Splice-region antisense composition and method

Abstract

Antisense compositions targeted against an mRNA sequence coding for a selected protein, at a region having its 5′ end from 1 to about 25 base pairs downstream of a normal splice acceptor junction in the preprocessed mRNA, are disclosed. The antisense compound is RNase-inactive, and is preferably a phosphorodiamidate-linked morpholino oligonucleotide. Such targeting is effective to inhibit natural mRNA splice processing, produce splice variant mRNAs, and inhibit normal expression of the protein.

It is claimed:  
     
         1 . An antisense compound having an uncharged morpholino backbone and a base sequence between 12 and 25 nucleotide bases in length which is complementary to a target region of a selected preprocessed mRNA coding for a protein selected from the group consisting of myc, myb, rel, fos, jun, ab1, bc1, p53, an integrin, a cathedrin, a telomerase, a cytokine, a kinase, a receptor protein, hCG, HIV rev, human papilloma virus, and human parvovirus B19, 
 where the 5′ end of the target region is 1-25 bases downstream of a normal splice acceptor site in said preprocessed mRNA.    
     
     
         2 . The compound of  claim 1 , having intersubunit linkages selected from the group consisting of the structures presented in FIGS.  2 AA- 2 EE.  
     
     
         3 . The compound of  claim 2 , wherein the linkage is a phosphorodiamidate linkage as represented at FIG. 2B-B, where X═NH 2 , NHR, or NRR′, Y═O, and Z═O, or where X═OR, Y═NH or NR′, and Z═O, and R and R′ are groups which do not interfere with target binding.  
     
     
         4 . The compound of  claim 3 , wherein R and R′ are moieties independently selected from alkyl, polyalkyleneoxy, and a combination thereof, which may be substituted with one or more groups selected from hydroxy, alkoxy, amino, alkylamino, thiol, alkanethiol, halogen, oxo, carboxylic acid, carboxylic ester, and inorganic ester.  
     
     
         5 . The compound of  claim 4 , wherein each said moiety R and R′, independent of substitution, is from 1 to 6 atoms long.  
     
     
         6 . The compound of  claim 3 , wherein NRR′ represents a nitrogen heterocycle having 5-7 ring atoms selected from nitrogen, carbon, oxygen, and sulfur, and having at least as many carbon ring atoms as non-carbon ring atoms.  
     
     
         7 . The compound of  claim 6 , wherein the 5′ end of the target region is 10-15 bases downstream of a normal splice acceptor site.  
     
     
         8 . The compound of  claim 1 , wherein the selected protein is human c-myc.  
     
     
         9 . The compound of  claim 8 , wherein the base sequence is selected from the group consisting of SEQ ID NOs: 16 through 32.  
     
     
         10 . The compound of  claim 9 , wherein the base sequence is SEQ ID NO: 25.  
     
     
         11 . The compound of  claim 8 , wherein the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 34.  
     
     
         12 . The compound of  claim 11 , wherein the base sequence is SEQ ID NO: 33.  
     
     
         13 . The compound of  claim 1 , wherein the selected protein is human androgen receptor, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 9 or SEQ ID NO: 13.  
     
     
         14 . The compound of  claim 13 , wherein the base sequence is SEQ ID NO: 8 or SEQ ID NO: 12.  
     
     
         15 . The compound of  claim 1 , wherein the selected protein is HCG-β subunit, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 15.  
     
     
         16 . The compound of  claim 15 , wherein the base sequence is SEQ ID NO: 14.  
     
     
         17 . The compound of  claim 1 , wherein the selected protein is human p53, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 36.  
     
     
         18 . The compound of  claim 17 , wherein the base sequence is SEQ ID NO: 35.  
     
     
         19 . The compound of  claim 1 , wherein the selected protein is human ab1, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 38.  
     
     
         20 . The compound of  claim 19 , wherein the base sequence is SEQ ID NO: 37.  
     
     
         21 . The compound of  claim 1 , wherein the selected protein is HIV-1 rev, and the base sequence is a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 41.  
     
     
         22 . The compound of  claim 21 , wherein the base sequence is SEQ ID NO: 40.  
     
     
         23 . A method of inhibiting normal splicing of mRNA in a eukaryotic cell, comprising 
 contacting the cell with an antisense compound having an uncharged morpholino backbone and a base sequence between 12 and 25 nucleotide bases in length which is complementary to a target region of a selected preprocessed mRNA coding for a selected protein; where the 5′ end of the target region is 1-25 bases downstream of the a normal splice acceptor site in said preprocessed mRNA,    wherein the compound:    is taken up by the cell;    hybridizes to the target region of preprocessed mRNA in the cell, and    being so hybridized, prevents splicing at said normal acceptor splice site, such that the splice mechanism proceeds to a downstream splice acceptor sequence in the mRNA, producing a splice variant processed mRNA with a truncated coding sequence.    
     
     
         24 . The method of  claim 23 , wherein the protein is selected from the group consisting of myc, myb, rel, fos, jun, ab1, bc1, p53, an integrin, a cathedrin, a telomerase, hCG, a receptor protein, a cytokine, a kinase, HIV rev, human papilloma virus, and human parvovirus B19.  
     
     
         25 . The method of  claim 24 , wherein the compound has intersubunit linkages selected from the group consisting of the structures presented in FIGS.  2 AA- 2 EE.  
     
     
         26 . The method of  claim 25 , wherein the linkage is the phosphorodiamidate linkage represented at FIG. 2B-B, where X═NH 2 , NHR, or NRR′, Y═O, and Z═O, or where X═OR, Y═NH or NR′, and Z═O, and R and R′ are groups which do not interfere with target binding.  
     
     
         27 . The method of  claim 26 , wherein R and R′ are moieties independently selected from alkyl, polyalkyleneoxy, and a combination thereof, which may be substituted with one or more groups selected from hydroxy, alkoxy, amino, alkylamino, thiol, alkanethiol, halogen, oxo, carboxylic acid, carboxylic ester, and inorganic ester.  
     
     
         28 . The method of  claim 27 , wherein each said moiety R and R′, independent of substitution, is from 1 to 12 atoms long.  
     
     
         29 . The method of  claim 26 , wherein NRR′ represents a nitrogen heterocycle having 5-7 ring atoms selected from nitrogen, carbon, oxygen, and sulfur, and having at least as many carbon ring atoms as non-carbon ring atoms.  
     
     
         30 . The method of  claim 23 , wherein the 5′ end of the target region is 10-15 bases downstream of a normal splice acceptor site.  
     
     
         31 . The method of  claim 23 , wherein said downstream splice acceptor site is a whole multiple of three bases downstream of the normal splice acceptor site, such that said splice variant mRNA has a coding sequence in frame with that of the processed mRNA when it is normally spliced.  
     
     
         32 . The method of  claim 23 , wherein the selected protein has multiple distinct binding regions, and said truncated coding sequence codes for a variant protein in which a binding region is disabled.  
     
     
         33 . The method of  claim 32 , wherein said variant protein is a dominant negative protein.  
     
     
         34 . The method of  claim 33 , wherein said selected protein is human c-myc, and said variant protein is an N-terminal truncated c-myc protein.  
     
     
         35 . The method of  claim 34 , wherein the antisense compound has a base sequence selected from the group consisting of SEQ ID NOs: 16 through 32.  
     
     
         36 . The method of  claim 35 , wherein the antisense compound has the base sequence SEQ ID NO: 25.  
     
     
         37 . The method of  claim 23 , wherein the selected protein and corresponding antisense base sequence are selected from the group consisting of: 
 (a) human chorionic gonadotropin, i subunit: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 15;    (b) human androgen receptor: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 9 or SEQ ID NO: 13;    (c) human c-myc: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 34;    (d) human p53: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 36;    (e) human ab1: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 38; and    (f) HIV-1 rev: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 41.    
     
     
         38 . The method of  claim 37 , wherein the selected protein and corresponding antisense base sequence are selected from the group consisting of: 
 (a) human chorionic gonadotropin, β subunit: SEQ ID NO: 14;    (b) human androgen receptor: SEQ ID NO: 8 or SEQ ID NO: 12;    (c) human c-myc: SEQ ID NO: 33;    (d) human p53: SEQ ID NO: 35;    (e) human ab1: SEQ ID NO: 37; and    (f) HIV-1 rev: SEQ ID NO: 40.