Use of triplex structure DNA in transferring nucleotide sequences

Abstract

The invention concerns a recombinant vector characterised in that it comprises a polynucleotide comprising a central initiation cis-active region (cPPT) and a termination cis-active region (CTS), said regions being of retroviral or retroviral-like origin, said vector further comprising a predetermined nucleotide sequence (transgene or nucleotide sequence of interest) and retranscription regulating, expressing and packaging signals of retroviral or retroviral-like origin.

1 - 40 . (canceled)  
     
     
         41 . A recombinant vector, comprising: 
 a polynucleotide comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS), wherein the cPPT and CTS are of retroviral-like origin and derived from a retrotransposon;    a defined nucleotide sequence (transgene or sequence of interest);    and regulatory signals for reverse transcription, expression, and packaging, wherein said regulatory signals are of retroviral or retroviral-like origin.    
     
     
         42 . A recombinant vector according to  claim 41 , wherein the transgene or the sequence of interest is contained in an expression cassette comprising regulatory signals for transcription and expression.  
     
     
         43 . A recombinant vector according to  claim 41 , wherein the regulatory signals for reverse transcription, expression, and packaging, and the polynucleotide comprising the cPPT and CTS regions are derived from an HIV-type retrovirus.  
     
     
         44 . A recombinant vector according to  claim 43 , wherein the HIV-type retrovirus is HIV-1 or HIV-2.  
     
     
         45 . A recombinant vector according to  claim 41 , wherein the polynucleotide is a DNA sequence comprising the cis-acting central initiation region (cPPT) and the termination region (CTS) of an HIV-1 retroviral genome.  
     
     
         46 . A recombinant vector according to  claim 41 , wherein the polynucleotide comprises the cPPT and CTS regions of a sequence which may be selected from the sequences shown in  FIG. 11  or one of these sequences, mutated by deletion or insertion of one or more nucleotides, provided that the polynucleotide permits the formation of a triplex on reverse transcription of the vector under the control of suitable regulatory elements.  
     
     
         47 . A recombinant vector according to  claim 41 , wherein the gag, pol, and env sequences are derived from sequences of an HIV retrovirus.  
     
     
         48 . A recombinant vector according to  claim 48 , wherein the HIV retrovirus is HIV-1 or HIV-2.  
     
     
         49 . A recombinant vector according to  claim 41 , wherein the gag and pol sequences are derived from the sequences of an HIV retrovirus and the env sequence is derived from a different HIV retrovirus or from a virus.  
     
     
         50 . A recombinant vector according to  claim 41 , wherein the regulatory signals for reverse transcription, expression and packaging, and the polynucleotide comprising the cPPT and CTS regions are derived from a yeast retrotransposon.  
     
     
         51 . A recombinant cell comprising a vector according to  claim 41 .  
     
     
         52 . A method of ex vivo transfection or ex vivo transduction of non-mitotic differentiated cells, comprising transfecting or transducing the recombinant vector as claimed in  claim 41  into non-mitotic differentiated cells.  
     
     
         53 . A method of ex vivo transfection or ex vivo transduction of primary cells or immortalized cell lines, comprising transfecting or transducing the recombinant vector as claimed in  claim 41  into primary cells or immortalized cell lines.  
     
     
         54 . A polynucleotide comprising a nucleotide sequence of about 98 nucleotides, wherein said nucleotide sequence is from an HIV genome, wherein said nucleotide sequence is flanked on one side by a cis-acting central initiation nucleotide sequence (cPPT) comprising at least 10 nucleotides and on the other side by a cis-acting central termination nucleotide sequence (CTS) comprising at least 15 nucleotides, and wherein said polynucleotide comprises a segment of single-stranded or double-stranded polynucleotides.  
     
     
         55 . A polynucleotide according to  claim 54 , wherein the HIV genome is an HIV-1 genome.  
     
     
         56 . A method of in vivo transduction, comprising providing a recombinant vector or a polynucleotide as claimed in  claim 41  and transducing the recombinant vector or a polynucleotide in vivo.  
     
     
         57 . The method of  claim 56 , wherein the in vivo transduction further comprises injection of the recombinant vector or a polynucleotide into a tissue.  
     
     
         58 . A polynucleotide according to  claim 55  combined with a nucleotide sequence of interest or with a transgene.  
     
     
         59 . A recombinant retroviral vector particle, comprising: 
 (a) a gag polypeptide corresponding to a nucleoprotein of a lentivirus, or to a functional polypeptide derivative (GAG polypeptide);    (b) a pol polypeptide constituted by the RT, PRO, and IN proteins of a lentivirus, or a functional polypeptide derivative (POL polypeptide);    (c) an envelope polypeptide or a functional polypeptide derivative (ENV polypeptide); and    (d) a recombinant nucleotide sequence, comprising: 
 a defined nucleotide sequence (transgene or a sequence of interest), placed under the control of regulatory signals for transcription and expression; a sequence containing regulatory signals for reverse transcription, expression, and packaging, wherein the regulatory signals are of lentiviral origin; and a polynucleotide comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS) of lentiviral origin, inserted in a functional orientation with said regulatory signals of retroviral or retroviral-like origin.  
   
     
     
         60 . A recombinant retroviral vector particle according to  claim 59 , wherein the regulatory signals for reverse transcription, expression, and packaging, and the polynucleotide comprising the cPPT and CTS regions are derived from an HIV-type retrovirus.  
     
     
         61 . A recombinant vector according to  claim 60 , wherein the HIV-type retrovirus is HIV-1 or HIV-2.