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.

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, ref fos, jun, abl, bcl, 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. 2AA-2EE . 
     
     
         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, alkylamine, 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 - 22 . (canceled) 
     
     
         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 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, ref fos, jun, abl, bcl, 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. 2AA-2EE . 
     
     
         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, alkylamine, 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 - 38 . (canceled)