Reagents for treatment of hepatitis B virus (HBV) infection and use thereof

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: 4;

and wherein the shmiR inhibits or reduces expression of one or more Hepatitis B virus (HBV) genes in a cell by more than 60% relative to a cell in which the shmiR is absent.

2. The nucleic acid according to 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:22 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:22;

a shmiR comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:21 and capable of forming a duplex therewith; and

a shmiR comprising an effector sequence set forth in SEQ ID NO:21 and an effector complement sequence set forth in SEQ ID NO:22.

3. The nucleic acid according to 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 according to 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 according to 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; and/or

(b) wherein the shmiR comprises a sequence set forth in SEQ ID NO: 48.

6. The nucleic acid according to claim 1 , wherein the DNA sequence which encodes the shmiR is set forth in SEQ ID NO: 64.

7. A DNA-directed RNA interference (ddRNAi) construct comprising the nucleic acid according to claim 1 .

8. The ddRNAi construct according to claim 7 , comprising:

(a) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 22; and

a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 34;

(b) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 22; and

a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 40; or

(c) a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 21 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 22;

a nucleic acid comprising a DNA sequence encoding a shmiR comprising an effector sequence comprising or consisting of the sequence set forth in SEQ ID NO: 39 and an effector complement sequence comprising or 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 comprising or consisting of the sequence set forth in SEQ ID NO: 33 and an effector complement sequence comprising or consisting of the sequence set forth in SEQ ID NO: 34.

9. The ddRNAi construct according to claim 7 , 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: 4; 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: 4; (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; and (iii) 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.

10. The ddRNAi construct according to claim 9 , said ddRNAi construct comprising:

(a) (i) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48;

(ii) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:57; and

(iii) 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) (i) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO:64;

(ii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO:73; and

(iii) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70; or

(c) (i) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:48; and

(ii) a nucleic acid comprising a DNA sequence encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:54; or

(d) (i) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO:64;

and

(ii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO:70.

11. The ddRNAi construct according to claim 7 , comprising a RNA pol III promoter upstream of the or each nucleic acid encoding a 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.

13. The ddRNAi construct of claim 12 , wherein the or each RNA pol III promoter is a U6 promoter selected from a U6-9 promoter, a U6-1 promoter and U6-8 promoter.

14. The ddRNAi construct according to claim 13 , said ddRNAi construct comprising:

(a) (i) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64;

(ii) U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:73; and

(iii) U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70; or

(b) (i) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:64; and

(ii) U6-8 promoter or H1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:70.

15. An expression vector comprising the ddRNAi construct of claim 7 .

16. The expression vector of claim 15 , wherein the expression vector is a (i) plasmid, (ii) minicircle, or (iii) 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 7 and one or more pharmaceutically acceptable carriers, excipients or diluents.

18. 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 .

19. The method according to claim 18 , wherein the subject is suffering from acute HBV infection.

20. The method according to claim 18 , wherein the subject is suffering from chronic HBV infection.

21. The method of claim 18 , wherein treating HBV infection comprises one or more of the following:

(i) reducing Hepatitis B viral load in a subject infected with HBV;

(ii) reducing severity of symptoms associated with HBV infection in a subject suffering therefrom;

(iii) reducing the infectivity of HBV in a subject infected therewith; and/or

(iv) inhibiting or reducing expression of one or more HBV genes.

22. The method according to claim 18 , wherein the composition is administered together with a further therapeutic agent for treatment of HBV infection.

23. The method according to claim 22 , wherein the further therapeutic agent for treatment of HBV infection is selected from the group consisting of entecavir, tenofovir, lamivudine, adefovir and pegylated interferon.