Zinc-finger nuclease and rna interference mediated inactivation of viral genomes

Abstract

Embodiments of the present invention provide methods for targeted inactivation of viral genomes. In one embodiment, zinc-finger proteins in which DNA binding sites are altered such that they recognize and bind different, desired DNA sequences contained in hepatitis B virus (HBV) and that include nuclease domains are used for inactivation. Other embodiments for targeted inactivation of viral genomes use small nucleic acid molecules, such as short micro-RNA molecules or short hairpin RNA molecules capable of mediating RNA interference (RNAi) against the hepatitis B virus.

1 - 20 . (canceled) 
     
     
         21 . A chimeric polypeptide comprising: engineered zinc finger polypeptides and a DNA-cleavage domain, wherein the engineered zinc finger polypeptides comprise two or more zinc finger domains; and at least one linker. 
     
     
         22 . The polypeptide of  claim 21 , wherein the polypeptide comprises one or more amino acid sequences of SEQ ID NOs: 25-28. 
     
     
         23 . The polypeptide of  claim 21 , wherein the DNA-cleavage domain further comprises a DNA-cleavage domain of a Type II restriction enzyme. 
     
     
         24 . The polypeptide of  claim 21 , further comprising a linker positioned between at least one zinc finger domains and the DNA-cleavage domain. 
     
     
         25 . A nucleic acid construct comprising a nucleic acid sequence encoding the chimeric polypeptides of  claim 21 . 
     
     
         26 . The construct of  claim 25 , wherein the construct is DNA. 
     
     
         27 . The construct of  claim 25 , wherein the construct is RNA. 
     
     
         28 . A molecule comprising, an RNA construct capable of inhibiting expression of a pathogenic virus comprising, an RNA interference (RNAi) trigger molecule of 19-29 nucleotides in length of one or more of SEQ ID NO: 9 through SEQ ID NO: 23. 
     
     
         29 . The molecule of  claim 28 , wherein the RNAi trigger molecule comprises one or more of SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO: 16. 
     
     
         30 . The molecule of  claim 28 , wherein the RNAi trigger molecule is embedded into a larger RNA. 
     
     
         31 . The molecule of  claim 30 , wherein the larger RNA comprises microRNA or a hairpin. 
     
     
         32 . A pharmaceutical composition for inhibiting expression of Hepatitis B virus (HBV) in a subject comprising one or more chimeric polypeptides of  claim 21 , or one or more nucleic acid encoding constructs thereof, and a pharmaceutically acceptable excipient. 
     
     
         33 . The pharmaceutical composition of  claim 32 , further comprising one or more anti-viral agents. 
     
     
         34 . A method of inactivating viral genomes in a subject in need thereof comprising: administering to the subject one or more chimeric polypeptides comprising engineered zinc finger polypeptides and a DNA-cleavage domain, wherein the engineered zinc finger polypeptides comprise two or more zinc finger domains; or one or more nucleic acid encoding constructs thereof, and inactivating viral genomes in the subject. 
     
     
         35 . The method of  claim 34 , wherein the viral genome is an episomal genome. 
     
     
         36 . The method of  claim 34 , wherein the viral genome is a hepatitis virus. 
     
     
         37 . The method of  claim 36 , wherein the hepatitis virus is Hepatitis virus B. 
     
     
         38 . The method of  claim 34 , wherein the DNA-cleavage domain includes a Type II restriction enzyme DNA-cleavage domain. 
     
     
         39 . The method of  claim 34 , wherein a pair of the polypeptides or nucleic acid encoding constructs thereof is administered to the subject. 
     
     
         40 . The method of  claim 39 , wherein the pair of the polypeptides or nucleic acid encoding constructs thereof binds to the viral genome and inactivates the viral genome. 
     
     
         41 . The method of  claim 34 , wherein administering is transient. 
     
     
         42 . The method of  claim 34 , wherein the one or more nucleic acid encoding constructs corresponds to a nucleic acid sequence of SEQ ID NO: 24. 
     
     
         43 . The method of  claim 34 , wherein the one or more chimeric polypeptides comprise one or more amino acid sequences of SEQ ID NOS: 25-28. 
     
     
         44 . A transgenic mouse model for evaluating therapies for viral hepatitis infection with the mouse having a hepatitis genome transfected into the mouse, wherein the transfected hepatitis genome is episomal, wherein the improvement comprising: transfecting the mouse with a construct encoding an engineered zinc finger protein and a DNA-cleavage domain, or an RNA interference trigger molecule, or both.