Antisense oligomers and methods for inducing immune tolerance and immunosuppression

Abstract

A method and composition for inducing human dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10 is disclosed. A population of dendritic cells is exposed to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD86 transcript identified, in its processed form, by SEQ ID NO:33, to form a duplex structure between said compound and transcript having a Tm of at least 45° C. Formation of the duplex blocks expression of full-length CD86 in said cells, which in turn leads to reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10.

1 . A method of inducing human dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10, comprising 
 (a) exposing a population of human dendritic cells to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C.,    (b) by said forming, blocking expression of full-length CD86 in said cells, and    (c) by said blocking, producing inhibition of the expression of full-length CD86 on the surface of dendritic cells, and enhanced expression of extracellular IL-10 by mature dendritic cells.    
     
     
         2 . The method of  claim 1 , wherein the antisense compound to which the dendritic cells are exposed is composed of 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 compound to which the dendritic 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, and said heteroduplex structure formed in step (a) has a Tm of at least 50° C.  
     
     
         4 . The method of  claim 3 , wherein X═NR 2 , where each R is independently hydrogen or methyl in the compound to which the T cells are exposed.  
     
     
         5 . The method of  claim 1 , wherein the dendritic cells which are exposed to said compound include mature dendritic cells, and said blocking step (b) is effective to enhance expression of extracellular IL-10 by the dendritic cells.  
     
     
         6 . The method of  claim 1 , wherein said compound is covalently linked, at one compound end, to an arginine-rich peptide effective to enhance uptake of said compound into the dendritic cells.  
     
     
         7 . The method of  claim 6 , wherein said arginine-rich peptide has a sequence selected from the group consisting of SEQ ID NOS: 1 and 2.  
     
     
         8 . The method of  claim 6 , wherein said dendritic cells which are exposed to said compound include a mixture of immature and mature dendritic cells, said arginine-rich peptide is an rTAT peptide having the sequence identified by SEQ ID NO: 1, and the rTAT peptide is effective to achieve a greater level of intracellular uptake of the antisense compound into the mature dendritic cells than is achieved (i) in the immature dendritic cells, or (ii) by exposing the mature dendritic cells to the antisense compound in the absence of the rTAT polypeptide.  
     
     
         9 . The method of  claim 1 , wherein said antisense compound is effective to hybridize to a target region adjacent the start site of the processed human CD86 transcript, the compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human CD86 transcript identified by SEQ ID NO:9, and said blocking step (b) is effective to block translation of said processed transcript.  
     
     
         10 . The method of  claim 9 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:21-23 and 32.  
     
     
         11 . The method of  claim 1 , wherein the antisense compound is effective to hybridize to a splice site of preprocessed human CD68, and has a base sequence that is complementary to at least 12 contiguous bases of a splice site in a preprocessed human CD86 transcript, and said blocking step (b) is effective to block processing of a preprocessed CD86 transcript to produce a full-length, processed CD86 transcript.  
     
     
         12 . The method of  claim 11 , wherein the splice site in the preprocessed CD86 transcript has a sequence selected from the group consisting of: SEQ ID NOS:10-14.  
     
     
         13 . The method of  claim 12 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:24-28.  
     
     
         14 . The method of  claim 1 , for use in inhibiting transplantation rejection in a human subject receiving an allograft tissue or organ, wherein said exposing includes administering the antisense compound to the subject, in an amount effective to inhibit the rate and extent of rejection of the transplant.  
     
     
         15 . The method of  claim 14 , wherein said administering is carried out both prior to and following the allograft tissue or organ transplantation in the subject.  
     
     
         16 . The method of  claim 14 , wherein said administering is carried out for a selected period of 1-3 weeks.  
     
     
         17 . The method of  claim 16 , which further includes further administering the antisense compound to the subject, as needed, to control the extent of transplantation rejection in the subject.  
     
     
         18 . The method of  claim 1 , for use in treating an autoimmune condition in a human subject, wherein said exposing includes administering the antisense compound to the subject, in an amount effective to reduce the severity of the autoimmune condition.  
     
     
         19 . The method of  claim 18 , wherein said administering is carried out over an extended period of time, as needed, to control the severity of the autoimmune condition in the subject.  
     
     
         20 . A method of inducing mature human dendritic cells to a condition of increased production of extracellular IL-10, comprising 
 (a) exposing the population of cells containing mature dendritic cells to a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD-86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C.,    (b) by said forming, blocking expression of full-length CD86 in said cells, and    (c) by said blocking, enhancing expression of extracellular IL-10 by the mature dendritic cells.    
     
     
         21 . A composition for use in inducing dendritic cells to a condition of reduced capacity for antigen-specific activation of T cells, and, in mature dendritic cells, increased production of extracellular IL-10, said conjugate comprising, 
 a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to an expression-sensitive region of a preprocessed or processed human CD-86 transcript identified, in its processed form, by SEQ ID NO:33, to form a heteroduplex structure between said compound and transcript having a Tm of at least 45° C.    
     
     
         22 . The composition of  claim 21 , wherein the antisense compound is composed of phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.  
     
     
         23 . The composition of  claim 22 , wherein the morpholino subunits in the compound to which the dendritic 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, and said heteroduplex structure has a Tm of at least 50° C.  
     
     
         24 . The composition of claim  27 , wherein X═NR 2 , where each R is independently hydrogen or methyl in the compound to which the T cells are exposed.  
     
     
         25 . The composition of  claim 21 , which further includes an arginine-rich peptide covalently linked to one end of said antisense compound, and said peptide is effective to promote uptake of the composition into dendritic cells.  
     
     
         26 . The composition of  claim 25 , wherein said arginine-rich peptide is an rTAT peptide having the sequence identified as SEQ ID NO: 1, and said peptide is effective to achieve a greater level of intracellular uptake of the antisense compound into the mature dendritic cells than is achieved (i) in the immature dendritic cells, or (ii) by exposing the mature dendritic cells to the antisense compound in the absence of the rTAT polypeptide.