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.

1 - 43 . (canceled) 
     
     
         44 . A method of expressing a protein of interest in a human cell in vivo comprising:
 a) providing a lentiviral particle encoding the protein of interest;   b) transducing a cell with the lentiviral particle;   c) expressing the protein of interest encoded by the lentiviral particle in the cell, and   d) administering the cell to an individual;   wherein the lentiviral particle comprises:   at least one copy of lentiviral cPPT and CTS regions;   a heterologous nucleic acid sequence encoding the protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence;   a lentiviral LTR that is deleted for the promoter and the enhancer of U3;   a lentiviral integrase protein;   a lentiviral reverse transcriptase protein;   a lentiviral Gag protein; and   an envelope protein.   
     
     
         45 . The method of  claim 44 , wherein the lentiviral cPPT and CTS regions are HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV cPPT and CTS regions. 
     
     
         46 . The method of  claim 45 , wherein the lentiviral cPPT and CTS regions are HIV-1 cPPT and CTS regions. 
     
     
         47 . The method of  claim 45 , wherein the lentiviral LTR is an HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV LTR. 
     
     
         48 . The method of  claim 46 , wherein the lentiviral LTR is an HIV-1 LTR. 
     
     
         49 . The method of  claim 47 , wherein the lentiviral integrase and reverse transcriptase proteins are HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV integrase and reverse transcriptase proteins. 
     
     
         50 . The method of  claim 48 , wherein the lentiviral integrase and reverse transcriptase proteins are HIV-1 integrase and reverse transcriptase proteins. 
     
     
         51 . The method of  claim 49 , wherein the lentiviral Gag protein is an HIV-1, HIV-2 VISNA, EIAV, FIV, or CAEV Gag protein. 
     
     
         52 . The method of  claim 50 , wherein the lentiviral Gag protein is an HIV-1 Gag protein. 
     
     
         53 . The method of  claim 44 , wherein the envelope protein is a VSV envelope protein. 
     
     
         54 . The method of  claim 44 , wherein the cell is a T cell. 
     
     
         55 . A method of expressing a protein of interest in vivo comprising:
 a) administering a lentiviral particle encoding the protein of interest to an individual;   b) transducing a cell of the individual with the lentiviral particle; and   c) expressing the protein of interest encoded by the lentiviral particle in the cell of the individual,   wherein the lentiviral particle comprises:   at least one copy of lentiviral cPPT and CTS regions;   a heterologous nucleic acid sequence encoding the protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence;   a lentiviral LTR that is deleted for the promoter and the enhancer of U3;   a lentiviral integrase protein;   a lentiviral reverse transcriptase protein;   a lentiviral Gag protein; and   an envelope protein.   
     
     
         56 . A lentiviral particle comprising:
 a) at least one copy of lentiviral cPPT and CTS regions;   b) a heterologous nucleic acid sequence encoding a protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence;   c) a lentiviral LTR that is deleted for the promoter and the enhancer of U3:   d) a lentiviral integrase protein;   e) a lentiviral reverse transcriptase protein;   f) a lentiviral Gag protein; and   g) an envelope protein.   
     
     
         57 . A method of introducing a heterologous nucleic acid sequence into the nucleus of a cell comprising transducing the cell in vitro with a lentiviral vector particle comprising a 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,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         58 . A method for producing a lentiviral gene transfer vector that promotes nuclear import of a gene of interest comprising generating a gene transfer 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,   (b) a gene of interest,   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3, and   (d) a cis-acting lentiviral encapsidation sequence.   
     
     
         59 . An isolated HIV-1 expression vector comprising:
 (a) an isolated or purified nucleic acid comprising one copy of the cPPT and CTS cis-acting regions of HIV-1, wherein said cPPT and CTS regions induce a three-stranded DNA structure,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         60 . An isolated nucleic acid comprising the nucleic acid sequence of the EcoRI/MluI insert of vector pTRIPΔU3 EF1αGFP in which the promoter and enhancer of the U3 sequence have been deleted, deposited at National Collection of Cultures of Microorganismes, Accession Number 1-2328. 
     
     
         61 . An isolated HIV-1 expression vector comprising:
 (a) an isolated or purified nucleic acid comprising 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,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         62 . A process for expressing at least one heterologous nucleic acid of interest, in vitro, wherein the process comprises:
 (a) exposing a target cell the expression vector of  claim 61  under conditions that permit uptake of the expression vector into the target cell to create a recombinant cell and   (b) culturing the recombinant cell under conditions that permit the heterologous nucleic acid sequence to be transferred to the nucleus of the recombinant cell and permit the heterologous nucleic acid sequence to be expressed.   
     
     
         63 . An isolated recombinant cell transduced with a lentiviral vector particle comprising a nucleic acid comprising:
 (a) at least one copy of the cPPT and CTS cis-acting regions of a lentivirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         64 . A method for producing an isolated recombinant cell comprising the transduction of a cell with a lentiviral vector particle,
 wherein the lentiviral vector particle comprises a nucleic acid comprising:   (a) at least one copy of the cPPT and CTS cis-acting regions of a lentivirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         65 . A recombinant lentiviral vector particle comprising Gag, Pol, and Env proteins and a 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.   
     
     
         66 . A method of introducing a heterologous nucleic acid sequence into the nucleus of a cell comprising transducing the cell in vivo with a lentiviral vector particle comprising a 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,   (b) a heterologous nucleic acid sequence, and   (c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         67 . A method of expressing a protein of interest in a human cell in vivo comprising:
 a) providing a lentiviral particle encoding the protein of interest;   b) transducing a cell with the lentiviral particle;   c) expressing the protein of interest encoded by the lentiviral particle in the cell, and   d) administering the cell to an individual;   wherein the lentiviral particle comprises at least one copy of lentiviral cPPT and CTS regions;   a heterologous nucleic acid sequence encoding the protein of interest under the control of an internal promoter not naturally associated with the heterologous nucleic acid sequence; and   a lentiviral LTR that is deleted for the promoter and the enhancer of U3.   
     
     
         68 . The method of  claim 67 , wherein the lentiviral cPPT and CTS regions are HIV-1 cPPT and CTS regions. 
     
     
         69 . The method of  claim 68 , wherein the cell is a T cell. 
     
     
         70 . 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.