Lentiviral triplex dna, and vectors and recombinant cells containing lentiviral triplex dna

Abstract

The present invention provides nucleic acid, vectors, viruses, and recombinant cells comprising triple-stranded structures, such as those resulting from central initiation and termination of HIV-1 reverse transcription at the center of HIV-1 linear DNA genomes. These triplex structures can act as a cis-determinant of HIV-1 DNA nuclear import, allowing infection of non-dividing target cells. In one aspect, the presence of the DNA triplex sequence in an HIV vector strongly stimulates gene transfer in hematopoietic stem cells. The invention also provides methods of using these triplex structures for making recombinant cells, as well as methods of using the recombinant cells to express proteins of interest both in vitro and in vivo.

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         44 . A kit comprising recombinant lentiviral vector particles comprising Gag, Pol, and Env proteins and at least one replication-deficient lentiviral vector comprising:
 (a) at least one copy of the cPPT (central polypurine tract) and CTS (central termination sequence) cis-acting regions of a lentivirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure upon reverse transcription,   (b) a transgene of interest, and   (c) a deletion of the promoter and the enhancer sequences of U3 region of the HIV-1 LTR.   
     
     
         45 . The kit of  claim 44 , wherein the cPPT region and the CTS region are derived from a human immunodeficiency virus type 1 genome. 
     
     
         46 . The kit of  claim 44 , wherein the recombinant vector particle comprises human immunodeficiency virus type 1 Gag, Pol, and Env proteins. 
     
     
         47 . The kit of  claim 44 , wherein the recombinant vector particle comprises human immunodeficiency virus type 1 Gag and Pol proteins, and vesicular somatitis virus Env proteins. 
     
     
         48 . The kit of  claim 44 , comprising at least two different recombinant lentiviral vector particles in different containers. 
     
     
         49 . The kit of  claim 45 , comprising at least two different recombinant lentiviral vector particles in different containers. 
     
     
         50 . A kit comprising at least one replication-deficient lentiviral vector comprising:
 (a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure upon reverse transcription,   (b) a transgene of interest, and   (c) a deletion of the promoter and the enhancer sequences of U3 region of the HIV-1 LTR.   
     
     
         51 . The kit of  claim 50 , wherein the cPPT region and the CTS region are derived from a human immunodeficiency virus type 1 genome. 
     
     
         52 . The kit of  claim 50 , comprising at least two different replication-deficient lentiviral vectors in different containers. 
     
     
         53 . The kit of  claim 51 , comprising at least two different replication-deficient lentiviral vectors in different containers. 
     
     
         54 . A kit comprising at least one nucleic acid comprising:
 (a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure upon reverse transcription,   (b) a transgene of interest, and   (c) a deletion of the promoter and the enhancer sequences of U3 region of the HIV-1 LTR.   
     
     
         55 . The kit of  claim 54 , wherein the cPPT region and the CTS region are derived from a human immunodeficiency virus type 1 genome. 
     
     
         56 . The kit of  claim 54 , comprising at least two different nucleic acids in different containers. 
     
     
         57 . The kit of  claim 55 , comprising at least two different nucleic acids in different containers.