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

Abstract

The invention provides antisense antiviral compounds and methods of their use in inhibition of growth of human immunodeficiency virus-1 (HOV-1), as in treatment of a viral infection. The antisense antiviral compounds have morpholino subunits linked by uncharged phosphorodiamidate linkages interspersed with cationic phosphorodiamidate linkages. An exemplary embodiment of the invention provides an antisense compound directed to the HIV Vif gene, causing the production of defective HIV- 1 virions in an infected individual.

1 . An antiviral compound directed against an human immunodeficiency virus (HIV-1), comprising 
 a morpholino oligonucleotide compound composed of 12 to 40 morpholino subunits (a) with a targeting base sequence that is substantially complementary to a viral target sequence composed of at least 12 contiguous bases in a region of HIV-1 positive strand RNA identified by one of the sequences selected from the group consisting of SEQ ID NOS:17-19, and (b) that are linked by uncharged phosphorodiamidate linkages interspersed with at least two and up to half positively charged phosphorodiamidate linkages.    
     
     
         2 . The compound of  claim 1 , wherein said 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 (where base-pairing moieties on different subunits may be the same or different), and X is alkyl, alkoxy, thioalkoxy, or alkyl amino of the form NR 2 , where each R is independently hydrogen or methyl, for the uncharged linkages, and the positively charged linkages are represented by the same structure, but where X is 1-piperazino.  
       
     
     
         3 . The compound of  claim 1 , which has a T m , with respect to binding to said viral target sequence, of greater than about 45° C., and said compound is actively taken up by mammalian cells.  
     
     
         4 . The compound of  claim 1 , wherein the antisense compound is capable of hybridizing with a sequence consisting of SEQ ID NO:17 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45 degrees C., and (ii) to inhibit the synthesis of the HIV Vif protein in the infected cells.  
     
     
         5 . The compound of  claim 4 , having a targeting sequence with at least 90% homology to a sequence selected from SEQ ID NOS.5-13  
     
     
         6 . The compound of  claim 1 , wherein said compound is a covalent conjugate of the oligonucloeotide and an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.  
     
     
         7 . The compound of  claim 1 , wherein the arginine-rich polypeptide has a sequence selected from the group consisting of SEQ ID NOS: 1-4.  
     
     
         8 . The compound of  claim 1 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS: 18 and 19 to form a heteroduplex structure having a Tm of dissociation of at least 45 degrees C.  
     
     
         9 . The compound of  claim 1 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOS: 14-16.  
     
     
         10 . A method of inhibiting infection by an HIV-1 virus in a subject, comprising: 
 administering to the subject, a therapeutically effective amount of a morpholino oligonucleotide compound composed of 12 to 40 morpholino subunits (a) with a targeting base sequence that is substantially complementary to a viral target sequence composed of at least 12 contiguous bases in in a region of HIV-1 positive strand RNA identified by one of the sequences selected from the group consisting of SEQ ID NOS:17-19, and (b) that are linked by uncharged phosphorodiamidate linkages interspersed with at least two and up to half positively charged phosphorodiamidate linkages.    
     
     
         11 . The method of  claim 10 , wherein said 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 (where base-pairing moieties on different subunits may be the same or different), and X is alkyl, alkoxy, thioalkoxy, or alkyl amino of the form NR 2 , where each R is independently hydrogen or methyl, for the uncharged linkages, and the positively charged linkages are represented by the same structure, but where X is 1-piperazino.  
       
     
     
         12 . The method of  claim 10 , wherein the compound has a T m , with respect to binding to said viral target sequence, of greater than about 50° C., and said compound is actively taken up by mammalian cells.  
     
     
         13 . The method of  claim 10 , wherein the antisense compound is capable of hybridizing with a sequence consisting of SEQ ID NO:17 (i) to form a heteroduplex structure having a Tm of dissociation of at least 45 degrees C., and (ii) to inhibit the synthesis of the HIV Vif protein in the infected cells.  
     
     
         14 . The method of  claim 10 , wherein the compound has a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NOS.5-13.  
     
     
         15 . The method of  claim 10 , wherein said compound is a covalent conjugate of the oligonucloeotide and an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.  
     
     
         16 . The method of  claim 10 , wherein the arginine-rich polypeptide has a sequence selected from the group consisting of SEQ ID NOS: 1-4.  
     
     
         17 . The method of  claim 10 , wherein the antisense compound is capable of hybridizing with a sequence selected from the group consisting of SEQ ID NOS:18 and 19 to form a heteroduplex structure having a Tm of dissociation of at least 45 degrees C.  
     
     
         18 . 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:14-16.