Reagents for treatment of hepatitis B virus (HBV) infection and use thereof
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.
1. A DNA-directed RNA interference (ddRNAi) construct comprising:
(a) a first nucleic acid comprising a DNA sequence which encodes a short hairpin RNA (shRNA) comprising an effector sequence of at least 19 nucleotides in length which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 4; and
(b) a second nucleic acid comprising a DNA sequence which encodes a shRNA comprising an effector sequence of at least 19 nucleotides in length which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 58.
2. The ddRNAi construct of claim 1 , comprising:
(a) a first nucleic acid comprising a DNA sequence which encodes a shRNA comprising an effector sequence of at least 19 nucleotides in length which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 4;
(b) a second nucleic acid comprising a DNA sequence which encodes a shRNA comprising an effector sequence of at least 19 nucleotides in length which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 9; and
(c) a third nucleic acid comprising a DNA sequence which encodes a shRNA comprising an effector sequence of at least 19 nucleotides in length which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 58.
3. The ddRNAi construct of claim 1 , wherein:
(a) the first nucleic acid comprises a DNA sequence which encodes a shRNA comprising an effector sequence which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 28; and
(b) the second nucleic acid comprises a DNA sequence which encodes a shRNA comprising an effector sequence which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 58.
4. The ddRNAi construct of claim 2 , wherein:
(a) the first nucleic acid comprises a DNA sequence which encodes a shRNA comprising an effector sequence which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 28;
(b) the second nucleic acid comprises a DNA sequence which encodes a shRNA comprising an effector sequence which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 48; and
(c) the third nucleic acid comprises a DNA sequence which encodes a shRNA comprising an effector sequence which is substantially complementary to a RNA transcript encoded by the sequence set forth in SEQ ID NO: 58.
5. The ddRNAi construct of claim 3 , wherein:
(a) the first nucleic acid comprises a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence set forth in SEQ ID NO:28; and
(b) the second nucleic acid comprises: (i) a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:47 and an effector complement sequence set forth in SEQ ID NO:48; or (ii) a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:57 and an effector complement sequence set forth in SEQ ID NO:58.
6. The ddRNAi construct of claim 4 , wherein:
(a) the first nucleic acid comprises a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence set forth in SEQ ID NO:28;
(b) the second nucleic acid comprises a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:47 and an effector complement sequence set forth in SEQ ID NO:48; and
(c) the third nucleic acid comprises a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:57 and an effector complement sequence set forth in SEQ ID NO:58.
7. The ddRNAi construct according to claim 5 , wherein:
(a) the first nucleic acid comprises a DNA sequence encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO:78; and
(b) the second nucleic acid comprises a DNA sequence encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO:92 or SEQ ID NO:101.
8. The ddRNAi construct according to claim 6 , wherein:
(a) the first nucleic acid comprises a DNA sequence encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO:78;
(b) the second nucleic acid comprises a DNA sequence encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO:92; and
(c) the third nucleic acid comprises a DNA sequence encoding a shRNA comprising or consisting of the sequence set forth in SEQ ID NO:101.
9. The ddRNAi construct according to claim 1 , comprising a RNA pol III promoter upstream of each nucleic acid encoding a shRNA, wherein each RNA pol Ill promoter is selected from a U6 and a H1 promoter.
10. An expression vector comprising the ddRNAi construct of claim 1 .
11. The expression vector of claim 10 , wherein:
(i) the expression vector is a plasmid or minicircle;
(ii) the expression vector is a plasmid or minicircle complexed with a cationic DNA binding polymer; or
(iii) the expression vector is 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.
12. A composition comprising:
(i) the ddRNAi construct according to claim 1 and one or more pharmaceutically acceptable carriers; or
(ii) the ddRNAi construct according to claim 1 , and one or more pharmaceutically acceptable carriers, wherein the ddRNAi construct is comprised within an expression vector.
13. A composition comprising:
(i) the ddRNAi construct according to claim 2 and one or more pharmaceutically acceptable carriers; or
(ii) the ddRNAi construct according to claim 2 and one or more pharmaceutically acceptable carriers, wherein the ddRNAi construct is comprised within an expression vector.
14. A method of treating Hepatitis B virus (HBV) infection in a subject, said method comprising administering to the subject a composition of claim 12 .
15. A method of treating Hepatitis B virus (HBV) infection in a subject, said method comprising administering to the subject a composition of claim 13 .
16. The method according to claim 14 , wherein the subject is suffering from chronic HBV infection.
17. The method according to claim 15 , wherein the subject is suffering from chronic HBV infection.
18. The method of claim 14 , wherein administering the nucleic acid to the subject reduces HBV viral load in the subject and/or reduces severity of symptoms associated with HBV infection in the subject and/or reduces infectivity of HBV in the subject.
19. The method of claim 15 , wherein administering the nucleic acid to the subject reduces HBV viral load in the subject and/or reduces severity of symptoms associated with HBV infection in the subject and/or reduces infectivity of HBV in the subject.
20. A pharmaceutical composition comprising a ddRNAi construct comprising:
(a) a first nucleic acid comprising a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:27 and an effector complement sequence set forth in SEQ ID NO:28;
(b) a second nucleic acid comprising a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:47 and an effector complement sequence set forth in SEQ ID NO:48; and
(c) a third nucleic acid comprising a DNA sequence encoding a shRNA comprising an effector sequence set forth in SEQ ID NO:57 and an effector complement sequence set forth in SEQ ID NO:58;
wherein each DNA sequence encoding a shRNA comprises a U6 and a H1 promoter,
wherein the ddRNAi construct is comprised with an adeno-associated viral (AAV) vector, and
wherein the composition comprises one or more pharmaceutically acceptable carriers.