Antisense compound for inducing immunological tolerance

Abstract

A method and conjugate for selectively killing antigen-activated T cells are disclosed. The conjugate is composed of a substantially uncharged antisense compound targeted against the human cFLIP protein, and a reverse TAT (rTAT) polypeptide coupled covalently to the antisense compound. The rTAT polypeptide is effective to produce selective uptake of the conjugate into antigen-activated T cells, relative to the uptake of the conjugate into non-activated T cells. The cFLIP antisense compound causes activation induced cell death (AICD) of activated lymphocytes. The method is useful in treating transplantation rejection and autoimmune conditions.

1 . A method of enhancing uptake of a substantially uncharged antisense compound selectively into antigen-activated mammalian T cells, antigen-activated B cells, or mature dendritic cells, comprising
 covalently attaching the oligonucleotide compound, an rTAT polypeptide having the polypeptide sequence identified as SEQ ID NO: 1.   
     
     
         2 . The method of  claim 1 , wherein the rTAT polypeptide is covalently coupled at its C terminus to the 3′ or 5′ end of the antisense compound. 
     
     
         3 . The method of  claim 1 , wherein said antisense compound is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. 
     
     
         4 . A method of achieving selective uptake of a substantially uncharged antisense compound into antigen-activated T cells, comprising
 (a) exposing a population of mammalian T cells that include antigen-activated and non-activated T cells to an rTAT-antisense conjugate composed of (i) the antisense compound and (ii) covalently coupled thereto, a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO:1; and   (b) by said exposing, achieving a greater level of intracellular uptake of the antisense compound into antigen-activated T cells than is achieved (i) by exposing non-activated T cells to the same rTAT-antisense conjugate, or (ii) by exposing antigen-activated T cells to the antisense compound in the absence of the rTAT polypeptide.   
     
     
         5 . The method of  claim 4 , wherein said antisense compound in the conjugate to which the T cells are exposed is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. 
     
     
         6 . The method of  claim 5 , wherein the morpholino subunits in the conjugate to which the T 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, including dialkylamino. 
       
     
     
         7 . A method of selectively killing activated T cells, comprising
 (A) exposing a population of mammalian T cells that include antigen-activated and non-activated T cells to an rTAT-antisense conjugate composed of   (i) a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to a region of preprocessed or processed human cFLIP transcript identified by SEQ ID NOS:4-16, respectively, and by said hybridizing, to block expression of cFLIP in T cells, and   (ii) a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO: 1 and covalently coupled to the antisense compound,   (B) by said exposing, achieving selective uptake of the antisense conjugate into antigen-activated T cells, relative to the uptake of the conjugate into non-activated T cells in said population, and   (C) by said selective uptake, promoting antigen activated cell death selectively in the antigen-activated T cells of said population.   
     
     
         8 . The method of  claim 7 , wherein the antisense compound in the conjugate to which the T 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. 
     
     
         9 . The method of  claim 8 , wherein the morpholino subunits in the conjugate to which the T 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. 
       
     
     
         10 . The method of  claim 9 , wherein X═NR 2 , where each R is independently hydrogen or methyl in the compound to which the T cells are exposed. 
     
     
         11 . The method of  claim 7 , wherein the antisense compound in the conjugate to which the T cells are exposed is effective to target the start site of the processed human cFLIP transcript, and has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human cFLIP transcript, and which includes at least 6 contiguous bases of the sequence selected from the group consisting of: SEQ ID NOS:4-6. 
     
     
         12 . The method of  claim 11 , wherein the antisense compound in the conjugate to which the T cells are exposed includes a base sequence selected from the group consisting of: SEQ ID NOS:17-19. 
     
     
         13 . The method of  claim 7 , wherein the antisense compound in the conjugate to which the T cells are exposed is effective to target a splice site of preprocessed human cFLIP and has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a preprocessed human cFLIP transcript, and which includes at least 6 contiguous bases of the sequence selected from the group consisting of: SEQ ID NOS:7-15. 
     
     
         14 . The method of  claim 13 , wherein the antisense compound in the conjugate to which the T cells are exposed includes a base sequence selected from the group consisting of: SEQ ID NOS:20-28. 
     
     
         15 . The method of  claim 7 , wherein the rTAT polypeptide in the conjugate to which the T cells are exposed is covalently coupled at its C terminus to the 3′ end of the antisense compound. 
     
     
         16 . The method of  claim 7 , for use in inhibiting transplantation rejection in a human subject receiving an allograft tissue or organ, wherein said exposing includes administering the antisense conjugate to the subject, in an amount effective to inhibit the rate and extent of rejection of the transplant. 
     
     
         17 . The method of  claim 16 , wherein said administering is carried out both prior to and following the allograft tissue or organ transplantation in the subject. 
     
     
         18 . The method of  claim 7 , for use in treating an autoimmune condition in a human subject, wherein said exposing includes administering the antisense conjugate to the subject, in an amount effective to reduce the severity of the autoimmune condition. 
     
     
         19 . A antisense conjugate for use in selectively killing activated T cells, comprising
 (i) a substantially uncharged antisense compound containing 12-40 subunits and a base sequence effective to hybridize to a region of preprocessed or processed human cFLIP transcript identified by SEQ ID NO:16, respectively, and by said hybridizing, to block expression of cFLIP in T cells, and   (ii) a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO: 1 and covalently coupled to the antisense compound.   
     
     
         20 . The conjugate of  claim 19 , wherein the antisense compound is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. 
     
     
         21 . The conjugate of  claim 20 , wherein the 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, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino. 
       
     
     
         22 . The conjugate of  claim 21 , wherein X═NR 2 , where each R is independently hydrogen or methyl in the compound to which the T cells are exposed. 
     
     
         23 . The conjugate of  claim 19 , for use in targeting the start site of the processed human cFLIP transcript, wherein said antisense compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human cFLIP transcript, and which includes at least 6 contiguous bases of the sequence selected from the group consisting of: SEQ ID NOS:4-6. 
     
     
         24 . The conjugate of  claim 23 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:17-19. 
     
     
         25 . The conjugate of  claim 19 , for use in targeting the a splice site of preprocessed human cFLIP, wherein said antisense compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a preprocessed human cFLIP transcript, and which includes at least 6 contiguous bases of the sequence selected from the group consisting of: SEQ ID NOS:7-15. 
     
     
         26 . The conjugate of  claim 25 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS:20-28. 
     
     
         27 . The conjugate of  claim 19 , wherein the rTAT polypeptide in the conjugate to which the T cells are exposed is covalently coupled at its C terminus to the 3′ end of the antisense compound.