Reagents for treatment of hepatitis b virus (hbv) infection and use thereof

Abstract

This disclosure relates to RNA interference (RNAi) reagents for treatment of hepatitis B virus (HBV) infection, compositions comprising same, and use thereof to treat individuals infected with HBV.

We claim: 
     
         1 . A nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising:
 an effector sequence of at least 17 nucleotides in length;   an effector complement sequence;   a stemloop sequence; and   primary micro RNA (pri-miRNA) backbone;   
       wherein the effector sequence is substantially complementary to a RNA transcript set forth in SEQ ID NO: 40. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the shmiR is selected from the group consisting of:
 a shmiR comprising an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:40;   a shmiR comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 and capable of forming a duplex therewith; and   a shmiR comprising an effector sequence set forth in SEQ ID NO:39 and an effector complement sequence set forth in SEQ ID NO:40.   
     
     
         3 . The nucleic acid of  claim 1 , wherein the shmiR comprises, in a 5′ to 3′ direction:
 (a) a 5′ flanking sequence of the pri-miRNA backbone;
 the effector complement sequence; 
 the stemloop sequence; 
 the effector sequence; and 
 a 3′ flanking sequence of the pri-miRNA backbone; or 
 
 (b) a 5′ flanking sequence of the pri-miRNA backbone;
 the effector sequence; 
 the stemloop sequence; 
 the effector complement sequence; and 
 a 3′ flanking sequence of the pri-miRNA backbone. 
 
 
     
     
         4 . The nucleic acid of  claim 1 , wherein:
 (a) the stemloop sequence is the sequence set forth in SEQ ID NO: 75; and/or   (b) the pri-miRNA backbone is a pri-miR-30a backbone.   
     
     
         5 . The nucleic acid of  claim 3 , wherein:
 (a) the 5′ flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 76 and the 3′ flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 77;   (b) the shmiR comprises a sequence set forth in SEQ ID NO: 57; and/or   (c) the DNA sequence which encodes the shmiR is set forth in SEQ ID NO: 73.   
     
     
         6 . A DNA-directed RNA interference (ddRNAi) construct comprising the nucleic acid according to  claim 1 . 
     
     
         7 . The ddRNAi construct of  claim 6 , comprising:
 (a) (i) a nucleic acid encoding a shmiR comprising or consisting of an effector sequence which is substantially complementary to a RNA transcript comprising the sequence set forth in SEQ ID NO: 40; and (ii) a nucleic acid encoding a shmiR comprising or consisting of an effector sequence which is substantially complementary to a RNA transcript comprising the sequence set forth in SEQ ID NO: 9; or   (b) (i) a nucleic acid encoding a shmiR comprising or consisting of an effector sequence which is substantially complementary to a RNA transcript comprising the sequence set forth in SEQ ID NO: 40; (ii) a nucleic acid encoding a shmiR comprising or consisting of an effector sequence which is substantially complementary to a RNA transcript comprising the sequence set forth in SEQ ID NO: 4.   
     
     
         8 . The ddRNAi construct of  claim 7 , comprising:
 (a) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 40; and
 a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 34; or 
   (b) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 40; and
 a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 22. 
   
     
     
         9 . The ddRNAi construct of  claim 8 , said ddRNAi construct comprising:
 (a) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:57; and
 a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:54; or 
   (b) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:57; and
 a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48. 
   
     
     
         10 . The ddRNAi construct of  claim 8 , said ddRNAi construct comprising:
 (a) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 73; and
 a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 70; or 
   (b) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 73; and
 a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 64. 
   
     
     
         11 . The ddRNAi construct of  claim 6 , comprising a RNA pol III promoter upstream of the nucleic acid encoding the or each shmiR. 
     
     
         12 . The ddRNAi construct of  claim 11 , wherein the or each RNA pol III promoter is selected from a U6 and a H1 promoter, optionally wherein the U6 promoter is selected from a U6-9 promoter, a U6-1 promoter and U6-8 promoter. 
     
     
         13 . An expression vector comprising the ddRNAi construct of  claim 6 . 
     
     
         14 . An expression vector comprising the ddRNAi construct of  claim 7 . 
     
     
         15 . The expression vector of  claim 13 , wherein the expression vector is a plasmid, a minicircle, or a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral (AdV) vector and a lentiviral (LV) vector. 
     
     
         16 . The expression vector of  claim 14 , wherein the expression vector is a plasmid, a minicircle, or a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral (AdV) vector and a lentiviral (LV) vector. 
     
     
         17 . A composition comprising a DNA Directed RNA interference (ddRNAi) construct according to  claim 6  or an expression vector comprising same and one or more pharmaceutically acceptable carriers, excipients or diluents. 
     
     
         18 . A composition comprising a DNA Directed RNA interference (ddRNAi) construct according to  claim 7  or an expression vector comprising same and one or more pharmaceutically acceptable carriers, excipients or diluents. 
     
     
         19 . A method of treating Hepatitis B virus (HBV) infection in a subject, said method comprising administering to the subject a therapeutically effective amount of a composition according to  claim 17 . 
     
     
         20 . The method of  claim 19 , wherein:
 (i) the subject is suffering from acute or chronic HBV infection;   (ii) expression of one or more HBV genes is reduced or inhibited in the subject;   (iii) treating the HBV infection comprises one or more of reducing HBV load in a subject infected with HBV or reducing severity of symptoms associated with HBV infection in a subject suffering therefrom or reducing the infectivity of HBV in a subject infected therewith;   (iv) treating the HBV infection comprises administering the nucleic acid, ddRNAi construct, expression vector or composition in combination with a further therapeutic agent for treatment of HBV infection; and/or   (v) treating the HBV infection comprises administering the nucleic acid, ddRNAi construct, expression vector or composition in combination with a further therapeutic agent selected from the group consisting of entecavir, tenofovir, lamivudine, adefovir and pegylated interferon.