Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders

Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

What is claimed is: 
     
         1 . A composition comprising:
 (A)—I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
 (a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, 
 (b) a tracr mate sequence, and 
 (c) a tracr sequence, and 
   II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,   or   (B) I. polynucleotides comprising:
 (a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, and 
 (b) at least one or more tracr mate sequences, 
   II. a polynucleotide sequence encoding a CRISPR enzyme, and   III. a polynucleotide sequence comprising a tracr sequence,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target hepatitis B virus (HBV) sequence, and   wherein the composition the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA;   whereby the composition edits an HBV sequence in a gene or genome.   
     
     
         2 . The composition of  claim 1 , wherein when present as RNA within the cell the guide sequence is capable of hybridizing to a sequence of the target viral nucleic acid which is comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism and wherein said manipulation is a manipulation of the episomal viral nucleic acid molecule, preferably wherein the episomal nucleic acid molecule is a double-stranded DNA polynucleotide molecule. 
     
     
         3 . The composition of  claim 2 , wherein the episomal viral nucleic acid molecule is a covalently closed circular DNA (cccDNA). 
     
     
         4 . The composition of  claim 2 , wherein the CRISPR/Cas complex is capable of reducing the amount of episomal viral nucleic acid molecule in a cell of the organism compared to the amount of episomal viral nucleic acid molecule in a cell of the organism in the absence of providing the complex. 
     
     
         5 . The composition of  claim 2 , wherein the CRISPR/Cas complex is capable of manipulating the episomal nucleic acid molecule to promote degradation of the episomal nucleic acid molecule. 
     
     
         6 . The composition of  claim 1 , wherein when present as RNA within in the cell the guide sequence is capable of hybridizing to a sequence of the target viral nucleic acid which is integrated into the genome of the organism and wherein said manipulation is a manipulation of the integrated target nucleic acid. 
     
     
         7 . The composition of  claim 6 , wherein when formed within the cell the CRISPR/Cas complex is capable of manipulating the integrated nucleic acid to promote excision of all or part of the target viral nucleic acid from the genome of the organism. 
     
     
         8 . The composition of  claim 6 , wherein said at least one target viral nucleic acid is comprised in a double-stranded DNA polynucleotide cccDNA molecule and/or viral DNA integrated into the genome of the organism and wherein said manipulation of the at least one target viral nucleic acid by the CRISPR-Cas complex comprises cleavage of viral cccDNA and/or integrated viral DNA. 
     
     
         9 . The composition of  claim 8 , wherein said cleavage comprises one or more double-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two double-strand break(s). 
     
     
         10 . The composition of  claim 8 , wherein said cleavage is via one or more single-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two single-strand break(s). 
     
     
         11 . The composition of  claim 9 , wherein said one or more double-strand break(s) or said one or more single-strand break(s) leads to the formation of one or more insertion and deletion mutations (INDELs) in the viral cccDNA sequences and/or integrated viral DNA sequences. 
     
     
         12 . The composition of  claim 1 , wherein the polynucleotides are comprised within a vector system comprising one or more vectors. 
     
     
         13 . The composition of  claim 1 , wherein the CRISPR-Cas system RNA is a chimeric RNA (chiRNA). 
     
     
         14 . The composition of  claim 1 , wherein the CRISPR-Cas system is a multiplexed CRISPR enzyme system further comprising multiple chimeras and/or multiple multiguide sequences and a single tracr sequence. 
     
     
         15 . The composition of  claim 1 , wherein the CRISPR enzyme is a nuclease directing cleavage of one or both strands at the location of the target sequence. 
     
     
         16 . The composition of  claim 1 , wherein the CRISPR enzyme comprises one or more mutations. 
     
     
         17 . The composition of  claim 15 , wherein the CRISPR enzyme is a nickase directing cleavage at the location of the target sequence. 
     
     
         18 . The composition of  claim 15 , wherein the nickase is a double nickase. 
     
     
         19 . The composition of  claim 1 , further comprising at least two or more NLS. 
     
     
         20 . The composition of  claim 1 , wherein the CRISPR enzyme has one or more mutations in a catalytic domain, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain. 
     
     
         21 . The composition of  claim 20 , wherein the functional domain is a transcriptional activation domain. 
     
     
         22 . The composition of  claim 21 , wherein the transcriptional activation domain is VP64. 
     
     
         23 . The composition of  claim 1 , wherein the CRISPR/Cas enzyme comprises a Cas9 enzyme or a biologically active fragment or derivative thereof. 
     
     
         24 . The composition of  claim 23 , wherein the Cas9 enzyme comprises a  Streptococcus pyogenes  Cas9 enzyme or a biologically active fragment or derivative thereof or  Streptococcus aureus  Cas9 enzyme or a biologically active fragment or derivative thereof. 
     
     
         25 . The composition of  claim 1 , wherein the CRISPR/Cas enzyme further comprises one or more nuclear localization sequences (NLSs) capable of driving the accumulation of the CRISPR/Cas enzyme to a detectible amount in the nucleus of the cell of the organism. 
     
     
         26 . The composition of  claim 25 , wherein the CRISPR/Cas enzyme comprises at least one NLS at or near the amino-terminus of the CRISPR/Cas enzyme and/or at least one NLS at or near the carboxy-terminus the CRISPR/Cas enzyme. 
     
     
         27 . A CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a guide RNA capable of hybridizing to a target sequence in an HBV viral genome within the cell; and (iii); a tracr mate sequence; and (iv) a tracr sequence,
 wherein, when expressed within the cell, the guide RNA directs sequence-specific binding of a CRISPR complex to the target sequence, and the CRISPR complex comprises (a) the tracr mate sequence hybridised to the tracr sequence and (b) a CRISPR enzyme bound to the guide RNA, such that the guide RNA can hybridise to its target sequence in the viral genome.   
     
     
         28 . A method of treating an HBV infection in an individual in need thereof comprising administering an effective amount of the system of  claim 28 . 
     
     
         29 . The method of  claim 29  including administering an additional HBV treatment. 
     
     
         30 . The method of  claim 30  wherein the additional treatment comprises an epigenetic modifier.