Peptide conjugated, inosine-substituted antisense oligomer compound and method

Abstract

A therapeutic oligomer-peptide conjugate, and methods of using the conjugate are disclosed. The conjugate includes (a) a substantially uncharged oligonucleotide analog compound having a base sequence that includes a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind, and (b) conjugated to the compound, an arginine-rich peptide effective to enhance the uptake of the compound into target cells. The string of bases in the compound includes at least one inosine base positioned in the string so as to limit the number of contiguous guanine bases in said string to three or fewer. The conjugate has greater cellular uptake than the compound alone, by virtue of the arginine-rich peptide, and substantially greater antisense activity greater activity than the conjugate in the absence of inosine-for guanine substitutions.

1 . In a method for enhancing the cellular uptake of a substantially uncharged oligonucleotide analog compound, by forming a conjugate of the compound and an arginine-rich peptide effective to enhance the uptake of the compound into target cells, and where the compound includes a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind, an improvement comprising substituting an inosine base for at least one guanine base in said string of bases in said compound so as to limit the number of contiguous guanine bases in said string to three or fewer.  
     
     
         2 . The improvement of  claim 1 , wherein the inosine substitution is effective to limit the number of contiguous guanine bases in said string to two or fewer.  
     
     
         3 . The improvement of  claim 1 , wherein at least two inosine bases are substituted in said string of bases.  
     
     
         4 . The improvement of  claim 1 , wherein said substituting is effective to enhance the water solubility of the conjugate during a purification step involving conjugate binding to and release from an cationic ion exchange resin, relative to the same conjugate in the absence of the inosine substitution.  
     
     
         5 . The improvement of  claim 1 , wherein said target nucleic acid region includes the start codon in an mRNA, and said substituting is effective to enhance the ability of the conjugate to block translation of the protein encoded by the mRNA, relative to the same conjugate in the absence of the inosine substitution.  
     
     
         6 . The improvement of  claim 1 , wherein said target nucleic acid region includes a donor or acceptor splice site in an preprocessed mRNA, and said substituting is effective to enhance the ability of the conjugate to mask mRNA splicing at said target region, relative to the same conjugate in the absence of the inosine substitution.  
     
     
         7 . The improvement of  claim 1 , wherein said target nucleic acid region includes a virally-encoded cis-acting element involved in viral replication, and said substituting is effective to enhance the ability of the conjugate to block viral replication, relative to the same conjugate in the absence of the inosine substitution.  
     
     
         8 . The improvement of  claim 1 , wherein said arginine-rich peptide comprises 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where >50% of said subunits are X subunits, and where 
 (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R 1 N═C(NH 2 )R 2 , where R 1  is H or R; R 2  is R, NH 2 , NHR, or NR 2 , where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R 1  and R 2  may together form a ring; and the side chain is linked to said amino acid via R 1  or R 2 ;    (b) each Y subunit independently represents a neutral amino acid —C(O)—(CHR) n —NH—, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and    (c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine.    
     
     
         9 . The improvement of  claim 8 , wherein said oligonucleotide analog compound is a morpholino oligomer composed of morpholino subunits linked by phosphorus-containing linkages between the morpholino nitrogen of one subunit and an exocyclic carbon at the morpholino 3-position of an adjacent subunit.  
     
     
         10 . The improvement of  claim 9 , wherein said morpholino subunits are joined by uncharged 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, or alkyl amino.  
     
     
         11 . A therapeutic oligomer-peptide conjugate, comprising 
 (a) a substantially uncharged oligonucleotide analog compound having a base sequence that includes a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind, and    (b) conjugated to said compound, an arginine-rich peptide effective to enhance the uptake of the compound into target cells, where    said string of bases in the compound includes at least one inosine base, positioned in said string so as to limit the number of contiguous guanine bases in said string to three or fewer.    
     
     
         12 . The conjugate of  claim 11 , wherein the position of one or more inosine bases in said compound is such as to limit the number of contiguous guanine bases in said string to two or fewer.  
     
     
         13 . The conjugate of  claim 12 , wherein said compound includes at least two inosine bases in said string of bases.  
     
     
         14 . The conjugate of  claim 11 , wherein said arginine-rich peptide comprises 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where >50% of said subunits are X subunits, and where 
 (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R 1 N═C(NH 2 )R 2 , where R 1  is H or R; R 2  is R, NH 2 , NHR, or NR 2 , where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R 1  and R 2  may together form a ring; and the side chain is linked to said amino acid via R 1  or R 2 ;    (b) each Y subunit independently represents a neutral amino acid —C(O)—(CHR) n —NH—, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and    (c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine.    
     
     
         15 . The conjugate of  claim 14 , wherein said oligonucleotide analog compound is a morpholino oligomer composed of morpholino subunits linked by phosphorus-containing linkages between the morpholino nitrogen of one subunit and an exocyclic carbon at the morpholino 3-position of an adjacent subunit.  
     
     
         16 . The conjugate of  claim 15 , wherein said morpholino subunits are joined by uncharged 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, or alkyl amino.  
     
     
         17 . The conjugate of  claim 11 , which has a greater water solubility during a purification step involving conjugate binding to and release from an cationic ion exchange resin than the same conjugate but for said one or more inosine bases.  
     
     
         18 . The conjugate of  claim 11 , for use in blocking processing of a preprocessed mRNA at a selected donor or acceptor splice site in the target nucleic acid region of the preprocessed mRNA, which is more active in blocking such mRNA splice-site processing than the same conjugate but for said one or more inosine bases.  
     
     
         19 . The conjugate of  claim 11 , for use in blocking viral replication in cells infected with a virus whose target nucleic acid region includes a cis-acting element essential for viral replication, which is more active in blocking such viral replication than the same conjugate but for said one or more inosine bases.  
     
     
         20 . The conjugate of  claim 11 , for use in blocking translation of a protein encoded by an mRNA whose target nucleic acid region includes a start codon, which is more active in blocking such translation than the same conjugate but for said one or more inosine bases.  
     
     
         21 . The conjugate of  claim 20 , wherein said compound (i) is targeted against a region including the AUG start site of human c-myc mRNA and (ii) includes a targeting sequence selected from the group consisting of SEQ ID NOS: 2-10.  
     
     
         22 . The conjugate of  claim 21 , wherein said peptide includes a sequence selected from group consisting of SEQ ID NOS: 16, 17 and 18.  
     
     
         23 . The conjugate of  claim 11 , wherein said peptide includes a sequence selected from group consisting of SEQ ID NOS: 16, 17 and 18.  
     
     
         24 . A method of treating a subject having a pathological condition responsive to inhibition of c-myc expression in subject target cells, comprising 
 administering to the subject, a therapeutically effective amount of an oligomer-peptide conjugate composed of    (a) a substantially uncharged oligonucleotide analog compound having a base sequence that includes a string of bases that are complementary to four or more contiguous cytosine bases in a target mRNA region that includes the mRNA start codon, and    (b) conjugated to said compound, an arginine-rich peptide effective to enhance the uptake of the compound into such target cells, where    (i) the base sequence of the compound includes, in said string of bases at least one inosine base, positioned in said string so as to limit the number of contiguous guanine bases in said string to three or fewer, and    (ii) the conjugate is more active in blocking such translation than the same conjugate but for said one or more inosine bases.    
     
     
         25 . The method of  claim 24 , wherein said arginine-rich peptide comprises 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where >50% of said subunits are X subunits, and where 
 (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R 1 N═C(NH 2 )R 2 , where R 1  is H or R; R 2  is R, NH 2 , NHR, or NR 2 , where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R 1  and R 2  may together form a ring; and the side chain is linked to said amino acid via R 1  or R 2 ;    (b) each Y subunit independently represents a neutral amino acid —C(O)—(CHR) n —NH—, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is a neutral side chain selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, and aralkyl, wherein said neutral side chain, when selected from substituted alkyl, alkenyl, and alkynyl, includes at most one heteroatom for every four carbon atoms; and    (c) each Z subunit independently represents an amino acid selected from alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine, and threonine.    
     
     
         26 . The method of  claim 25 , wherein said oligonucleotide analog compound is a morpholino oligomer composed of morpholino subunits linked by phosphorus-containing linkages between the morpholino nitrogen of one subunit and an exocyclic carbon at the morpholino 3-position of an adjacent subunit.  
     
     
         27 . The method of  claim 26 , wherein said morpholino subunits are joined by uncharged 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, or alkyl amino.  
     
     
         28 . The method of  claim 27 , wherein said compound includes a targeting sequence selected from the group consisting of SEQ ID NOS: 2-10.  
     
     
         29 . The method of  claim 27 , wherein said peptide includes a sequence selected from group consisting of SEQ ID NOS: 16, 17 and 18.  
     
     
         30 . The method of  claim 29 , wherein said compound includes a targeting sequence selected from the group consisting of SEQ ID NOS: 2-10.  
     
     
         31 . The method of  claim 24 , for use in treating bladder cancer, wherein said conjugate is administered by transurethral delivery, and the method further includes administered a cis-platin anti-cancer compound to the patient.  
     
     
         32 . The method of  claim 24 , for use in reducing the risk of coronary-artery restenosis at the site of vascular injury following an angioplasty procedure, wherein said conjugate is administered by intravascular delivery.  
     
     
         33 . The method of  claim 32 , wherein said angioplasty procedure includes placement of a stent at the site of vascular injury, and said conjugate is administered by release from the stent.  
     
     
         34 . The method of  claim 24 , for use in protecting an saphenous vein placed during a coronary bypass operation, and said conjugate is administered by exposing the vein to the conjugate prior to its surgical placement.  
     
     
         35 . The method of  claim 24 , for treating polycistic kidney disease, wherein said conjugate is administered to the subject by oral or parenteral administration.