DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING VIRAL COMPONENTS
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.
1 - 38 . (canceled)
39 . A method of modifying a eukaryotic organism in vivo, comprising delivering to the eukaryotic organism one or more adeno-associated viral (AAV) vectors, wherein the one or more AAV vectors comprise heterologous nucleic acid molecule(s) engineered for expression in at least one eukaryotic cell of the eukaryotic organism, wherein the heterologous nucleic acid molecule(s) encodes a Cas9 and a CRISPR-Cas system RNA, wherein the Cas9 and the CRISPR-Cas system RNA are expressed in the eukaryotic cell and form a CRISPR-Cas complex, wherein the CRISPR-Cas system RNA targets the CRISPR-Cas complex to a target genomic locus in nucleus of the eukaryotic cell.
40 . The method of claim 39 , wherein the CRISPR-Cas system RNA is a chimeric RNA (chiRNA) comprising (a) a guide sequence that hybridizes to a target sequence at the target genomic locus, (b) a tracr mate sequence, and (c) a tracr sequence, wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation.
41 . The method of claim 39 , wherein the one or more AAV vectors encode two or more CRISPR-Cas system RNAs each targeting a different target genomic locus.
42 . The method of claim 39 , wherein the one or more AAV vectors further comprise a repair template for homology-directed repair.
43 . The method of claim 39 , wherein the Cas9 is a Cas9 ortholog of a genus selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Fihfactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
44 . The method of claim 39 , wherein the Cas9 is S. aureus Cas9 or S. pyogenes Cas9.
45 . The method of claim 39 , wherein the Cas9 is a nuclease directing cleavage of both DNA strands at the target genomic locus.
46 . The method of claim 39 , wherein the Cas9 comprises one or more mutations in a catalytic domain and is a nickase directing cleavage of one DNA strand at the target genomic locus.
47 . The method of claim 39 , wherein the Cas9 is linked to at least one nuclear localization signal (NLS).
48 . The method of claim 39 , wherein the Cas9 is linked to at least two NLSs.
49 . The method of claim 39 , wherein the Cas9 comprises one or more mutations in a catalytic domain and is fused to a heterologous protein domain.
50 . The method of claim 49 , wherein the heterologous protein domain is a transcriptional activation domain or a transcriptional repression domain.
51 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV1/2, AAV2/8 or a combination thereof.
52 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 or a combination thereof, and wherein the target genomic locus is associated with a neurological or neuronal disease or disorder.
53 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV6, AAV7, AAV8, AAV9 or a combination thereof, and wherein the target genomic locus is associated with a muscular or skeletal disease or disorder, optionally Duchenne Muscular Dystrophy (DMD).
54 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV2, AAV5, AAV6, AAV9 or a combination thereof, and wherein the target genomic locus is associated with a lung disease or disorder, optionally cystic fibrosis or alpha-1 antitrypsin deficiency (AAD).
55 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV5, AAV7, AAV8, AAV2/8, or AAV9 or a combination thereof, wherein the target genomic locus is associated with a liver disease or disorder, optionally glycogen storage disease or alpha-1 antitrypsin deficiency (AAD).
56 . The method of claim 39 , wherein the serotype of the AAV vector is AAV6 or AAV8 or a combination thereof, wherein the target genomic locus is associated with a pancreatic disease or disorder.
57 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV2, AAV5, AAV6, AAV9 or a combination thereof, wherein the target genomic locus is associated with a epithelial cell disease or disorder.
58 . The method of claim 39 , wherein the serotype of the AAV vector is AAV1, AAV2, AAV4, AAV5, or AAV8 or a combination thereof, wherein the target genomic locus is associated with an ocular disease or disorder, optionally Leber Congenital Amaurosis (LCA) or Usher Syndrome.