Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells

Abstract

A method and conjugate for selectively targeting activated hematopoietic cells, e.g., macrophage or T-lymphocyte cells, are disclosed. The conjugate is composed of a substantially uncharged antisense compound targeted against HIV, and a reverse TAT (rTAT) polypeptide coupled covalently to the antisense compound. The rTAT polypeptide is effective to produce selective uptake of the conjugate into activated, HIV-infected cells, e.g., activated, HIV-infected macrophage and T-lymphocyte cells. An exemplary embodiment of the invention provides an antisense compound directed to the HIV Vif gene, causing the production of defective HIV virions in an infected individual.

1 . A method of achieving selective uptake of a substantially uncharged antisense compound into activated human macrophage or T lymphocyte cells, comprising 
 (a) exposing a population of human macrophage or T lymphocyte cells that include activated human macrophage or T lymphocyte cells to an rTAT-antisense conjugate composed of (i) the antisense compound and (ii) covalently coupled thereto, a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO:1; and    (b) by said exposing, achieving a greater level of intracellular uptake of the oligonucleotide analog into activated macrophage or T-lymphocyte cells than is achieved (i) by exposing non-activated macrophage or T-lymphocyte cells to the same antisense conjugate, or (ii) by exposing activated macrophage or T-lymphocyte cells to the antisense compound in the absence of the rTAT polypeptide.    
     
     
         2 . The method of  claim 1 , wherein said antisense compound in the conjugate to which the cells are exposed is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.  
     
     
         3 . The method of  claim 2 , wherein the morpholino subunits in the conjugate to which the cells are exposed are joined by phosphorodiamidate linkages, in accordance with the structure:  
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino.  
       
     
     
         4 . The method of  claim 1 , wherein the rTAT polypeptide in the conjugate to which the cells are exposed is covalently coupled at its C terminus to the 3′ end of the antisense compound.  
     
     
         5 . A method of selectively inhibiting HIV replication in activated, HIV-infected human macrophage or T lymphocyte cells, comprising 
 (a) exposing the activated cells to an rTAT-antisense conjugate composed of:    (i) a substantially uncharged antisense compound capable of hybridizing with an HIV sense-strand RNA sequence, to inhibit HIV replication in HIV-infected cells, and    (ii) covalently attached to the agent, an rTAT polypeptide having the polypeptide sequence identified as SEQ ID NO: 1, and    (b) by said exposing, (i) achieving a greater level of intracellular uptake of the oligonucleotide analog into activated, HIV-infected macrophage or T-lymphocyte cells than is achieved (ia) by exposing non-activated, non-HIV-infected macrophage or T-lymphocyte cells to the same conjugate compound, or (ib) by exposing activated, HIV-infected macrophage or T-lymphocyte cells to the oligonucleotide analog in the absence of the rTAT polypeptide, and (ii) thereby achieving selective inhibition of HIV in the activated, HIV-infected cells.    
     
     
         6 . The method of  claim 5 , wherein said antisense compound in the conjugate to which the cells are exposed is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.  
     
     
         7 . The method of  claim 6 , wherein the morpholino subunits in the conjugate to which the cells are exposed are joined by phosphorodiamidate linkages, in accordance with the structure:  
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino.  
       
     
     
         8 . The method of  claim 1 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 26-47 to form a heteroduplex structure having a Tm of dissociation of at least 45° C.  
     
     
         9 . The method of  claim 8 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS: 4-25.  
     
     
         10 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 26-31 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to inhibit the synthesis of the HIV Vif protein in the infected cells.  
     
     
         11 . The method of  claim 10 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS: 4-9.  
     
     
         12 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with SEQ ID NO:32 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to inhibit the synthesis of HIV Gag-pol precursor polyprotein.  
     
     
         13 . The method of  claim 12 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NO:10.  
     
     
         14 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with SEQ ID NOS: 33, 45 and 46, (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block inhibit the synthesis of the HIV Rev phosphoprotein.  
     
     
         15 . The method of  claim 14 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NOS:11, 23 and 24.  
     
     
         16 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 35-37, and 40-44 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block cis-acting tRNA-primer binding and activation sites and Psi-packaging and dimerization elements.  
     
     
         17 . The method of  claim 16 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS:13-15 and 18-22.  
     
     
         18 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 38 and 39 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block the major splice donor site.  
     
     
         19 . The method of  claim 18 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS:16 and 17.  
     
     
         20 . The method of  claim 8 , wherein the antisense compound is capable of hybridizing with SEQ ID NO:47 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block reverse transcription of viral RNA by blocking the minus-strand transfer step.  
     
     
         21 . The method of  claim 20 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NO:25.  
     
     
         22 . A antisense conjugate for use in selectively inhibiting HIV replication in activated, HIV-infected human macrophage or T lymphocyte cells, comprising 
 (i) a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize with an HIV sense-strand RNA sequence, to inhibit replication of HIV replication in HIV-infected cells, and    (ii) a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO: 1 and covalently coupled to the antisense compound.    
     
     
         23 . The conjugate of  claim 22 , wherein the antisense compound is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.  
     
     
         24 . The conjugate of  claim 23 , wherein the morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:  
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl, and dialkyl amino.  
       
     
     
         25 . The conjugate of  claim 24 , wherein X=NR 2 , where each R is independently hydrogen or methyl in the compound to which the T cells are exposed.  
     
     
         26 . The conjugate of  claim 22 , wherein the rTAT polypeptide is covalently coupled at its C terminus to the 3′ end of the antisense compound.  
     
     
         27 . The conjugate of  claim 22 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS:26-47 to form a heteroduplex structure having a Tm of dissociation of at least 45° C.  
     
     
         28 . The conjugate of  claim 27 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS: 4-17.  
     
     
         29 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 26-31 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to inhibit the synthesis of the HIV Vif protein in the infected cells.  
     
     
         30 . The conjugate of  claim 29 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS: 4-9.  
     
     
         31 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with SEQ ID NO:32 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to inhibit the synthesis of HIV Gag-Pol precursor polyprotein.  
     
     
         32 . The conjugate of  claim 31 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NO:10.  
     
     
         33 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with SEQ ID NOS: 33, 45 and 46, (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block inhibit the synthesis of the HIV Rev phosphoprotein.  
     
     
         34 . The conjugate of  claim 33 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NOS:11, 23 and 24.  
     
     
         35 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 35-37, and 40-44 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block cis-acting tRNA-primer binding and activation sites and Psi-packaging and dimerization elements.  
     
     
         36 . The conjugate of  claim 35 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS:13-15 and 18-22.  
     
     
         37 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 38 and 39 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block the major splice donor site.  
     
     
         38 . The conjugate of  claim 37 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS:16 and 17.  
     
     
         39 . The conjugate of  claim 27 , wherein the antisense compound is capable of hybridizing with SEQ ID NO:47 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45° C., and (ii) to block reverse transcription of viral RNA by blocking the minus-strand transfer step.  
     
     
         40 . The conjugate of  claim 39 , wherein the antisense compound has at least 12 contiguous bases from the sequence identified as SEQ ID NO:25.