Immunomodulatory agents and methods of use

Abstract

An antisense oligonucleotide compound, composition, vaccine and methods for treating a variety of conditions characterized by up-regulation of IL-10 in a mammalian subject are disclosed. The compound (i) 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, (ii) is capable of uptake by monocytes, lymphocytes, and dendritic cells in a mammalian subject, (iii) contains between 10-40 nucleotide bases, and (iv) has a base sequence effective to hybridize to at least 12 contiguous bases of a target sequence contained in an exon-2 or exon-4 slice site region of human IL-10 pre-mRNA.

1 . A method of treating a mammalian subject infected with a pathogen which acts to up-regulate IL-10 during infection in a mammalian host, as evidenced by increased serum levels of IL-10, comprising administering to the subject, a therapeutically effective amount of an antisense composition containing an antisense oligonucleotide compound
 (i) 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,   (ii) capable of uptake by monocytes, lymphocytes, and dendritic cells in a mammalian subject,   (iii) containing between 10-40 nucleotide bases, and   (iv) having a base sequence effective to hybridize to at least 12 contiguous bases of the 5′-most 25 bases of exon 2 or exon 4 of the preprocessed human IL-10 transcript contained within SEQ ID NO:6, and identified by SEQ ID NOS:2 and 5, respectively.   
     
     
         2 . The method of  claim 1 , wherein the compound administered has a base sequence effective to hybridize to at least 12 contiguous bases of SEQ ID NO:5 in SEQ ID NO:6. 
     
     
         3 . The method of  claim 1 , wherein the compound administered has a base sequence effective to hybridize to at least 12 contiguous bases of SEQ ID NO:2 in SEQ ID NO:6. 
     
     
         4 . The method of  claim 1 , wherein the target region to which the oligonucleotide compound hybridizes is contained entirely within SEQ ID NOS:2 or 5. 
     
     
         5 . The method of  claim 1 , wherein the composition administered contains a second oligonucleotide antisense compound having a base sequence effective to hybridize to at least 12 contiguous bases of another splice-site target sequence of the preprocessed human IL-10 transcript contained within SEQ ID NO:6. 
     
     
         6 . The method of  claim 1 , wherein the antisense oligonucleotide compound which is administered is conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into monocytes, lymphocytes, and dendritic cells. 
     
     
         7 . The method of  claim 1  for use in treating a mammalian subject infected with a viral pathogen, which further includes administering to the subject, an anti-viral compound effective to inhibit replication of the viral pathogen in the mammalian host. 
     
     
         8 . The method of  claim 1  for use in treating a mammalian subject infected with a bacterial pathogen, which further includes administering to the subject, an anti-bacterial compound effective to inhibit replication of the bacterial pathogen in the mammalian host. 
     
     
         9 . A vaccine against a pathogen which acts to up-regulate IL-10 during infection in a mammalian host, as evidenced by increased serum levels of IL-10, comprising
 (a) a pathogen antigenic component capable of eliciting an immune response against the pathogen,   (b) an antisense oligonucleotide compound   (i) 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,   (ii) capable of uptake by monocytes, lymphocytes, and dendritic cells in a mammalian subject,   (iii) containing between 10-40 nucleotide bases, and   (iv) having a base sequence effective to hybridize to at least 12 contiguous bases of the 5′-most 25 bases of exon 2 or exon 4 of the preprocessed human IL-10 transcript contained within SEQ ID NO:6, and identified by SEQ ID NOS:2 and 5, respectively, and   (c) an adjuvant in which the antigenic component and antisense compound are formulated.   
     
     
         10 . The vaccine of  claim 9 , which further includes a second antisense oligonucleotide compound having a base sequence effective to hybridize to at least 12 contiguous bases of another splice-site target sequence of the preprocessed human IL-10 transcript contained within SEQ ID NO:6. 
     
     
         11 . The vaccine of  claim 9 , wherein the antisense oligonucleotide compound is conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into monocytes, lymphocytes, and dendritic cells. 
     
     
         12 . The vaccine of  claim 9 , wherein the arginine-rich polypeptide has the sequence defined by SEQ ID NO:36 or SEQ ID NO:40. 
     
     
         13 . The vaccine of  claim 9 , wherein the intersubunit linkages linking the morpholino subunits are phosphorodiamidate linkages having 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 e.g., wherein X=NR 2 , where each R is independently hydrogen or methyl. 
       
     
     
         14 . An antisense oligonucleotide compound
 (i) 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,   (ii) capable of uptake by monocytes, lymphocytes, and dendritic cells in a mammalian subject,   (iii) containing between 10-40 nucleotide bases, and   (iv) having a base sequence effective to hybridize to at least 12 contiguous bases of a target sequence composed of 5′-end 25 bases of exon 2 or exon 4 of the preprocessed human IL-10 transcript contained within SEQ ID NO:6, and identified by SEQ ID NOS:2 and 5, respectively.   
     
     
         15 . The compound of  claim 14 , wherein the compound administered has a base sequence effective to hybridize to at least 12 contiguous bases of SEQ ID NO:5 in SEQ ID NO:6. 
     
     
         16 . The compound of  claim 14 , wherein the compound administered has a base sequence effective to hybridize to at least 12 contiguous bases of SEQ ID NO:2 in SEQ ID NO:6. 
     
     
         17 . The compound of  claim 14 , wherein the target region to which the oligonucleotide compound hybridizes is contained entirely within SEQ ID NOS:2 or 5. 
     
     
         18 . The compound of  claim 14 , which is conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into monocytes, lymphocytes, and dendritic cells. 
     
     
         19 . The compound of  claim 18 , wherein the arginine-rich polypeptide has the sequence defined by SEQ ID NO:36 or SEQ ID NO:40. 
     
     
         20 . The compound of  claim 14 , wherein the intersubunit linkages linking the morpholino subunits are phosphorodiamidate linkages having 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 e.g., wherein X=NR 2 , where each R is independently hydrogen or methyl. 
       
     
     
         21 . The compound of  claim 20 , wherein the intersubunit linkages are uncharged linkages interspersed with linkages that are positively charged at physiological pH, where the total number of positively charged linkages is between 2 and no more than half of the total number of linkages. 
     
     
         22 . The compound of  claim 21 , wherein the positively charged linkages have the structure X is 1-piperazine. 
     
     
         23 . Use of the compound of  claim 14  for the treatment of a mammalian subject infected with a pathogen which acts to up-regulate IL-10 during infection in a mammalian host.