 150, from 1-294, from 1-489, from 1-697, or from 1-854. 
     
     
         25 . Retroviral gag or gagpol based vector particle, whereby the packaging proteins are derived from retroviruses and are encoded by nucleic acids according to  claim 21  and the transgene construct is derived from wildtyp retroviruses. 
     
     
         26 . Retroviral gag or gagpol based vector particle according to  claim 25 , wherein the retrovirus is selected from the group of oncoviruses, HTLV-1 or -2, spumaviruses, lentiviruses, in particular HIV, in particular HIV-1, HIV-1 IIIB , HIV-2, and SIV, in particular SIV mac239 . 
     
     
         27 . Retroviral gag or gagpol based vector particle whereby the packaging proteins are derived from a first retrovirus and the transgene construct is derived from a different second retrovirus and whereby the packaging proteins are encoded by nucleic acids according to  claim 21 . 
     
     
         28 . Retroviral gag or gagpol based vector particle according to  claim 27 , wherein the first and second retrovirus are selected from the group of oncoviruses, HTLV-1 and 2, spumaviruses, lentiviruses, in particular HIV, in particular HIV-1, HIV- 1I   IIB , HIV-2, and SIV, in particular SIV mac239 . 
     
     
         29 . Retroviral gag or gagpol based vector particle generated by nucleic acid molecules comprising the nucleic acid sequence according to  claim 21  having at least a 25 fold reduced incorporation rate of said nucleic acid molecules encoding the gag and pol polypeptides compared to retroviral particles generated by wildtyp nucleic acid sequences encoding the gag and pol polypeptides. 
     
     
         30 . Retroviral gag or gagpol based vector particle produced by nucleic acid molecules comprising the nucleic acid sequence according to  claim 21  having at least a 100 fold reduced recombination rate between said nucleic acid molecules encoding the gag and pol polypeptides and a wildtype genome based transgene construct derived from the same or another retrovirus. 
     
     
         31 . Retroviral packaging cell for the generation of the retroviral vector particles according to  claim 25  transformed with nucleic acid molecules comprising the nucleic acid sequence according to  claim 21 . 
     
     
         32 . Nucleic acid sequence as depicted in SEQ ID NO:4. 
     
     
         33 . Nucleic acid molecule according to  claim 21  comprising an amino acid substitution wherein the myristilation of the gag-precursor is inhibited. 
     
     
         34 . Nucleic acid molecule according to  claim 33 , wherein the HIV or SIV N-terminal amino acid glycin is substituted to alanin. 
     
     
         35 . Nucleic acid molecule according to any  claim 32 , wherein one nucleotide is added or two nucleotides are deleted to introduce a ribosomal frameshift so that the gag and pol coding regions are using the same reading frame. 
     
     
         36 . Nucleic acid molecule according to  claim 32  comprising a deletion of the complete protease gene or of a part of the protease gene or one or more point mutations in the protease gene so that the protease is rendered inactive. 
     
     
         37 . Nucleic acid molecule according to  claim 32  comprising a deletion of the complete reverse transcriptase gene or of a part of the reverse transcriptase gene or one or more point mutations in the reverse transcriptase gene so that the reverse transcriptase is rendered inactive. 
     
     
         38 . Nucleic acid molecule according to  claim 32  comprising a deletion of the complete integrase gene or of a part of the integrase gene or one or more point mutations in the integrase gene so that the integrase is rendered inactive. 
     
     
         39 . Nucleic acid molecules comprising the nucleic acid sequence according to  claim 21  for use as an active pharmaceutical substance. 
     
     
         40 . Use of nucleic acid molecules comprising the nucleic acid sequence according to  claim 21  for the preparation of a pharmaceutical composition for the treatment and prophylactic of diseases caused by retroviruses.