Sequence of natural or synthetic retroelements enabling nucleotide sequence insertion into a eukaryotic cell

Abstract

A retroviral vector that eliminates cis-acting retroviral elements, which are not useful to and may cause problems for, an integrated provirus utilizes a recombination system, such as the bacteriophage P1 cre-lox recombination system. A gene of interest and a loxP site are inserted into a long terminal repeat, which is then duplicated. The enzyme cre is inserted between the long terminal repeats and combines them. The structure of the resulting provirus in the host genome is that of a single, long terminal repeat carrying a single copy of the gene of interest. Following infection, the retrovirus produces proviral structures having only a single long terminal repeat.

1 . A sequence of synthetic or natural retroelements, which comprises an insertion sequence incorporated in a region that can be transferred into a target cell and integrated into a recombinant provirus when said target cell is infected by a retrovirus comprising said sequence of retroelements, said insertion sequence comprising: 
 (a) a nucleotide sequence of interest which can be integrated into the genome of the target cell; and    (b) a recombinase recognition sequence;    wherein said retroelements comprise a 3′ and/or 5′ LTR region and said insertion sequence is incorporated into said 3′ LTR region or 5′ LTR region.    
     
     
         2 - 24 . (canceled)  
     
     
         25 . A nucleic acid molecule comprising retroelements that comprise a recombinant provirus when a target cell is infected by a retrovirus containing said retroelements, wherein said retroelements comprise an insertion sequence, said insertion sequence comprising: 
 (a) a nucleotide sequence of interest, which can be expressed in the target cell and which can be transferred with said retroelements into the target cell and integrated into the recombinant provirus; and    (b) a recombinase recognition site for the elimination of proviral sequences in the recombinant provirus, which are not necessary for expression of the nucleotide sequence of interest in the target cell after integration of the recombinant provirus into the target cell;    wherein said retroelements comprise a 3′ and/or 5′ LTR region and said insertion sequence is incorporated into said 3′ LTR region or 5′ LTR region.    
     
     
         26 - 29 . (canceled)  
     
     
         30 . The nucleic acid molecule as claimed in claim  28 , wherein the recombinase recognition sequence is situated upstream or downstream from the nucleotide sequence of interest.  
     
     
         31 . The nucleic acid molecule as claimed in  claim 25 , wherein the recombinase recognition sequence is located upstream or downstream from the nucleotide sequence of interest.  
     
     
         32 . The nucleic acid molecule as claimed in  claim 25 , wherein said nucleic acid molecule comprises a nucleotide sequence coding for a recombinase that recognizes said recombinase recognition site.  
     
     
         33 . The nucleic acid molecule as claimed in  claim 32 , wherein the retroelements comprise 5′ LTR and 3′ LTR regions and the nucleotide sequence coding for the recombinase is situated between the 5′ LTR and 3′ LTR regions.  
     
     
         34 . The nucleic acid molecule as claimed in  claim 32 , wherein the nucleotide sequence coding for the recombinase encodes CRE protein.  
     
     
         35 . The nucleic acid molecule as claimed in  claim 33 , wherein the nucleotide sequence coding for the recombinase encodes CRE protein.  
     
     
         36 . The nucleic acid molecule as claimed in any one of claims  33 ,  34 , or  35 , wherein the recombinase recognition sequence comprises a Lox P recognition site.  
     
     
         37 . The nucleic acid molecule as claimed in  claim 25 , wherein the nucleotide sequence of interest encodes a polypeptide or RNA.  
     
     
         38 . The nucleic acid molecule as claimed in  claim 37 , wherein the RNA is antisense RNA or a ribozyme sequence.  
     
     
         39 . A nucleic acid molecule comprising retroelements that comprise a recombinant provirus when a target cell is infected by a retrovirus containing said retroelements; said retroelements comprise a 3′ and/or 5′ LTR region; an insertion sequence located in the 3′ LTR region or the 5′ LTR region; said insertion sequence comprises a nucleotide sequence of interest, which can be expressed in the target cell and which can be transferred with said retroelements into the target cell and integrated into the recombinant provirus; and a recombinase recognition site for the elimination of proviral sequences in the recombinant provirus, which are not necessary for expression of the nucleotide sequence of interest in the target cell after integration of the recombinant provirus into the target cell, wherein the nucleic acid molecule is contained in a plasmid deposited under C.N.C.M. Accession No. I-1599.  
     
     
         40 . A retroviral vector comprising a nucleic acid molecule according to  claim 25 .  
     
     
         41 . The sequence of retroelements as claimed in  claim 1 , wherein the recombinase recognition sequence is a single recognition sequence.  
     
     
         42 . The sequence of retroelements as claimed in  claim 1 , wherein the sequence is comprised of retroviral DNA.  
     
     
         43 . (canceled)  
     
     
         44 . The sequence of retroelements as claimed in  claim 41 , wherein said retroelements comprise U3 of 3′ LTR and/or U5 of 5′ LTR and the insertion sequence is incorporated into U3 or U5.  
     
     
         45 . The nucleic acid molecule as claimed in  claim 25 , wherein said retroelements comprise U3 of a 3′ LTR and/or U5 of a 5′ LTR, and the sequence of interest and the recombinase recognition sequence are incorporated into U3 or U5.  
     
     
         46 . The nucleic acid molecule as claimed in  claim 1 , wherein the 3′ LTR region comprises a U3 region and an R region, wherein the sequence of interest and the recombinase recognition sequence are incorporated into one of said U3 region and R region.  
     
     
         47 . The nucleic acid molecule as claimed in  claim 1 , wherein the 5′ LTR region comprises a U5 region and an R region, wherein the sequence of interest and the recombinase recognition sequence are incorporated into one of said U5 region and R region.  
     
     
         48 . The sequence of retroelements as claimed in  claim 25 , wherein the 3′ LTR region comprises a U3 region and an R region, wherein the sequence of interest and the recombinase recognition sequence are incorporated into one of said U3 region and R region.  
     
     
         49 . The sequence of retroelements as claimed in  claim 25 , wherein the 5′ LTR region comprises a U5 region and an R region, wherein the sequence of interest and the recombinase recognition sequence are incorporated into one of said U5 region and R region.