Delivery, Use, and Therapeutic Applications of CRISPR-Cas Systems and Compositions for Genome Editing

Abstract

To provide delivery, engineering, and optimization of systems, methods, and compositions for manipulation of sequences and / or activities of target sequences.SOLUTION: 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] RELATED APPLICATIONS AND INCORPORATION BY REFERENCE This application claims priority to U.S. Provisional Patent Applications Nos. 61 / 915,176; 61 / 915,192; 61 / 915,215; 61 / 915,107; 61 / 915,145; 61 / 915,148; and 61 / 915,153, each of which was filed on December 12, 2013.

[0002] The above applications, and all documents cited in these applications or cited during the prosecution of these applications ("application cited documents"), and all documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products described herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be utilized in the practice of the present invention. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] The present invention relates generally to the delivery, engineering, optimization and therapeutic applications of systems, methods and compositions used for the control of gene expression, such as genome perturbation or gene editing, involving sequence targeting involving clustered regularly interspaced short palindromic repeats (CRISPR) and its components. In particular, the present invention relates to in vitro, ex vivo and / or in vivo systems, methods and compositions for the delivery of CRISPR-Cas systems for therapeutic benefit by genome editing in animals, including mammals.

[0004] Federally Sponsored Research Statement This invention was made with federal support under an NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health and grant 1DP1OD009552 also awarded by the National Institutes of Health. The federal government has certain rights in this invention. [Background technology]

[0005] Recent advances in genome sequencing technology and analysis have significantly accelerated the ability to classify and map genetic factors associated with a diverse range of biological functions and diseases. Accurate genome targeting technology is needed to enable the selective perturbation of individual genetic elements, thereby enabling systematic reverse engineering of causative gene variants, and to advance synthetic biology, biotechnology applications, and pharmaceutical applications. Although genome editing technologies, such as designer zinc finger (ZFN), transcription activator-like effector (TALE), or homing meganuclease, are available for the production of targeted genome perturbations, there is still a need for new genome engineering technologies that are inexpensive, easy to set up, scalable, and amenable to targeting multiple locations in eukaryotic genomes. Summary of the Invention [Means for solving the problem]

[0006] Despite effective therapeutic hypotheses and strong efforts in drug development, there are only limited successful cases of using small molecules to treat diseases with strong genetic involvement. Therefore, there is an urgent need for alternative and robust systems for therapeutic strategies that can modify nucleic acids in disease-affected cells and tissues. The addition of CRISPR-Cas systems to the repertoire of therapeutic genome engineering methods greatly simplifies methodology and facilitates the ability to enumerate and map genetic factors associated with various biological functions and diseases, develop animal models of genetic diseases, and develop safe and effective therapeutic options. In order to effectively utilize CRISPR-Cas systems for genome editing without adverse effects, it is critical to understand the operation, optimization, and cell type / tissue / organ specific delivery of these genome engineering tools, which are the aspects of the claimed invention. Aspects of the present invention address this need and provide related advantages.

[0007] An exemplary CRISPR complex may include a CRISPR enzyme (e.g., Cas9) complexed with a guide sequence that hybridizes to a target sequence in a target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to the tracr sequence. Applicants have optimized the components of the CRISPR-Cas genome engineering system, including using SaCas9 from Staphylococcus aureus. Various delivery methods may be used to deliver the components of the CRISPR-Cas system to cells, tissues, and organs ex vivo and / or in vivo. Applicants have successfully packaged CRISPR-Cas system components (including, for example, SaCas9) into viral delivery vectors, such as AAV, and have demonstrated that they can be used to modify endogenous genomic sequences in mammalian cells in vivo. An important feature of the applicants' invention is that it effectively addresses the problems of low in vivo delivery efficiency (of therapeutic components) and low homology-dependent repair (HDR) efficiency, in particular, the problems associated with co-delivery are solved by a small Cas9, SaCas9 from Staphylococcus aureus, which can be easily packaged into a single adeno-associated virus (AAV) vector to express both the Cas9 protein and its corresponding one or more sgRNAs. Moreover, importantly, applicants show that the introduction of the small SaCas9 reduces the number of viral vectors required to perform HDR from three to two vectors. In an embodiment of the invention, particles can be used to deliver one or more components of the CRISPR-Cas system. And the number of particles to be contacted can be one or two. In one embodiment, the invention provides a method of using one or more elements of the CRISPR-Cas system. The CRISPR complexes of the present invention provide an effective means of modifying target polynucleotides at genomic loci, where the genomic loci are associated with mutations, including mutations associated with aberrant protein expression or disease conditions or states.The CRISPR complexes of the present invention have a wide range of utility, including modifying (e.g., deleting, inserting, transposing, inactivating, activating) target polynucleotides within genomic loci, such as within coding, non-coding, or regulatory elements of such target loci. Thus, the CRISPR complexes of the present invention have a wide range of applications, for example, in gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis. Aspects of the invention relate to Cas9 enzymes with improved targeting specificity in CRISPR-Cas9 systems with optimally active guide RNAs that are shorter in length compared to wild-type Cas9 enzymes and nucleic acid molecules encoding same, and chimeric Cas9 enzymes, as well as methods for improving targeting specificity of Cas9 enzymes or designing CRISPR-Cas9 systems, comprising designing or preparing optimally active guide RNAs and / or selecting or preparing Cas9 enzymes that are smaller in size or length compared to wild-type Cas9, and therefore less coding thereof in the delivery vector compared to wild-type Cas9, making it easier to package the nucleic acid encoding it into a delivery vector, and / or creating chimeric Cas9 enzymes. Also provided are the uses of the sequences, vectors, enzymes or systems of the invention in medicine. Also provided are the uses thereof in gene or genome editing.

[0008] In the present invention, the Cas enzyme may be a wild-type Cas9, including any naturally occurring bacterial Cas9. Cas9 orthologs typically share a common organization of 3-4 RuvC domains and an HNH domain. The 5'-most RuvC domain cleaves the non-complementary strand, and the HNH domain cleaves the complementary strand. All designations are relative to the guide sequence. The catalytic residues of the 5' RuvC domain are identified by homology comparison of the Cas9 of interest to other Cas9 orthologs (from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus), and the Cas9 is converted into a complementary strand nicking enzyme by mutating the conserved Asp residue (D10) to alanine. Similarly, mutating the conserved His and Asn residues in the HNH domain to alanine converts Cas9 into a non-complementary strand nicking enzyme. In some embodiments, both sets of mutations can be made to convert Cas9 into a non-cleaving enzyme. Thus, the Cas enzyme can be wild-type Cas9, including any naturally occurring bacterial Cas9. The CRISPR, Cas or Cas9 enzyme can be codon optimized for a particular type of human cell, or modified forms, including any chimera, mutant, homolog or ortholog. In further aspects of the invention, the Cas9 enzyme can include one or more mutations and can be used as a general DNA binding protein with or without fusion to a functional domain. The mutations can be artificially introduced mutations or can be gain-of-function or loss-of-function mutations. The mutations can include, but are not limited to, mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively. Further mutations have been characterized. In one aspect of the invention, the transcription activation domain can be VP64. In other aspects of the invention, the transcriptional repressor domain may be KRAB or SID4X.Other aspects of the invention relate to mutant Cas9 enzymes fused to domains including, but not limited to, transcription activators, repressors, recombinases, transposases, histone remodelers, demethylases, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains, or chemical-inducible / regulatory domains. The invention may involve guides or chimeric guide sequences that enhance the performance of sgRNA or tracrRNA or these RNAs in cells. The CRISPR enzyme may be a type I or type III CRISPR enzyme, preferably a type II CRISPR enzyme. The type II CRISPR enzyme may be any Cas enzyme. The preferred Cas enzyme may be identified as Cas9, as Cas9 may refer to a general class of enzymes that share homology with the largest nucleases containing multiple nuclease domains of the type II CRISPR system. Most preferably, the Cas9 enzyme is from or derived from spCas9 or saCas9. By derived, Applicants mean that the derived enzyme is primarily based on the wild-type enzyme in the sense of having a high degree of sequence homology to the wild-type enzyme, but that it has been mutated (modified) in some way as described herein.

[0009] It will be understood that the terms Cas and CRISPR enzyme are generally used interchangeably herein unless otherwise clear. As mentioned above, many of the residue numberings used herein are based on the Cas9 enzyme from the type II CRISPR locus of Streptococcus pyogenes. However, it will be understood that the present invention includes many more Cas9s from other microbial species, such as SpCas9, SaCas9, St1Cas9, etc. Further examples are provided herein. Those skilled in the art will be able to determine the appropriate corresponding residues in Cas9 enzymes other than SpCas9 by comparison of related amino acid sequences. Thus, when a particular amino acid substitution is referred to using SpCas9 numbering, the present disclosure is intended to encompass the corresponding modifications in other Cas9 enzymes, unless the context makes clear that it is not intended to refer to other Cas9 enzymes. In this case, examples of human-optimized (i.e., optimized for expression in humans) codon-optimized sequences are provided herein (see SaCas9 human codon-optimized sequence). While this is preferred, it is understood that other examples are possible, and codon optimization for host species is known. The present invention encompasses methods in which the Cas9 is a chimeric Cas9 protein. These methods may include one or more N-terminal fragments of one or more other or alternative Cas9 homologs together with one or more C-terminal fragments of one or more other or alternative Cas9 homologs. In the methods, it will be understood that when the organism is an animal, the modification may be performed ex vivo or in vitro, for example in cell culture, and in some cases other than in vivo. In other embodiments, the modification may be performed in vivo. The present invention encompasses, in some embodiments, the compositions of the present invention or the CRISPR enzymes thereof (including or instead of the mRNA encoding the CRISPR enzyme), in which the target sequence is flanked at its 3' end by a PAM (protospacer adjacent motif) sequence that includes a 5'-motif, particularly when the Cas9 is (or is derived from) S. pyogenes or S. aureus Cas9.For example, a suitable PAM is 5'-NRG or 5'-NNGRR (where N is any nucleotide) for the SpCas9 or SaCas9 enzyme (or derived enzyme). It will be understood that the SpCas9 or SaCas9 is derived from or derived from S. pyogenes or S. aureus Cas9.

[0010] In one aspect, the present invention provides a method for producing A) I. CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequences, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) tracr mate sequence, and (c) tracr sequence A polynucleotide sequence comprising: II. Polynucleotide sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence [(a), (b) and (c) are arranged in the 5' to 3' direction, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and delivering a non-naturally occurring or engineered composition comprising a CRISPR complex comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to a tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA. The present invention provides a method for modifying an organism or non-human organism by manipulation of a target sequence in a genomic locus of interest associated with a mutation associated with aberrant protein expression or a disease condition or state, comprising: The method can also optionally include a step of delivering an HDR template, e.g., via a viral delivery vector or particle, the HDR template, where the HDR template results in expression of a normal or a less aberrant form of a protein; "normal" is with respect to wild type, and "aberrant" can be a protein expression that causes a pathology or disease state; and Optionally, the method may include the steps of isolating or obtaining cells expressing the aberrant protein from an organism or non-human organism, optionally expanding this cell population, contacting the cells with one or more viral vectors or particles to obtain a modified cell population, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism.

[0011] In one aspect, the invention provides a method of modifying an organism or non-human organism by manipulation of a target sequence in a genomic locus of interest associated with a mutation associated with aberrant protein expression or a disease condition or state, comprising contacting a cell with a viral vector or particle containing a non-naturally occurring or engineered composition comprising: I. (a) a guide sequence hybridizable to a target sequence in HSCs, and (b) at least one or more tracr mate sequences, II. a CRISPR enzyme, optionally with one or more NLS, and III. a polynucleotide sequence comprising a tracr sequence, wherein 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 the CRISPR complex comprises (1) the guide sequence that hybridizes to the target sequence, and (2) the CRISPR enzyme complexed with the tracr mate sequence that hybridizes to the tracr sequence; and The method can also optionally include a step of delivering an HDR template, e.g., via a viral delivery vector or particle, the HDR template, where the HDR template results in expression of a normal or a less aberrant form of a protein; "normal" is with respect to wild type, and "aberrant" can be a protein expression that causes a pathology or disease state; and Optionally, the method may include the steps of isolating or obtaining cells expressing the aberrant protein from an organism or non-human organism, optionally expanding this cell population, contacting the cells with one or more viral vectors or particles to obtain a modified cell population, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism.

[0012] This delivery may be, for example, delivery of one or more polynucleotides encoding any one or more or all of the CRISPR complexes, preferably linked to one or more regulatory elements for in vivo expression, via one or more particles containing a vector containing one or more polynucleotides operably linked to one or more regulatory elements. Some or all of the polynucleotide sequences encoding the CRISPR enzyme, guide sequences, tracr mate sequences or tracr sequences may be RNA. When a polynucleotide is referred to that is RNA and is said to "comprise" such a tracr mate sequence feature, it will be understood that the RNA sequence comprises the feature. When a polynucleotide is DNA and is said to include such a tracr mate sequence feature, the DNA sequence is or can be transcribed into RNA that includes the feature in question. When the feature is a protein, such as a CRISPR enzyme, the DNA or RNA sequence referred to is or can be translated (in the case of DNA, first transcribed and then translated).

[0013] In certain embodiments, the invention provides a method of modifying an organism, e.g., a mammal, including a human, or a non-human mammal or organism, by manipulation of a target sequence at a genomic locus of interest, e.g., associated with a mutation associated with aberrant protein expression or a disease condition or state, comprising the step of delivering, e.g., by contacting, a non-naturally occurring or engineered composition to a cell or population of cells, wherein the composition comprises one or more delivery vectors or particles comprising one or more viruses, plasmids, or nucleic acid molecule vectors (e.g., RNA) operably encoding the composition for expression, the composition comprising: (A) a first regulatory element operably linked to a I. CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, the polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracrRNA polynucleotide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (c) a tracrRNA polynucleotide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (d) a tracrRNA polynucleotide sequence capable of hybridizing to a target sequence in a eukaryotic cell; and (c) a tracr sequence; and II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally at least one or more nuclear localization sequences, since some embodiments may not involve an NLS), wherein (a), (b) and (c) are arranged in a 5' to 3' direction, components I and II are located on the same or different vectors of the system, when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex comprises (1) a guide sequence hybridizing to the target sequence, and (2) a CRISPR enzyme complexed with the tracr mate sequence hybridizing to the tracr sequence; or (B) a non-naturally occurring or engineered composition, comprising I. (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences. a first regulatory element operably linked to a mate sequence, II. a second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, and III.A third regulatory element operably linked to the tracr sequence [components I, II, and III are located on the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex is composed of (1) the guide sequence that hybridizes to the target sequence, and (2) the tracr sequence that hybridizes to the tracr sequence. The composition includes a vector system comprising one or more vectors comprising a CRISPR enzyme complexed with a mate sequence; the method can optionally also include a step of delivering an HDR template, for example via a delivery vector or particle contacting the cell or cell population, or by contacting the cell, cell or cell population with another delivery vector or particle containing the HDR template, where the HDR template results in expression of a normal or less aberrant form of a protein; "normal" is with respect to wild type, and "aberrant" can be a protein expression that causes a pathology or disease state; and optionally the method can include a step of isolating or obtaining a cell expressing the aberrant protein from an organism or non-human organism, optionally a step of expanding the cell population, a step of performing contacting one or more delivery vectors or particles with the cells expressing the aberrant protein to obtain a modified cell population, optionally a step of expanding the population of modified cells, and optionally a step of administering the modified cells to an organism or non-human organism. In some embodiments, components I, II, and III are located on the same vector. In other embodiments, components I and II are located on the same vector while component III is located on another vector. In other embodiments, components I and III are located on the same vector while component II is located on another vector. In other embodiments, components II and III are located on the same vector while component I is located on another vector. In other embodiments, each of components I, II and III are located on a different vector. The present invention also provides a viral or plasmid vector system as described herein.

[0014] By engineering a target sequence, applicants also mean epigenetic engineering of the target sequence. This may be engineering the chromatin state of the target sequence, such as by modifying the methylation state of the target sequence (i.e. adding or removing methylation or methylation patterns or CpG islands), histone modification, increasing or decreasing the accessibility of the target sequence, or by promoting three-dimensional folding. When referring to a method of modifying an organism or mammal, including a human, or a non-human mammal or organism, by engineering a target sequence at a genomic locus of interest, it will be understood that this may apply to the organism (or mammal) as a whole, or (if the organism is a multicellular organism) to just a single cell or cell population of the organism. In the case of humans, for example, applicants particularly envisage single cells or cell populations, which may be modified preferably ex vivo and then reintroduced. In this case, a biopsy or other tissue or biological fluid sample may be required. Stem cells are also particularly preferred in this regard. However, of course, in vivo embodiments are also envisaged. And the invention is particularly advantageous with respect to ocular cells, retinal cells, vascular cells, epithelial cells, endothelial cells and cochlear cells.

[0015] The present invention, in some embodiments, includes, for example, I. A first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, (a) a first guide sequence capable of hybridizing to a first target sequence; (b) a first tracr mate sequence, and (c) First tracr sequence A first polynucleotide sequence comprising: II. A second CRISPR-Cas system chiRNA polynucleotide sequence, (a) a second guide sequence capable of hybridizing to a second target sequence; (b) a second tracr mate sequence, and (c) Second tracr sequence a second polynucleotide sequence comprising III. A polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations, (a), (b) and (c) arranged in a 5' to 3' direction; or IV. One or more expression products of one or more of I.-III., e.g., a first and second tracr mate sequence, a CRISPR enzyme; [Upon transcription, the first and second tracr mate sequences hybridize to the first and second tracr sequences, respectively, and the first and second guide sequences direct sequence-specific binding of first and second CRISPR complexes to the first and second target sequences, respectively, wherein the first CRISPR complex comprises a CRISPR enzyme complexed with (1) the first guide sequence that hybridizes to the first target sequence, and (2) the first tracr mate sequence that hybridizes to the first tracr sequence, and the second CRISPR complex comprises (1) the second guide sequence that hybridizes to the second target sequence, and (2) the second tracr mate sequence that hybridizes to the second tracr sequence. a CRISPR enzyme complexed with a mate sequence, the polynucleotide sequence encoding the CRISPR enzyme being DNA or RNA, and a first guide sequence directing cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence directing cleavage of the other strand near a second target sequence to create a double strand break, thereby modifying the organism or non-human organism, comprising contacting the cell or cell population with a delivery vector, e.g., a viral vector or particle, comprising a non-naturally occurring or engineered composition comprising: and the mthod optionally also includes a step of delivering an HDR template, e.g., via a delivery vector contacting a cell or cell population containing the HDR template, or by contacting a cell or cell population with another delivery vector containing the HDR template, where the HDR template results in expression of a normal or less aberrant form of the protein; "normal" being with respect to wild type, and "aberrant" can be a protein expression that causes a pathology or disease state;and optionally the method may include isolating or obtaining a cell or cell population from the organism or non-human organism, optionally expanding the cell population, contacting the cell or cell population with one or more delivery vectors or particles to obtain a modified cell population, and optionally expanding the population of modified cells.A method of modeling a disease associated with a genomic locus in a eukaryotic organism or a non-human organism, comprising the steps of delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors functionally encoding a composition for expression of the composition, the composition comprising: (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence being (a) a guide sequence capable of hybridizing to a target sequence; (b) tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to the enzyme coding sequence encoding SaCas9, optionally including at least one nuclear localization sequence. [(a), (b) and (c) are arranged in the 5' to 3' direction, Components I and II are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and the CRISPR complex comprises (1) a guide sequence that hybridizes to a target sequence, and (2) SaCas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element, (a) a guide sequence capable of hybridizing to a target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding SaCas9; and III. A third regulatory element operably linked to the tracr sequence [wherein components I, II and III are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The method includes the steps of: (1) a composition comprising a vector system comprising one or more vectors comprising a CRISPR complex comprising SaCas9 complexed with a guide sequence that hybridizes to the target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence; and optionally administering the modified cell to an organism or a non-human organism. In some methods of the invention, some or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences, or the first and second tracr sequences are RNA. In a further embodiment of the invention, the polynucleotide encoding the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences, or the first and second tracr sequences are RNA and are delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, delivery is advantageously by viral vectors or particles. In certain embodiments of the present invention, the first and second tracr mate sequences share 100% identity, and / or the first and second tracr sequences share 100% identity. In some embodiments, the polynucleotide may be included in a vector system comprising one or more vectors. In a preferred embodiment of the present invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9 or SaCas9. In an aspect of the present invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, the one or more mutations being selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A, based on SpCas9, for example, a D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations that make it a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations that make it a non-complementary strand nicking enzyme. Alternatively, the first enzyme may be a non-complementary strand nicking enzyme and the second enzyme may be a complementary strand nicking enzyme.In a preferred method of the invention, a first guide sequence induces cleavage of one strand of a DNA duplex near a first target sequence, and a second guide sequence induces cleavage of the other strand near a second target sequence, resulting in a 5' overhang. In an embodiment of the invention, the 5' overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In an embodiment of the invention, the 5' overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.

[0016] With respect to mutations in CRISPR enzymes, if the enzyme is not SpCas9, the mutations can occur at any or all of the residues corresponding to SpCas9 positions 10, 762, 840, 854, 863, and / or 986 (which can be ascertained, for example, by standard sequence comparison tools). In particular, in SpCas9, any or all of the following mutations are preferred: D10A, E762A, H840A, N854A, N863A, and / or D986A; further, it is envisioned that any substituted amino acids are conservative substitutions. In one aspect, the invention provides any or each or all of the embodiments described herein, wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutations or the at least two or more mutations are D10, E762, H840, N854, N863, or D986 in an SpCas9 protein, e.g., D10A, E762A, H840A, N854A, N863A, and / or D986A in SpCas9, e.g., D10A, E762A, H840A, N854A, N863A, and / or D986A in SaCas9. or N580A, or any corresponding mutation in a Cas9 orthologue of Sp or Sa, or the CRISPR enzyme comprises at least one mutation, at least H840 or N863A in SpCas9, or N580A in SaCas9; for example, the CRISPR enzyme comprises H840A, or D10A and H840A, or D10A and N863A in an SpCas9 protein, or any corresponding mutation in a Cas9 orthologue of an Sp protein or Sa protein.

[0017] The present invention, in some embodiments, includes, for example, I. A first regulatory element, (a) a first guide sequence capable of hybridizing to a first target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element, (a) a second guide sequence capable of hybridizing to a second target sequence, and (b) at least one tracr mate sequence a second regulatory element operably linked to III. A third regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme; and IV. A fourth regulatory element operably linked to the tracr sequence, One or more expression products of one or more of VI-IV., e.g., the first and second tracr mate sequences, a CRISPR enzyme;

[0023] Components I, II, III and IV are located on the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the first and second guide sequences direct sequence-specific binding of first and second CRISPR complexes to first and second target sequences, respectively, wherein the first CRISPR complex comprises a CRISPR enzyme complexed with (1) the first guide sequence hybridized to the first target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence, and the second CRISPR complex comprises (1) the second guide sequence hybridized to the second target sequence, and (2) the tracr mate sequence hybridized to the tracr sequence. a CRISPR enzyme complexed with a mate sequence, the polynucleotide sequence encoding the CRISPR enzyme being DNA or RNA, and a first guide sequence directing cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence directing cleavage of the other strand near a second target sequence creating a double stranded break, thereby modifying the organism or non-human organism, by contacting the cell or cell population with one or more delivery vectors or particles comprising the non-naturally occurring or engineered composition comprising: encompasses a method of modifying an organism or non-human organism by manipulation of first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest associated with a condition-associated mutation; and the method can optionally also include the step of delivering the HDR template, e.g., via a delivery vector or particle that contacts the cell or cell population containing the HDR template, or by contacting the cell or cell population with another particle that contains the HDR template, where the HDR template results in expression of a normal or less aberrant form of the protein; "normal" is with respect to wild type, and "aberrant" can be a protein expression that causes a pathology or disease state;and optionally the method may include isolating or obtaining a cell or cell population from the organism or non-human organism, optionally expanding the cell, contacting the cell or cell population with one or more delivery vectors or particles to obtain a modified cell population, optionally expanding the population of modified cells, and optionally administering the modified HSC to the organism or non-human organism;

[0018] The present invention also provides a vector system as described herein. The system may include one, two, three or four different vectors. Thus, components I, II, III and IV may be located on one, two, three or four different vectors, and all possible combinations of component locations are envisaged herein, such as: components I, II, III and IV may be located on the same vector, components I, II, III and IV may each be located on a different vector, components I, II, III and IV may be located on a total of two or three different vectors, etc., in all envisaged combinations of locations. In some methods of the present invention, some or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or the first and second tracr sequences are RNA. In further embodiments of the present invention, the first and second tracr mate sequences share 100% identity, and / or the first and second tracr sequences share 100% identity. In a preferred embodiment of the present invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9. In an aspect of the present invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, and the one or more mutations are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A based on SpCas9; for example, D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations that make it a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations that make it a non-complementary strand nicking enzyme. Alternatively, the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In a further embodiment of the present invention, one or more of the viral vectors are delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, viral or particle delivery is advantageous.

[0019] In a preferred method of the invention, a first guide sequence induces cleavage of one strand of a DNA duplex near a first target sequence, and a second guide sequence induces cleavage of the other strand near a second target sequence, resulting in a 5' overhang. In an embodiment of the invention, the 5' overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In an embodiment of the invention, the 5' overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.

[0020] The invention encompasses, in some embodiments, a method of modifying a genomic locus of interest in a cell or cell population, e.g., associated with a mutation associated with aberrant protein expression or a disease condition or state, by introducing into a cell or cell population one or more delivery vectors or particles comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand, respectively, of a DNA molecule in the cell or cell population, e.g., by contacting the cell or cell population, whereby the guide RNA targets the DNA molecule and the Cas protein nicks each of the first strand and the second strand of the DNA molecule, thereby altering the target in the cell or cell population (and the Cas protein and the two guide RNAs do not naturally occur together), and the method optionally includes, e.g., administering to a cell or cell population containing an HDR template a genomic locus of interest associated with a mutation associated with a disease condition or state, e.g., administering to a cell or cell population containing an HDR template a genomic locus of interest associated with a mutation associated with a mutation in a cell or cell population. The method may also include a step of delivering the HDR template via a delivery vector or particle contacted with a 5' poulation of the HDR template or by contacting the cell or population with another delivery vector or particle containing the HDR template, where the HDR template results in expression of a normal or less aberrant form of the protein; "normal" is with respect to wild type, and "aberrant" may be a protein expression that causes a pathology or disease state; and optionally the method may include a step of isolating or obtaining cells from the organism or non-human organism, optionally expanding the cell population, performing contact of the cells with one or more delivery vectors or particles to obtain a modified cell population, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism. In a preferred method of the invention, the Cas protein nicks each of the first and second strands of the DNA molecule, resulting in a 5' overhang. In an embodiment of the invention, the 5' overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In an embodiment of the invention, the 5' overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.The embodiments of the present invention also encompass a guide RNA comprising a guide sequence and a tracr sequence fused to a tracr mate sequence. In an aspect of the present invention, the Cas protein is codon-optimized for expression in eukaryotic cells, preferably mammalian or human cells. In a further embodiment of the present invention, the Cas protein is a type II CRISPR-Cas protein, such as a Cas9 protein. In a highly preferred embodiment, the Cas protein is a Cas9 protein, such as a SpCas9 or SaCas9. In an aspect of the present invention, the Cas protein is based on SpCas9 and has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A; for example, the D10A mutation. Aspects of the present invention relate to reducing expression of a gene product, or further introducing a template polynucleotide into a DNA molecule encoding a gene product, or allowing reannealing and ligation of two 5' overhangs to allow precise excision of the intervening sequence, or altering the activity or function of a gene product, or increasing expression of a gene product. In certain embodiments of the invention, the gene product is a protein.

[0021] The present invention, in some embodiments, includes, for example, a) a first regulatory element operably linked to each of two CRISPR-Cas system guide RNAs that target a first strand and a second strand, respectively, of a double-stranded DNA molecule of a cell or a cell population; and b) a second regulatory element operably linked to the Cas protein; or c) one or more expression products of a) or b); wherein components (a) and (b) are located on the same or different vectors of the system], by contacting the cell or cell population with the cell or cell population, whereby the guide RNA targets a DNA molecule of a cell within the cell or cell population, and the Cas protein nicks each of the first and second strands of the DNA molecule of the cell or cell within the cell (and the Cas protein and the two guide RNAs do not naturally occur together), thereby modifying a genomic locus of interest in the cell or cell population, e.g., a genomic locus of interest associated with a mutation associated with aberrant protein expression or a disease condition or state; and the method optionally includes contacting a cell or cell population containing, e.g., an HDR template. The method may also include a step of delivering the HDR template via a contacting delivery vector or particle or by contacting the cell or cell population with another particle containing the HDR template, where the HDR template results in expression of a normal or less aberrant form of the protein; "normal" may be with respect to wild type, and "aberrant" may be a protein expression that causes a pathology or disease state; and optionally, the method may include a step of isolating or obtaining a cell from an organism or a non-human organism, a step of optionally expanding said cell population, a step of performing contact of the cell with one or more delivery vectors or particles to obtain a modified cell population, a step of optionally expanding the population of modified cells, and a step of optionally administering the modified cells to the organism or a non-human organism. In an embodiment of the invention, the guide RNA may include a guide sequence and a tracr sequence fused to a tracr mate sequence. In an embodiment of the invention, the Cas protein is a type II CRISPR-Cas protein. In an embodiment of the invention, the Cas protein is codon-optimized for expression in eukaryotic cells, preferably mammalian cells or human cells. In a further embodiment of the invention, the Cas protein is a type II CRISPR-Cas protein, such as a Cas9 protein. In a highly preferred embodiment, the Cas protein is a Cas9 protein, such as SpCas9 or SaCas9.In an embodiment of the invention, the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A relative to SpCas9; for example, the D10A mutation. Aspects of the invention relate to reducing expression of a gene product, or introducing a template polynucleotide further into the DNA molecule encoding the gene product, or allowing reannealing and ligation of two 5' overhangs to precisely excise the intervening sequence, or altering the activity or function of the gene product, or increasing expression of the gene product. In an embodiment of the invention, the gene product is a protein. In a preferred embodiment of the invention, the vector of the system is a viral vector. In a further embodiment, the vector of the system is delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; and particles are preferred. In one aspect, the invention provides a method of modifying a target polynucleotide in a cell or cell population. In some embodiments, the method includes binding a CRISPR complex to a target polynucleotide, causing cleavage of the target polynucleotide, thereby modifying the target polynucleotide, where the CRISPR complex includes a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within the target polynucleotide, the guide sequence linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. In some embodiments, the cleavage includes cleaving one or both strands at the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of a target gene. In some embodiments, the method further includes repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, where the repair results in a mutation including an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene that includes a target sequence.In some embodiments, the method further comprises delivering one or more vectors or one or more expression products thereof to the cell or cell population, e.g., via one or more delivery vectors or particles, where the one or more vectors drive expression of one or more of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence. In some embodiments, the vector is delivered to the cell or cell population of a subject. In some embodiments, the modifying step is performed on the cell or cell population in a cell culture. In some embodiments, the method further comprises isolating the cell or cell population from a subject prior to the modifying step. In some embodiments, the method further comprises returning the cell or cell population and / or cells derived therefrom to the subject.

[0022] In one aspect, the invention provides a method of generating a cell or cell population comprising a mutated disease gene. In some embodiments, the disease gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises: (a) introducing one or more vectors or one or more expression products thereof into a cell or cell population, e.g., via one or more delivery vectors or particles, wherein the one or more vectors drive expression of one or more of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence; and (b) binding a CRISPR complex to a target polynucleotide, causing cleavage of a target polynucleotide within the disease gene, the CRISPR complex comprising a CRISPR enzyme complexed with (1) a guide sequence hybridized to a target sequence within the target polynucleotide, and (2) a tracr mate sequence hybridized to a tracr sequence, thereby generating a cell or cell population comprising a mutated disease gene. In some embodiments, the cleavage comprises cleavage of one or both strands at the location of the target sequence by the CRISPR enzyme. In some embodiments, the truncation results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing the truncated target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide. In some embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the modified cell or cell population is administered to an animal, thereby creating an animal model.

[0023] In one aspect, the present invention provides a method for modifying a target polynucleotide in a cell or cell population. In some embodiments, the method comprises a step of binding a CRISPR complex to a target polynucleotide, causing cleavage of the target polynucleotide, thereby modifying the target polynucleotide, the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence in the target polynucleotide, the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. In another embodiment, the present invention provides a method for modifying expression of a polynucleotide in a eukaryotic cell resulting from a cell or cell population expressing an abnormal protein. The method comprises a step of increasing or decreasing expression of a target polynucleotide using a CRISPR complex that binds to a polynucleotide in a cell or cell population; advantageously, the CRISPR complex is delivered via one or more viral delivery vectors or particles.

[0024] In some methods, altered expression in a cell or cell population can occur by inactivating a target polynucleotide, for example, when a CRISPR complex binds to a target sequence in a cell, the target polynucleotide is inactivated, such that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function like the wild-type sequence.

[0025] In some embodiments, the functional domain is a transcription activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is a SID, or a concatemer of SIDs (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, thus providing an epigenetic modification enzyme. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

[0026] The present invention further encompasses the use of the composition of the present invention or its CRISPR complex or enzyme or its RNA (including or instead of the mRNA encoding the CRISPR enzyme) in medicine or therapy. In some embodiments, the present invention encompasses the use of the composition of the present invention or its components in the method of the present invention. In some embodiments, the present invention provides the use of the composition of the present invention or its CRISPR complex or enzyme or its RNA (including or instead of the mRNA encoding the CRISPR enzyme) in ex vivo gene or genome editing, particularly in a cell or cell population that may then optionally be introduced into the organism or non-human organism from which the cell or cell population was obtained or into another organism or non-human organism of the same species. In certain embodiments, the present invention encompasses the use of the composition of the present invention or its CRISPR complex or enzyme or its RNA (including or instead of the mRNA encoding the CRISPR enzyme) in the manufacture of an ex vivo gene or genome editing agent or agent used in the method of the present invention. In certain embodiments, the present invention provides a method of treating or inhibiting a pathology caused by a defect in a target sequence in a genomic locus of interest in a subject (e.g., a mammal or human) or non-human subject (e.g., a mammal) in need thereof, the method comprising modifying a cell or cell population of the subject or non-human subject by manipulation of the target sequence in the cell or cell population, and administering the modified cell to the subject or non-human subject, advantageously, modifying the cell is via contacting the cell with a delivery vector (e.g., a viral delivery vector) or particle containing a CRISPR complex or a component thereof, advantageously in certain embodiments the delivery vector (viral delivery vector) or particle also provides the HDR template, or a separate particle or vector provides the HDR template, and the pathology is amenable to treatment or inhibition by manipulation of the target sequence.

[0027] The specific RNA of the CRISPR Cas complex is also known and is called sgRNA (single guide RNA). In an advantageous embodiment, the RNA of the CRISPR Cas complex is sgRNA. The CRISPR-Cas9 system is engineered to target one or more loci in a cell or cell population. Advantageously, Cas9 proteins codon-optimized for eukaryotic cells and particularly mammalian cells, such as human cells (e.g., ocular cells, vascular cells, cochlear cells, etc.), and sgRNAs targeting one or more loci in cells, such as genes RHO, ATOH1, VEGFA, have been prepared and are exemplified herein. These are advantageously delivered by viral delivery (AAV). In the case of particle delivery, the particle is formed by mixing Cas9 protein and sgRNA. The sgRNA and Cas9 protein mixture is mixed with a mixture comprising or consisting essentially of or consisting of surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols, thereby forming particles containing the sgRNA and Cas9 protein. The invention encompasses the particles so produced and the particles from such methods and their uses. More generally, the particles are formed using an efficient process. First, the Cas9 protein and the sgRNA targeting the gene or the control gene LacZ are mixed together, advantageously in a sterile nuclease-free buffer, such as 1×PBS, in a suitable molar ratio, such as 3:1 to 1:3 or 2:1 to 1:2 or 1:1, at a suitable temperature, such as 15-30° C., such as 20-25° C., such as room temperature, for a suitable time, such as 15-45, such as 30 minutes. Separately, particle components such as or including surfactants, e.g., cationic lipids, e.g., 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); phospholipids, e.g., dimyristoylphosphatidylcholine (DMPC); biodegradable polymers, e.g., ethylene glycol polymers or PEG, and lipoproteins, e.g., low density lipoproteins, e.g., cholesterol, are dissolved in alcohol, preferably C 1~6Dissolved in an alkyl alcohol, such as methanol, ethanol, isopropanol, for example 100% ethanol. These two solutions are mixed together to form particles containing Cas9-sgRNA complexes. In certain embodiments, the particles can contain HDR template. This can be a particle that is co-administered with sgRNA+Cas9 protein-containing particles, or that is, in addition to contacting a cell or cell population with sgRNA+Cas9 protein-containing particles, the cell or cell population is contacted with particles containing HDR template; or HSC is contacted with particles containing all of sgRNA, Cas9 and HDR template. HDR template can be administered by a separate vector, whereby in the first example, the particle enters the HSC cell, and a separate vector also enters the cell, where the HSC genome is modified by sgRNA+Cas9, and the HDR template is also present, whereby the genomic locus is modified by HDR; for example, this can result in the correction of a mutation. The particles in the discussion herein are advantageously obtained or obtainable by mixing a mixture of one or more sgRNAs and Cas9 protein (optionally containing one or more HDR templates or such a mixture containing only one or more HDR templates in case separate particles for one or more templates are desired) with a mixture comprising or consisting essentially of or consisting of surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols, where one or more sgRNAs target one or more genetic loci associated with aberrant protein expression or mutations associated with a disease condition or state.

[0028] In one aspect, the invention provides a method of modeling a disease associated with a genomic locus in a eukaryotic or non-human organism, the method comprising: (A)-I. A CRISPR-Cas system RNA polynucleotide sequence, (a) a guide sequence capable of hybridizing to a target sequence; (b) tracr mate sequence, and (c) tracr sequence A polynucleotide sequence comprising: II. Polynucleotide sequences encoding Cas9, optionally including at least one nuclear localization sequence [(a), (b) and (c) are arranged in the 5' to 3' direction, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to a target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence, and the polynucleotide sequence encoding Cas9 is DNA or RNA. or (B) I. A polynucleotide comprising: (a) a guide sequence capable of hybridizing to a target sequence, and (b) at least one tracr mate sequence A polynucleotide comprising II. Polynucleotide sequences encoding Cas9, and III. Polynucleotide sequences comprising the tracr sequence [When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence, and the polynucleotide sequence encoding Cas9 is DNA or RNA.

[0029] In certain preferred embodiments, the Cas9 is SaCas9.

[0030] In one aspect, the invention provides a method of modeling a disease associated with a genomic locus in a eukaryotic or non-human organism, the method comprising manipulating a target sequence within a coding, non-coding or regulatory element of said genomic locus comprising delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors functionally encoding said composition for expression of said composition, wherein the composition comprises: (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence being (a) a guide sequence capable of hybridizing to a target sequence; (b) tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to the enzyme coding sequence encoding Cas9 (preferably SaCas9), optionally including at least one nuclear localization sequence. [(a), (b) and (c) are arranged in the 5' to 3' direction, Components I and II are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and the CRISPR complex comprises (1) a guide sequence that hybridizes to a target sequence, and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element, (a) a guide sequence capable of hybridizing to a target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to the enzyme coding sequence encoding Cas9; and III. A third regulatory element operably linked to the tracr sequence, [wherein components I, II and III are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex includes a composition comprising a vector system comprising one or more vectors comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence.

[0031] In one aspect, the invention provides a method of treating or inhibiting a condition or disease caused by one or more mutations in a genomic locus in a eukaryotic or non-human organism, the method comprising modifying a subject or non-human subject by manipulation of a target sequence, the method comprising manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a target sequence in a subject or non-human subject in need thereof, and the condition or disease is Delivering a non-naturally occurring or engineered composition comprising an AAV or lentiviral vector system, comprising one or more AAV or lentiviral vectors functionally encoding the composition for expression of the composition. wherein the target sequence is amenable to treatment or inhibition by manipulation of the target sequence comprising providing a treatment comprising the step of: (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence being (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to an enzyme coding sequence encoding Cas9, preferably SaCas9, comprising at least one nuclear localization sequence. [(A), (b) and (c) are arranged in the 5' to 3' direction, Components I and II are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and the CRISPR complex comprises one or more vectors comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding Cas9, preferably SaCas9; and III. A third regulatory element operably linked to the tracr sequence, [wherein components I, II and III are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex includes a composition comprising a vector system comprising one or more vectors comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence.

[0032] In certain embodiments, the invention provides a method of preparing an AAV or lentivirus vector for use in any of the methods of the invention, the method comprising the steps of transfecting an AAV-infected or lentivirus-infected cell with one or more plasmids containing or consisting essentially of one or more nucleic acid molecules encoding the AAV or lentivirus, and providing the AAV AAV or lentivirus rep and / or cap and / or helper nucleic acid molecules essential for AAV or lentivirus replication and packaging.

[0033] In one aspect, the invention provides a composition for use in any of the methods of the invention (e.g., a method of modeling a disease associated with a genetic locus in a eukaryotic or non-human organism) that comprises manipulation of a target sequence within a coding, non-coding, or regulatory element of the genetic locus. In certain embodiments, the invention provides for the use of the composition in ex vivo or in vivo gene or genome editing, including therapeutic applications.

[0034] In one aspect, the invention provides a composition for use in the manufacture of an in vitro, ex vivo or in vivo gene or genome editing agent or agent for use in a method of modifying an organism or non-human organism by manipulation of a target sequence at a genomic locus associated with a disease, or for use in a method of treating or inhibiting a condition or disease caused by one or more mutations at a genomic locus in a eukaryotic organism or non-human organism.

[0035] In one aspect, the present invention provides a method for producing (A)-I. A CRISPR-Cas system RNA polynucleotide sequence, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) tracr mate sequence, and (c) tracr sequence A polynucleotide sequence comprising: II. Polynucleotide sequence encoding Cas9, preferably Sa Cas9, optionally including at least one nuclear localization sequence [(a), (b) and (c) are arranged in the 5' to 3' direction, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to a target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence, and the polynucleotide sequence encoding Cas9 is DNA or RNA. or (B) I. A polynucleotide comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one tracr mate sequence A polynucleotide comprising II. Polynucleotide sequences encoding Cas9, preferably SaCas9, and III. Polynucleotide sequences comprising the tracr sequence [When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex comprises SaCas9 complexed with (1) a guide sequence that hybridizes to a target sequence, and (2) a tracr mate sequence that hybridizes to a tracr sequence, and the polynucleotide sequence encoding Cas9 is DNA or RNA; The present invention provides compositions for use in medicine or therapy; or for use in methods of modifying organisms or non-human organisms by manipulation of target sequences at genomic loci associated with a disease or disorder; or for use in methods of treating or inhibiting a pathology caused by one or more mutations at a genomic locus associated with a disease in a eukaryotic organism or non-human organism; or for use in in vitro, ex vivo or in vivo gene or genome editing.

[0036] In one aspect, the invention provides a therapeutic genome editing method for treating or inhibiting a condition or disease caused by one or more mutations in a genomic locus in a eukaryotic or non-human organism, the method comprising modifying a subject or non-human subject by manipulation of a target sequence, the target sequence comprising manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a subject or non-human subject in need thereof, and the condition or disease is Delivering a non-naturally occurring or engineered composition comprising an AAV or lentiviral vector system, comprising one or more AAV or lentiviral vectors functionally encoding the composition for expression of the composition. wherein the target sequence is amenable to treatment or inhibition by manipulation of the target sequence comprising providing a treatment comprising the step of: (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) tracr mate sequence, and (c) tracr sequence A first regulatory element comprising: II. A second regulatory element operably linked to an enzyme coding sequence encoding Cas9, preferably SaCas9, comprising at least one nuclear localization sequence. [(a), (b) and (c) are arranged in the 5' to 3' direction, Components I and II are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and the CRISPR complex comprises one or more vectors comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to a tracr sequence; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element, (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to an enzyme coding sequence encoding SaCas9; and III. A third regulatory element operably linked to the tracr sequence [wherein components I, II and III are located on the same or different vectors of the system, When transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex includes a composition comprising a vector system comprising one or more vectors comprising: (1) a guide sequence that hybridizes to a target sequence; and (2) a Cas9 complexed with a tracr mate sequence that hybridizes to tracr.

[0037] In one aspect, the present invention provides a method for individualized or personalized treatment of a genetic disease in a subject in need of such treatment, the method comprising: (a) introducing multiple mutations ex vivo in a tissue, organ or cell line comprising one or more Cas9-expressing eukaryotic cells (preferably Sa Cas9), or in vivo in a transgenic non-human mammal having cells expressing Cas9, comprising delivering a vector as discussed herein to one or more cells of the tissue, organ, cell or mammal, wherein the specific mutations or precise sequence substitutions are or have been associated with a genetic disease; (b) testing one or more treatments for the genetic disease on cells to which the vector has been delivered that have the specific mutation or precise sequence substitution associated with the genetic disease; and (c) treating the subject based on the results of the one or more treatments tested in step (b).

[0038] In certain embodiments of any of the foregoing aspects and embodiments of the invention, the viral vector may be AAV, such as AAV1, AAV2, AAV5, AAV7, AAV8, AAV DJ, or any combination thereof.

[0039] In any discussion herein of a target being associated with a mutation or disease state, such mutation or disease state may be, for example, a neurological disease; an ophthalmic disease (e.g., a retinal disease, e.g., retinitis pigmentosa; achromtaopsia; age-related macular degeneration; visual impairment), an auditory disease (e.g., a cochlear cell-related disease, hearing impairment, hearing loss), and the like.

[0040] Accordingly, it is the object of the present invention not to include within its scope any product, process for making a product, or method for using a product that has already been described, and the applicants reserve the right to any known product, process, or method, but hereby expressly disclaim it. It is further noted that the present invention does not include within its scope any product, process, or method for making such a product, or method for using such a product, that does not meet the description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (EPC, Section 83), and the applicants reserve the right to any product, process for making a product, or method for using a product that has already been described, but hereby expressly disclaim it.

[0041] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, words such as "comprises," "comprised," and "comprising" may have the meaning given to them by U.S. Patent Law; for example, these words mean "includes," "included," "including," and the like; and words such as "consisting essentially of" and "consists essentially of" have the meaning given to them by U.S. Patent Law, for example, allowing for elements not expressly recited, but excluding elements found in the prior art or that affect the basic or novel characteristics of the invention. In the practice of the present invention, compliance with Article 53(c) EPC and Rules 28(b) and (c) EPC will be advantageous. No covenants are present herein.

[0042] These and other embodiments are disclosed or will be apparent from and encompassed by the following detailed description. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained from the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0043] [Figure 1A-1H]Figure 1A-H show CRISPR-Cas9 system delivery and targeting of Mecp2 locus in mouse brain. (a) AAV-SpCas9 and AAV-SpGuide(Mecp2) expression vectors. The sgRNA vector contains a coding sequence for a GFP-KASH fusion protein to identify transduced neurons. (b) Expression of HA-Cas9 and GFP-KASH in the dorsal dentate gyrus (DG) of mouse hippocampus. Scale bar, 100 μm. (c) Quantification of cells efficiently targeted by the dual vector Cas9-CRISPR system. (d) Schematic representation of mouse Mecp2 locus showing Cas9 target position; sgRNA is shown in blue. PAM sequence is shown in purple. Representative mutation patterns detected by sequencing of Mecp2 locus are shown below: green - wild type sequence; red dash symbol - deleted base; red base: insertion or mutation; red triangular arrow indicates CRISPR-Cas9 cleavage site. (e) SURVEYOR™ assay gel showing alteration of the Mecp2 locus 2 weeks after AAV delivery to the DG region. (f) Western blot analysis of MeCP2 protein expression in targeted brain regions and quantification of MeCP2 protein levels in the dorsal DG (t-test, **p<0.001, n=4 from 3 animals, error bars: sem). (g) Image of the dorsal DG region 2 weeks after targeting of the Mecp2 locus with CRISPR-Cas9. Scale bar, 150 μm. (h) Quantification of the MeCP2 positive cell population (DAPI staining) among all cells detected in the targeted brain region compared to control collateral sites (t-test, ****p<0.0001, n=290 and 249 cells from 2 animals, respectively; error bars: sem). (ITR-inverted terminal repeat; HA-hemagglutinin tag; NLS-nuclear localization signal; spA-synthetic polyadenylation signal; U6-PolIII promoter; sgRNA-single guide RNA; hSyn-human synapsin 1 promoter; GFP-green fluorescent protein; KASH-Klarsicht, ANC1, Syne homology nuclear transmembrane domain; bGH pA-bovine growth hormone polyadenylation signal; WPRE-woodchuck hepatitis virus posttranscriptional regulatory element). [Figure 2A-2B] Figures 2A-B show analysis of gene expression in Cas9-mediated MeCP2 knockdown neurons. (a) Nuclei purification strategy of CRISPR-Cas9 targeted cells from mouse brain. (b) Hierarchical clustering of differentially expressed genes detected by RNAseq (t-test, p<0.01, n=19 populations of sorted nuclei from 8 animals). Relative log2(TPM+1) expression levels of genes per row are normalized and displayed on a red-blue color scale. Each column represents a population of 100 targeted neuronal nuclei FACS sorted from isolated dentate gyrus cell populations derived from either control or Mecp2 sgRNA-transduced animals as indicated. [Figure 3A-3E] Figures 3A-E show cell-autonomous defects in cellular response properties of neurons after CRISPR-mediated MeCP2 knockdown. (a) Diagram showing in vivo experimental setup and visual stimulation parameters from mouse visual cortex. GFP+ neurons are shown. Scale bar, 20 μm. (b) Diagram showing recording setup in layer 2 / 3 excitatory neurons receiving specific inputs to both the contralateral and ipsilateral eyes. Genome-modified GFP+ cells are green, whereas unmodified cells are gray. Normalized spike shapes show excitatory neurons that spike regularly. (c, d) Mean OSI (c) and evoked FR (d) were measured from GFP+ cells expressing Mecp2 and control sgRNA, respectively (t-test, *p<0.05; numbers in graphs indicate number of recorded cells; n=2-3 animals; error bars: sem). [Figure 4A-4F]4A-F show simultaneous multiple gene editing in mouse brain. (a) Schematic of the CRISPR-Cas9 system designed for multiplex genome targeting. (b) Illustrated representation of targeted DNMT mouse loci. Guide RNAs are shown in blue. PAM sequences are shown in purple. (c) SURVEYOR™ assay gel showing modification of DNMT loci in FACS-sorted GFP-KASH positive cells 4 weeks after AAV delivery into the DG region. (d) Deep sequencing-based analysis of DNMT locus modification in single cells showing simultaneous modification of multiple loci. (e) Western blot analysis of Dnmt3a and Dnmt1 proteins (top) after in vivo delivery of the CRISPR-Cas9 system targeting DNMT family genes. Western blot quantification of Dnmt3a and Dnmt1 protein levels in the DG after in vivo CRISPR-Cas9 targeting (bottom; t-test, **p<0.001, *p<0.05, Dnmt3a: n=7; Dnmt1: n=5 from 5 animals; error bars: sem). (f) Contextual learning impairment 8 weeks after targeting DNMT genes with SpCas9 in the DG region of the hippocampus, tested in training context and context shift (t-test, ***p<0.0001, n=18 animals, 2 independent experiments; error bars: sem). [Figure 5A-5F]Figure 5A-F show the cloning and expression of HA-tagged SpCas9 (HA-SpCas9) for AAV packaging. (a) Schematic of different cloning strategies using a short rat Map1b promoter (pMap1b), a truncated mouse Mecp2 promoter (pMecp2), and a short polyA motif (spA) to minimize SpCas9 expression cassette size. (b) Western blot analysis of primary cortical neuron cultures expressing HA-SpCas9 using different SpCas9 expression cassettes. (c) The Mecp2 promoter drives HA-SpCas9 (red) expression in neurons (Map1b, NeuN; arrows) but not in astroglia (GFAP, triangular arrow). Co-expression of HA-SpCas9 with GFP-KASH is shown (bottom). Nuclei were labeled with DAPI (blue). Scale bar, 20 μm. (d) Schematic of GFP labeling. Enhanced green fluorescent protein (GFP) fused to a nuclear transmembrane KASH domain and integration of GFP-KASH into the nuclear envelope are shown. (e) Superinfection efficiency calculation showing the population of cells expressing both HA-SpCas9 and GFP-KASH (n=973 neurons from three cultures; error bars: sem). (f) Cells were stained with the LIFE / DEAD® kit 7 days after virus delivery. Quantification of DAPI+ and dead (DEAD+) cells (control n=518 DAPI+ nuclei; SpCas9 / GFP-KASH n=1003 DAPI+ nuclei from two cultures; error bars: sem). (ITR-inverted terminal repeat; HA-hemagglutinin tag; NLS-nuclear localization signal; spA-synthetic polyadenylation signal; U6-PolIII promoter; sgRNA-single guide RNA; hSyn-human synapsin 1 promoter; GFP-green fluorescent protein; KASH-Klarsicht, ANC1, Syne homology nuclear transmembrane domain; bGH pA-bovine growth hormone polyadenylation signal; WPRE-woodchuck hepatitis virus posttranscriptional regulatory element). [Figure 6A-6B]6A-6B show targeting of Mecp2 in Neuro-2a cells. (a) Mecp2 targeting sequence and corresponding protospacer adjacent motif (PAM). (b) Evaluation of six Mecp2 sgRNAs co-transfected with SpCas9 into Neuro-2a cells. Locus modification efficiency was analyzed 48 hours post-transfection using the SURVEYOR™ assay. [Figure 7A-7D] 7A-7D show targeting of Mecp2 by CRISPR-SpCas9 in primary cortical neurons. (a) Immunofluorescence staining of MeCP2 (red) in cultured neurons 7 days after AAV-CRISPR transduction (green, GFP-KASH). Nuclei were labeled with DAPI (blue). Scale bar, 20 μm. (b) Assessment of Mecp2 gene targeting using SpCas9 or dSpCas9 with Mecp2 sgRNA or control (targeting bacterial lacZ gene) sgRNA using SURVEYOR™ assay gel. (c) Quantification of MeCP2 positive nuclei (GFP+) in the targeted neuronal population. (d) Western blot of MeCP2 protein levels and quantification of MeCP2 protein levels after targeting of the Mecp2 locus by CRISPR-SpCas9 (t test, **p<0.001, n=5 from 3 cultures, error bars: sem). [Figure 8A-8E]Figures 8A-E show morphological changes in the dendritic tree of neurons after SpCas9-mediated MeCP2 knockdown in vitro. (a) Reduction in dendritic tree complexity in neurons after targeting of the Mecp2 locus with CRISPR-SpCas9. Scale bar, 20 μm. (b) Changes in dendritic spine morphology in neurons targeted with SpCas9 and Mecp2 sgRNA. Scale bar, 10 μm. Cell morphology was visualized by co-transfection with mCherry constructs. Cells for morphological analysis were selected based on the results of Mecp2 staining. (c) Dendritic tree morphology assessed by the number of dendritic terminals and (d) Sholl analysis (t test, ***p<0.0001, n=40 from two cultures). (e) Spine density quantification (t test, ***p<0.0001, n=40 from two cultures, error bars: sem). [Figure 9] Figure 9 shows RNAseq of neural nuclei from control animals and SpCas9-mediated Mecp2 knockdown. Box plot showing the number of genes detected in all RNA-seq libraries (100 each of nuclei taken from control sgRNA or 19 libraries of nuclei transduced with Mecp2 sgRNA; n=4 animals / group) per quantile of expression level. All genes were divided into 10 quantiles by their mean log2(TPM+1) expression level, and then for each quantile, the number of genes detected (log2(TPM+1)>2) per sample was counted. The three target sequences shown are SEQ ID NO: ___, SEQ ID NO: ___, and SEQ ID NO: ___ for Dnmt3a, Dnmt1, and Dnmt3b, respectively. [Figure 10A-10B] 10A-10B show multiplex genomic targeting of DNMT family members in vitro. (a) Dnmt3a, Dnmt1, and Dnmt3b targeting sequences and corresponding protospacer adjacent motifs (PAMs). (b) SURVEYOR™ nuclease assay analysis of Neuro-2a cells 48 hours after transfection of SpCas9 and DNMT 3xsgRNA vectors targeting Dnmt3a, Dnmt1, and Dnmt3b loci. Efficient genome editing of all three targeted genes is shown. [Figures 11A-11C] 11A-11C show next generation sequencing of targeted Dnmt3a, Dnmt1 and Dnmt3b loci. Examples of sequencing results of mutant Dnmt3a (a), Dnmt1 (b) and Dnmt3b (c) loci after in vivo delivery of SpCas9 and DNMT 3xsgRNA to mouse dentate gyrus. Green: wild type sequence, red dashes: deleted bases, red bases: insertions or mutations. Red triangular arrows indicate CRISPR-SpCas9 cleavage sites. The complete sequences used in this figure are provided as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 for Dnmt3a, Dnmt1 and Dnmt3b loci, respectively. These are: SEQ ID NO: (Dnmt3a): CCT CCG TGT CAG CGA CCC ATG CCA A, SEQ ID NO: (Dnmt1): CCA GCG TCG AAC AGC TCC AGC CCG and SEQ ID NO: (Dnmt3b): AGA GGG TGC CAG CGG GTA TAT GAG G [Figure 12] FIG. 12 shows a comparison of different programmable nuclease platforms. [Figures 13A-13C]Figures 13A-C show types of therapeutic genome modifications. The specific type of genome editing therapy depends on the nature of the disease-causing mutation. a, In gene disruption, the pathogenic function of a protein is silenced by targeting the gene locus with NHEJ. The formation of indels in the gene of interest often results in frameshift mutations creating premature stop codons and nonfunctional protein products or nonsense-mediated decay of the transcript, suppressing gene function. b, Deleterious mutations can be corrected using HDR gene correction. DSBs are targeted in the vicinity of the mutation site in the presence of an exogenously provided correction HDR template. HDR repair of this break site with the exogenous template corrects the mutation and restores gene function. c, An alternative to gene correction is gene addition. This therapy introduces a therapeutic transgene into a safe harbor locus in the genome. A DSB is targeted to the safe harbor locus and an HDR template containing homology to the break site, a promoter and a transgene are introduced into the nucleus. HDR repair copies the promoter-transgene cassette into the safe harbor locus, restoring gene function, but without true physiological control over gene expression. [Figure 14] FIG. 14 shows a schematic of ex vivo vs. in vivo editing therapy. In ex vivo editing therapy, cells are removed from the patient, edited, and then reimplanted (top panel). For this therapy to be successful, the target cells must be able to survive outside the body and home to the target tissue after transplantation. In vivo therapy involves genome editing of cells in situ (bottom panel). For in vivo systemic therapy, a delivery agent that is relatively independent of the identity or state of the cell may be used to effect editing in a wide range of tissue types. This editing therapy approach may be possible in the future, but currently there is no delivery system efficient enough to make this feasible. In vivo targeted therapy, in which a delivery agent with tropism for a specific organ system is administered to the patient, is feasible using clinically relevant viral vectors. [Figure 15]FIG. 15 shows the SaCas9 system for ocular gene therapy. [Figure 16] FIG. 16 shows a schematic diagram of gene therapy using Cas9 homologous recombination (HR) vectors. [Figure 17] FIG. 17 shows an exemplary protocol for ocular gene therapy. [Figure 18A-18B] Figures 18A-B show the human RHO locus (alleles showing the P23H mutation). Figure 7A shows guide design for the RHO locus. Figure 7B shows in vitro guide screening results using the SURVEYOR assay. [Figure 19] FIG. 19 shows the RHO HR AAV vector. [Figure 20A-20B] 20A-20B show guide selection for CNGA3 and CNGB3. (a) shows the human CNGA3 locus (alleles showing two disease mutations) and guide selection. (b) shows the human CNGB3 locus (alleles showing disease mutations) and guide selection. [Figure 21] FIG. 21 shows the CNGA3 HR AAV vector. [Figure 22] FIG. 22 shows the CNGB3 HR AAV vector. [Figure 23A-23B] Figures 23A-B show guide selection of VEGFA. (a) shows the human VEGFA locus (lcous) (common region 1); (b) shows the human VEGFA locus (lcous) (common region 2). [Figure 24] Figure 24 shows the design of a dCas9-based epigenetic modulation system (the three components of the system are shown: dSaCas9, fusion effector, and sgRNA). [Fig. 25A-25C] Figures 25A-C show guide selection for ATOH1. (a) shows two selected highly accessible regions; (b) shows highly accessible region 1 - blue line indicates guide sequence, magenta line indicates PAM; (c) shows highly accessible region 2 - blue line indicates guide sequence, magenta line indicates PAM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The drawings herein are for illustrative purposes only and are not necessarily drawn to scale.

[0045] For general information regarding CRISPR-Cas systems, their components, and delivery of such components, as well as methods, materials, delivery vehicles, vectors, particles, AAVs, and the production and use, as well as amounts and formulations thereof, all of which are useful in practicing the present invention, see U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, and 8,895,308;US Patent Application Publication No. 2014-0310830 (US Patent Application No. 14 / 105,031), US Patent Application Publication No. 2014-0287938 A1 (US Patent Application No. 14 / 213,991), US Patent Application Publication No. 2014-0273234 A1 (US Patent Application No. 14 / 293,674), US Patent Application Publication No. 2014-0273232 A1 (US Patent Application No. 14 / 290,575), US Patent Application Publication No. 2014-0273231 (US Patent Application No. 14 / 259,420), US Patent Application Publication No. 2014-0256046 A1 (U.S. Patent Application No. 14 / 226,274), U.S. Patent Application Publication No. 2014-0248702 A1 (U.S. Patent Application No. 14 / 258,458), U.S. Patent Application Publication No. 2014-0242700 A1 (U.S. Patent Application No. 14 / 222,930), U.S. Patent Application Publication No. 2014-0242699 A1 (U.S. Patent Application No. 14 / 183,512), U.S. Patent Application Publication No. 2014-0242664 A1 (U.S. Patent Application No. 14 / 104,990), U.S. Patent Application Publication No. 2014-0234972 A1 (U.S. Patent Application No. 14 / 183,471), U.S. Patent Application Publication No. 2014-0227787 A1 (U.S. Patent Application No. 14 / 256,912), U.S. Patent Application Publication No. 2014-0189896 A1 (U.S. Patent Application No. 14 / 105,035), U.S. Patent Application Publication No. 2014-0186958 (U.S. Patent Application No. 14 / 105,017), U.S. Patent Application Publication No. 2014-0186919 A1 (U.S. Patent Application No. 14 / 104,977), U.S. Patent Application Publication No. 2014-0186843 A1 (U.S. Patent Application No. 14 / 104,900), U.S. Patent Application Publication No. 2014-0179770 A1 (U.S. Patent Application No. 14 / 104,837), and U.S. Patent Application Publication No. 2014-0179006 A1 (U.S. Patent Application No. 14 / 183,486), U.S. Patent Application Publication No. 2014-0170753 (U.S. Patent Application No. 14 / 183,429);European Patent Application No. 2771468 (EP 13818570.7), European Patent Application No. 2764103 (EP 13824232.6), and European Patent Application No. 2784162 (EP 14170383.5); and International Publication No. WO 2014 / 093661 (International Application No. PCT / US2013 / 074743). Detailed Description), International Application No. PCT / US2013 / 074790 (Specification), International Application No. PCT / US2014 / 093694 (Specification), International Application No. PCT / US2013 / 074611 (Specification), International Application No. PCT / US2014 / 093595 (Specification), International Application No. PCT / US2013 / 074611 (Specification), International Application No. PCT / US2014 / 093718 (Specification), International Application No. PCT / US2013 / 074825 (Specification), International Application No. PCT / US2014 / 093709 (Specification), Brochure (International Application No. PCT / US2013 / 074812), Brochure No. 2014 / 093622 (International Application No. PCT / US2013 / 074667), Brochure No. 2014 / 093635 (International Application No. PCT / US2013 / 074691), Brochure No. 2014 / 093655 (International Application No. PCT / US2 No. 013 / 074736, No. 2014 / 093712 (International Application No. PCT / US2013 / 074819), No. 2014 / 093701 (International Application No. PCT / US2013 / 074800), and No. 2014 / 018423 (International Application No. PCT / US2013 / 051418). See also U.S. Provisional Patent Application Nos. 61 / 758,468, filed January 30, 2013; 61 / 802,174, filed March 15, 2013; 61 / 806,375, filed March 28, 2013; 61 / 814,263, filed April 20, 2013; and 61 / 819,803, filed May 6, 2013;and 61 / 828,130, filed May 28, 2013. See also U.S. Provisional Patent Application No. 61 / 836,123, filed June 17, 2013. See also U.S. Provisional Patent Application Nos. 61 / 835,931, 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 836,080, and 61 / 835,973, each filed June 17, 2013. See also U.S. Provisional Patent Applications Nos. 61 / 862,468 and 61 / 862,355, filed August 5, 2013; 61 / 871,301, filed August 28, 2013; 61 / 960,777, filed September 25, 2013; and 61 / 961,980, filed October 28, 2013. Further, see International Application Nos. PCT / US2014 / 041803, PCT / US2014 / 041800, PCT / US2014 / 041809, PCT / US2014 / 041804, and PCT / US2014 / 041806, each filed on June 10, 2014; International Application No. PCT / US2014 / 041808, filed on June 11, 2014; International Application No. PCT / US2014 / 62558, filed on October 28, 2014; and U.S. Provisional Application Nos. 61 / 915,150, 61 / 915,301, and 61 / 915,267, each filed on December 12, 2013. and 61 / 915,260; 61 / 757,972, filed January 29, 2013, and 61 / 768,959, filed February 25, 2013; 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / Nos. 62 / 010,888 and 62 / 010,879, both filed June 11, 2014; Nos. 62 / 010,329 and 62 / 010,441, each filed June 10, 2014;See US Patent Nos. 61 / 939,228 and 61 / 939,242, filed February 12, 2014, respectively; US Patent No. 61 / 980,012, filed April 15, 2014; US Patent No. 62 / 038,358, filed August 17, 2014; US Patent Nos. 62 / 054,490, 62 / 055,484, 62 / 055,460, and 62 / 055,487, filed September 25, 2014; and US Patent No. 62 / 069,243, filed October 27, 2014. See also U.S. Provisional Patent Applications Nos. 62 / 055,484, 62 / 055,460, and 62 / 055,487, filed September 25, 2014; U.S. Provisional Patent Application No. 61 / 980,012, filed April 15, 2014; and U.S. Provisional Patent Application No. 61 / 939,242, filed February 12, 2014. See in particular International Application PCT / US14 / 41806, filed June 10, 2014, designating the United States. See U.S. Provisional Patent Application No. 61 / 930,214, filed January 22, 2014. U.S. Provisional Patent Application Nos. 61 / 915,251, each filed December 12, 2013;See U.S. Patent Application Nos. 61 / 915,260 and 61 / 915,267. See U.S. Provisional Patent Application No. 61 / 980,012, filed April 15, 2014. See specifically International Application No. PCT / US14 / 41806, filed June 10, 2014, designating the United States. Each of these patents, patent publications, and patent applications, and all documents cited in or during the prosecution of these patents ("application citations"), and all documents cited or referenced in the application citations, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products described in these patents or in any document of these patents, which are incorporated by reference herein, are incorporated by reference in these patents and may be utilized in the practice of the present invention. All documents (e.g., these patents, patent publications, and applications, and the documents cited in the applications) are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference;

[0046] Also, for general information regarding CRISPR-Cas systems, see (also incorporated herein by reference): Each of which is incorporated herein by reference and is briefly described below: Multiplex genome engineering using CRISPR / Cas systems.Cong, L.,Ran,FA,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,PD,Wu,X.,Jiang,W.,Marraffini,LA,& Zhang,F.Science Feb 15;339(6121):819-23(2013); RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Jiang W.,Bikard D.,Cox D.,Zhang F,Marraffini LA.Nat Biotechnol Mar;31(3):233-9(2013); One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR / Cas-Mediated Genome Engineering.Wang H.,Yang H.,Shivalila CS.,Dawlaty MM.,Cheng AW.,Zhang F.,Jaenisch R.Cell May 9;153(4):910-8(2013); Optical control of mammalian endogenous transcription and epigenetic states.Konermann S,Brigham MD,Trevino AE,Hsu PD,Heidenreich M,Cong L,Platt RJ,Scott DA,Church GM,Zhang F.Nature.2013 Aug 22;500(7463):472-6.doi:10.1038 / Nature12466.Epub 2013 Aug 23; Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.Ran,FA.,Hsu,PD.,Lin,CY.,Gootenberg,JS.,Konermann,S.,Trevino,AE.,Scott,DA.,Inoue,A.,Matoba,S.,Zhang,Y.,& Zhang,F.Cell Aug 28.pii:S0092-8674(13)01015-5.(2013); DNA targeting specificity of RNA-guided Cas9 nucleases.Hsu,P.,Scott,D.,Weinstein,J.,Ran,FA.,Konermann,S.,Agarwala,V.,Li,Y.,Fine,E.,Wu,X.,Shalem,O.,Cradick,TJ.,Marraffini,LA.,Bao,G.,& Zhang,F.Nat Biotechnol doi:10.1038 / nbt.2647(2013); Genome engineering using the CRISPR-Cas9 system.Ran,FA.,Hsu,PD.,Wright,J.,Agarwala,V.,Scott,DA.,Zhang,F.Nature Protocols Nov;8(11):2281-308.(2013); Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.Shalem,O.,Sanjana,NE.,Hartenian,E.,Shi,X.,Scott,DA.,Mikkelson,T.,Heckl,D.,Ebert,BL.,Root,DE.,Doench,JG.,Zhang,F.Science Dec 12.(2013).[Epub ahead of print]; Crystal structure of cas9 in complex with guide RNA and target DNA.Nishimasu,H.,Ran,FA.,Hsu,PD.,Konermann,S.,Shehata,SI.,Dohmae,N.,Ishitani,R.,Zhang,F.,Nureki,O.Cell Feb 27.(2014).156(5):935-49; Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Wu X.,Scott DA.,Kriz AJ.,Chiu AC.,Hsu PD.,Dadon DB.,Cheng AW.,Trevino AE.,Konermann S.,Chen S.,Jaenisch R.,Zhang F.,Sharp PA.Nat Biotechnol.(2014)Apr 20.doi:10.1038 / nbt.2889, CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling,Platt et al.,Cell 159(2):440-455(2014)DOI:10.1016 / j.cell.2014.09.014, Development and Applications of CRISPR-Cas9 for Genome Engineering,Hsu et al,Cell 157,1262-1278(June 5,2014)(Hsu 2014), Genetic screens in human cells using the CRISPR / Cas9 system,Wang et al.,Science.2014 January 3;343(6166):80-84.doi:10.1126 / science.1246981, Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench et al., Nature Biotechnology, published online 3 September 2014; doi:10.1038 / nbt.3026, and In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech et al, Nature Biotechnology; Published online 19 October 2014; doi:10.1038 / nbt.3055. Cong et al. engineered a type II CRISPR / Cas system for use in eukaryotic cells based on both Streptococcus thermophilus Cas9 and Streptococcus pyogenes Cas9 to demonstrate that Cas9 nuclease can be guided by short RNA to induce precise cleavage of DNA in human and mouse cells. Their work further demonstrated that Cas9 converted into a cleavage enzyme can be used to facilitate homologous recombination repair in eukaryotic cells with minimal mutagenic effects. In addition, their work demonstrated that multiple guide sequences can be encoded into a single CRISPR array, thereby enabling several simultaneous edits at endogenous genomic locus sites within mammalian genomes, demonstrating the readily programmable and broad applicability of RNA-guided nuclease technology. This ability to program sequence-specific DNA cleavage with RNA in cells defined a new class of genome engineering tool. These studies further demonstrated that other CRISPR loci are likely transplantable into mammalian cells and may also mediate mammalian genome cleavage. Importantly, it can be contemplated that several aspects of the CRISPR / Cas system can be further improved to increase its efficiency and versatility. Jiang et al. used clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with a duplex RNA to introduce precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli. This approach relied on duplex RNA:Cas9-dependent cleavage at the targeted genomic site to kill non-mutated cells, circumventing the need for a selectable marker or counter-selection system. This study reported reprogramming of duplex RNA:Cas9 specificity by altering the sequence of a short CRISPR RNA (crRNA) such that the editing template had single and multiple nucleotide changes. This study showed that the simultaneous use of two crRNAs allows for multiplex mutagenesis. Furthermore, when this approach was used in combination with recombineering in S. pneumoniae, nearly 100% of cells recovered using the described approach contained the desired mutations, and in E. coli, 65% of the recoveries contained mutations. Konermann et al. addressed the need in the art for a versatile and robust technology that enables optical and chemical modulation of DNA-binding domain-based CRISPR Cas9 enzymes and transcription activator-like effectors. The Cas9 nuclease from the microbial CRISPR-Cas system targets specific genomic loci with a 20-nt guide sequence that can tolerate certain mismatches to the DNA target, thus promoting undesired off-target mutations. To address this, Ran et al. described an approach in which Cas9 nickase mutations are combined with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking by appropriately offset guide RNAs is required for double-strand breaks, increasing the number of bases that are specifically recognized for targeted cleavage. The authors demonstrated that paired nicking can be used to facilitate gene knockout in mouse zygotes, reducing off-target activity by 50- to 1,500-fold in cell lines without sacrificing on-target cleavage efficacy. This versatile strategy enables diverse genome editing applications that require high specificity. Hsu et al. characterized SpCas9 targeting specificity in human cells to inform target site selection and avoid off-target effects. The study assessed over 700 guide RNA variants and SpCAs9-induced indel mutation levels at over 100 putative genomic off-target loci in 293T and 293FT cells. The authors reported that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, and is affected by the number, position, and distribution of mismatches. The authors further showed that SpCas9-mediated cleavage is not affected by DNA methylation, and that the amount of SpCas9 and sgRNA can be increased or decreased to minimize off-target modifications. In addition, to expand the applications of mammalian genome engineering, the authors reported providing a web-based software tool to guide target sequence selection and evaluation and off-target analysis. Ran et al. described a set of tools for Cas9-mediated genome editing by non-homologous end joining (NHEJ) or homology-dependent repair (HDR) in mammalian cells, and the generation of engineered cell lines for the study of downstream functions. To minimize off-target cleavage, the authors further described a double-nicking method using Cas9 nickase mutations with paired guide RNAs. The protocol provided by the authors empirically derived guidelines for the selection of target sites, evaluation of cleavage efficiency, and analysis of off-target activity. This study showed that starting from the design of the target, genetic modification can be achieved in as little as 1-2 weeks, and engineered clonal cell lines can be obtained within 2-3 weeks. Shalem et al. described a novel method to interrogate gene function on a genome-wide scale. Their study showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screening in human cells. First, the authors demonstrated the identification of genes essential for cell survival in cancer cells and pluripotent stem cells using the GeCKO library. Second, in a melanoma model, the authors screened for genes whose reduction affects resistance to vemurafenib, a therapeutic agent that inhibits the mutant protein kinase BRAF. Their study showed that the top candidates included the already evaluated genes NF1 and MED12, as well as the novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of consistency between independent guide RNAs targeting the same genes and a high hit confirmation rate, thus demonstrating that genome-wide screening with Cas9 is promising. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with an sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a two-lobe structure consisting of a target recognition lobe and a nuclease lobe that accommodates the sgRNA:DNA heteroduplex in a positively charged groove at its interface. The recognition lobe is essential for binding of the sgRNA to DNA, while the nuclease lobe contains the HNH and RuvC nuclease domains, with the HNH nuclease domain appropriately positioned to cleave the complementary strand of the target DNA and the RuvC nuclease domain appropriately positioned to cleave the non-complementary strand. The nuclease lobe also contains a carboxy-terminal domain involved in the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analysis reveal the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of novel, versatile genome editing technologies. Wu et al. mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes tagged with single guide RNA (sgRNA) in mouse embryonic stem cells (mESCs). The authors showed that each of the four sgRNAs tested targeted dCas9 between tens to thousands of genomic sites frequently characterized by the 5-nucleotide seed region of the sgRNA and a NGG protospacer adjacent motif (PAM). Chromatin inaccessibility reduced binding of dCas9 to other sites with matching seed sequences; thus, 70% of off-target sites were gene-associated. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one mutation site above background levels. The authors proposed a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but requires extensive pairing with the target DNA for cleavage. Hsu 2014 is a review article generally discussing the history of CRISPR-Cas9 from yogurt to genome editing, including genetic screening of cells within the information, data, and knowledge of applications in the line of this application filed before June 5, 2014. The general teachings of Hsu 2014 are not related to the particular models, animals herein.

[0047] Reference is also made to Tsai et al., “Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing,” Nature Biotechnology 32(6):569-77 (2014), which is not considered prior art to the present invention or application, but may be considered in the practice of the present invention.

[0048] Additionally, there is disclosed a method of preparing sgRNA·Cas9 protein-containing particles, the method comprising mixing a mixture comprising sgRNA and Cas9 protein (and optionally HDR template) with a mixture comprising, consisting essentially of, or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol; and with respect to particles from such methods, a reference is made to "DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DE ... Reference is made to co-filed PCT application entitled "PARTICLE DELIVERY COMPOUNDS," Attorney Docket Nos. 47627.99.2060 and BI-2013 / 107 (which claim priority from one or more or all of U.S. Provisional Patent Applications Nos. 62 / 054,490, filed September 24, 2014; 62 / 010,441, filed June 10, 2014; and 61 / 915,118, 61 / 915,215, and 61 / 915,148, each filed December 12, 2013) ("Particle Delivery PCT"), which are incorporated herein by reference. For example, the Cas9 protein and sgRNA are mixed together, advantageously in a sterile nuclease-free buffer, such as 1×PBS, in a suitable molar ratio, such as 3:1 to 1:3 or 2:1 to 1:2 or 1:1, at a suitable temperature, such as 15-30° C., such as 20-25° C., such as room temperature, for a suitable time, such as 15-45, such as 30 minutes. Separately, particle components such as or including surfactants, such as cationic lipids, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); phospholipids, such as dimyristoylphosphatidylcholine (DMPC); biodegradable polymers, such as ethylene glycol polymers or PEG, and lipoproteins, such as low density lipoproteins, such as cholesterol, are mixed together, advantageously in a sterile nuclease-free buffer, such as 1×PBS, in a suitable molar ratio, such as 1:1 to 1:3 or 2:1 to 1:2 or 1:1 ...1:1 molar ratio, such as 1:1 to 1:3 or 1:1 molar 1~6The sgRNA was dissolved in an alkyl alcohol, such as methanol, ethanol, isopropanol, e.g., 100% ethanol. These two solutions were mixed together to form particles containing the Cas9-sgRNA complex. Thus, the sgRNA may be pre-complexed with the Cas9 protein before forming the entire complex as a particle. Formulations may be made with various molar ratios of various components known to facilitate delivery of nucleic acids to cells, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol, e.g., the molar ratios of DOTAP:DMPC:PEG:cholesterol may be DOTAP100, DMPC0, PEG0, cholesterol0; or DOTAP90, DMPC0, PEG10, cholesterol0; or DOTAP90, DMPC0, PEG5, cholesterol5, DOTAP100, DMPC0, PEG0, cholesterol0. The application thus encompasses the step of mixing sgRNA and Cas9 protein with components that form particles; and particles from such mixing steps. Aspects of the invention may include particles; for example, particles using methods similar to particle delivery PCT, for example, by mixing a mixture containing sgRNA and / or Cas9 as in the invention with components that form particles, as in particle delivery PCT, to form particles, and particles from such mixing steps (or, of course, other particles that contain sgRNA and / or Cas9 as in the invention).

[0049] The present invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression with sequence targeting involving CRISPR-Cas systems and their components, such as genome perturbation or gene editing. In an advantageous embodiment, the Cas enzyme is Cas9, preferably SpCas9 or SaCas9.

[0050] The advantage of this method is that the CRISPR system avoids off-target binding and the resulting side effects, which is achieved by using a system that is engineered to have a high degree of sequence specificity for the target DNA.

[0051] Recent advances in the development of genome editing technologies based on zinc finger nucleases, transcription activators such as effector nucleases, and programmable nucleases such as CRISPR-Ca9 have greatly improved Applicants' ability to precisely modify the genome of eukaryotic cells. Genome editing has already expanded Applicants' ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly intriguing application of programmable nucleases is the possibility of directly correcting genetic mutations in diseased tissues and cells to treat genetic diseases that are refractory to conventional therapies. Applicants provide herein a discussion of the current progress and future prospects and challenges toward the development of programmable nuclease-based therapeutics.

[0052] Of the approximately 25,000 annotated genes in the human genome, mutations in over 3,000 genes have already been associated with disease phenotypes (www.omim.org / statistics / geneMap), and more disease-related genetic variants are becoming evident at an astonishing rate. Now, with the cost of sequencing dropping sharply, the Human Genome Project being completed, and genome sequencing data from patients increasing exponentially, the role of genetics in human health has become a major focus area in research, clinical medicine, and development of targeted therapeutics [Lander, ES Nature 470, 187-197 (2011)]. These advances in our understanding of the genetic basis of disease have improved our understanding of disease mechanisms and focused attention on potential therapeutic strategies. However, despite effective therapeutic hypotheses and intensive efforts in drug development, there have been only limited successful cases of using small molecules to treat diseases with strong genetic involvement [Thoene, JG Small molecule therapy for genetic disease, (Cambridge University Press, Cambridge, UK; New York, 2010)]. Therefore, alternative approaches are needed. New and emerging therapeutic strategies that can modify nucleic acids in disease-affected cells and tissues have great therapeutic potential. Highly penetrant single gene diseases, such as severe combined immunodeficiency (SCID), hemophilia, and certain enzyme deficiencies, are the focus of such treatments due to their well-defined genetics and the lack of safe and effective treatment options in many cases.

[0053] Two of the most powerful gene therapy strategies developed to date are viral gene therapy, which can complement defective gene function by transgene expression, and RNA interference (RNAi), which mediates targeted suppression of defective genes by knockdown of target mRNA (reviewed in Kay, MA Nature reviews. Genetics 12, 316-328 (2011) and Vaishnaw, AK, et al. Silence 1, 14 (2010)). Viral gene therapy has been successfully used to treat monogenic recessive disorders affecting the hematopoietic system, such as SCID and Wiskott-Aldrich syndrome, by semi-randomly integrating functional copies of the affected genes into the genome of hematopoietic stem / progenitor cells [Gaspar, HB, et al. Science translational medicine 3, 97ra79(2011), Howe, SJ, et al. The Journal of clinical investigation 118, 3143-3150(2008), Aiuti, A., et al. Science 341, 1233151(2013)]. RNAi has been used to suppress the function of genes involved in cancer, age-related macular degeneration, and TTR amyloidosis, among others, with therapeutic benefits in clinical trials (www.clinicaltrials.gov, trial numbers: NCT00689065, NCT01961921, and NCT00259753). Despite the promise and recent successes, viral gene therapy and RNAi have limitations that prevent their usefulness for many diseases. For example, viral gene therapy can cause insertional mutagenesis and dysregulation of transgene expression [Howe, SJ, et al. The Journal of clinical investigation 118, 3143-3150 (2008)]. Alternatively, RNAi can only suppress the expression of target genes, thus limiting its use to targets whose knockdown is beneficial. Also, RNAi often cannot completely suppress gene expression, and is therefore unlikely to benefit diseases in which complete loss of gene function is required for treatment.A radical alternative that could overcome these limitations would be the precise modification of the genome of target cells resulting in the removal or correction of deleterious mutations or the insertion of protective mutations. Cartier.

[0054] Watts, "Hematopoietic Stem Cell Expansion and Gene Therapy," Cytotherapy 13(10):1164-1171. doi:10.3109 / 14653249.2011.620748 (2011) (herein incorporated by reference as if set forth in its entirety, along with all citations therein) discusses hematopoietic stem cell (HSC) gene therapy, e.g., viral-mediated hematopoietic stem cell (HSC) gene therapy (thereapy), as a highly attractive treatment option for many disorders, including hematological conditions, immunodeficiencies including HIV / AIDS, and other genetic disorders such as lysosomal storage diseases, e.g., SCID-X1, ADA-SCID, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

[0055] Williams, "Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies," Cell Stem Cell 13:263-264 (2013) (incorporated herein by reference as if set forth in its entirety, along with its citations) report lentivirus-mediated gene transfer into HSC / P cells from patients with the lysosomal storage disease Metachromatic Leukodystrophy Disease (MLD), a genetic disorder caused by a deficiency in arylsulfatase A (ARSA) resulting in neuronal demyelination; and into HSCs from patients with Wiskott-Aldrich Syndrome (WAS), who have a defect in the WAS protein, an effector of the small GTPase CDC42 that regulates cytoskeletal function in blood cell lineages, and therefore suffer from immunodeficiency with recurrent infections, autoimmune symptoms, and thrombocytopenia with abnormally small, dysfunctional platelets that result in excessive bleeding and an increased risk of leukemia and lymphoma. In contrast to the use of lentivirus, those skilled in the art can, based on the knowledge in the art and the teachings of this disclosure, correct HSCs for MLD (deficiency of arylsulfatase A (ARSA)) using a CRISPR-Cas9 system (e.g., with a suitable HDR template that delivers the coding sequence of ARSA) to target and correct the mutation (deficiency of arylsulfatase A (ARSA)). In contrast to the use of lentivirus, those skilled in the art can, based on the knowledge in the art and the teachings of this disclosure, correct HSCs for WAS using a CRISPR-Cas9 system (e.g., with a suitable HDR template that delivers the coding sequence of WAS protein) to target and correct the mutation (deficiency of WAS protein); specifically, sgRNA can target the mutation that causes WAS (deficient WAS protein), and HDR can result in coding of the appropriate WAS protein expression.

[0056] Based on the knowledge in the art and the teachings of this disclosure, a person skilled in the art can correct HSCs for immune deficiency disease conditions such as HIV / AIDS, which includes contacting HSCs with a CRISPR-Cas9 system that targets and knocks out CCR5. sgRNA that targets and knocks out CCR5 (and advantageously a dual guide approach, e.g., a pair of different sgRNAs; e.g., sgRNAs targeting two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs)) and Cas9 protein can be introduced into HSCs. These cells can be administered; and optionally treated / expanded; e.g., Cartier. See also, Kiem, "Hematopoietic stem cell-based gene therapy for HIV disease," Cell Stem Cell. Feb 3, 2012;10(2):137-147 (incorporated herein by reference along with its cited references); Mandal et al, "Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR / Cas9," Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 November 2014 (incorporated herein by reference along with its cited references).Also mentioned is Ebina, "CRISPR / Cas9 system to suppress HIV-1 expression by editing HIV-1 integrated proviral DNA," SCIENTIFIC REPORTS | 3:2510 | DOI:10.1038 / srep02510 (herein incorporated by reference along with its cited references), as another means of using the CRISPR-Cas9 system to combat HIV / AIDS.

[0057] Genome editing technologies based on programmable nucleases such as zinc finger nucleases (reviewed in Urnov, FD, et al. Nature reviews. Genetics 11, 636-646 (2010)), transcription activator-like effector nucleases (reviewed in Bogdanove, AJ & Voytas, DF Science 333, 1843-1846 (2011)), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 (reviewed in Hsu, PD, et al. Cell 157, 1262-1278 (2014)) have opened the possibility of achieving therapeutic genome editing in diseased cells and tissues. Applicants provide a recent review herein.

[0058] Genome editing technology Programmable nucleases enable precise genome editing by introducing targeted DNA double-strand breaks (DSBs) at specific genomic loci, which then signal DNA damage and recruit endogenous repair machinery to the DSB site for either non-homologous end joining (NHEJ) or homology-dependent repair (HDR) to mediate genome editing.

[0059] To date, there are three major nuclease classes: zinc finger nucleases (ZFNs, Figure 12, left panel) [Kim, YG, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); Wolfe, SA, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Bibikova, M., et al. Science 300, 764 (2003); Bibikova, M., et al. Genetics 161, 1169-1175 (2002); Miller, J., et al. The EMBO journal 4, 1609-1614 (1985); Miller, JC, et al. Nature biotechnology 25,778-785(2007)], transcription activator-like effector nucleases (TALENs, Fig. 1, center panel) [Boch, J., et al. Science 326,1509-1512(2009); Moscow, MJ & Bogdanove, AJ Science 326,1501(2009); Christian, M., et al. Genetics 186,757-761(2010); Miller, JC, et al. Nature biotechnology 29,143-148(2011)], and the CRISPR-associated nuclease Cas9 (Fig. 1, right panel) [Bolotin, A., et al. Microbiology 151,2551-2561(2005); Barrangou, R., et al. Science 315,1709-1712(2007);Garneau,JE,et al.Nature 468,67-71(2010);Deltcheva,E.,et al.Nature 471,602-607(2011);Sapranauskas,R.,et al.Nucleic acids research 39,9275-9282(2011);Jinek,M.,et al.Science 337,816-821(2012);Gasiunas,G.,et al.Proceedings of the National Academy of Sciences of the United States of America 109,E2579-2586(2012);Cong,L.,et al.Science 339,819-823(2013);Mali,P.,et al.Science 339,823-826(2013)] have been developed to enable site-specific genome editing. These three nuclease systems can be broadly divided into two categories based on their DNA recognition modes: ZFN and TALEN achieve specific DNA binding through protein-DNA interactions, while Cas9 is targeted to specific DNA sequences via small RNA guide molecules that directly base pair with the target DNA (Figure 13). ZFNs and TALENs are chimeric enzymes consisting of a DNA-binding domain fused to the sequence-independent nuclease domain FokI [Kim, YG, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); Christian, M., et al. Genetics 186, 757-761 (2010)]. Retargeting of ZFNs and TALENs requires protein engineering of the DNA-binding domain, which is particularly challenging for ZFNs and even more so for TALENs [Isalan, M. Nature methods 9, 32-34 (2012); Sun, N. & Zhao, H. Biotechnology and bioengineering 110, 1811-1821 (2013)]. In contrast, the Cas9 protein is immutable and can be easily retargeted to new genomic loci by changing the sequence of a small portion of the associated RNA guide. All three nucleases have been demonstrated to achieve efficient genome editing in a wide range of model organisms and mammalian cells, and efforts are currently underway in both industry and academia to develop these tools as therapeutics [Tebas, P.,et al.The New England journal of medicine 370,901-910(2014);Genovese,P.,et al.Nature 510,235-240(2014);Li,H.,et al.Nature 475,217-221(2011);Yin,H.,et al.Nature biotechnology 32,551-553(2014)]. .

[0060] Once a DSB is created, the lesion can be repaired by either NHEJ or HDR depending on the cell condition and the presence of a repair template. NHEJ can repair the lesion by directly rejoining the two DSB ends in a process that does not require a repair template. NHEJ-mediated DSB repair can be accurate, but repeated repair of the same DSB by the NHEJ machinery due to nuclease activity eventually leads to the formation of small insertion or deletion mutations across the break site [Bibikova, M., et al. Genetics 161, 1169-1175 (2002)]. Such insertions or deletions (indels) introduced into the coding sequence of a gene can cause frameshift mutations, which can lead to mRNA decay via nonsense-mediated decay, depleting the functional gene or producing a nonfunctional truncated protein [Hentze, MW & Kulozik, AECell 96, 307-310 (1999)]. Thus, NHEJ can be used to silence gene function similarly to RNAi; however, NHEJ introduces permanent covalent modifications into the genome that continue to silence gene expression in target cells.

[0061] In comparison, HDR allows researchers to specify the outcome of DSB repair using exogenous DNA templates [Bibikova, M., et al. Science 300, 764 (2003); Choulika, A., et al. Molecular and cellular biology 15, 1968-1973 (1995); Bibikova, M., et al. Molecular and cellular biology 21, 289-297 (2001); Krejci, L., et al. Nucleic acids research 40, 5795-5818 (2012); Plessis, A., et al. Genetics 130, 451-460 (1992); Rouet, P., et al. Molecular and cellular biology 14, 8096-8106 (1994); Rudin, N., et al. Genetics 122, 519-534 (1989)]. Upon introduction of a targeted DSB, the HDR machinery can use an exogenously provided single- or double-stranded DNA template with sequence homology to the break site to synthesize DNA in a process that incorporates any changes encoded in the template DNA and use this DNA to repair the lesion. For example, HDR can be used with appropriately designed repair templates to directly correct deleterious mutations, thereby restoring gene function while maintaining physiological regulation of gene expression.

[0062] Treatment application considerations A primary consideration in genome editing therapy is the selection of a sequence-specific nuclease, as each nuclease platform has its own set of strengths and weaknesses, many of which must be balanced to maximize therapeutic benefit in the therapeutic context (Figure 12).

[0063] To date, two therapeutic editing approaches with nucleases have shown remarkable promise: gene disruption and gene correction. Gene disruption involves the creation of targeted indels in genetic elements by stimulating NHEJ, often resulting in loss-of-function mutations that are beneficial to patients (Figure 13A). In contrast, gene correction uses HDR to directly revert disease-causing mutations, restoring function while maintaining physiological regulation of the corrected element (Figure 13B). HDR can also be used to insert therapeutic transgenes into defined "safe harbor" loci in the genome to restore defective gene function (Figure 13C).

[0064] For a particular editing therapy to be effective, a sufficiently high level of modification must be achieved in the target cell population to reverse disease symptoms, with this therapeutic modification "threshold" determined by the fitness of the edited cells following treatment and the amount of gene product required to reverse symptoms.

[0065] Cell fitness and outcomes In terms of fitness, editing can result in three outcomes for treated cells compared to their unedited counterparts: increased fitness, intermediate fitness, or decreased fitness. In the case of increased fitness (e.g., in the treatment of SCID-X1), modified hematopoietic progenitor cells are selectively expanded compared to their unedited counterparts. SCID-X1 is a disease caused by mutations in the IL2RG gene, whose function is required for normal development of hematopoietic and lymphoid lineages [Leonard, WJ, et al. Immunological reviews 138, 61-86 (1994); Kaushansky, K. & Williams, WJ Williams hematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trials with patients who received viral gene therapy for SCID-X1, and in rare cases of spontaneous correction of SCID-X1 mutations, the corrected hematopoietic progenitor cells were able to mediate the treatment by reversing this developmental block and expanding relative to their diseased counterparts [Bousso, P., et al. Proceedings of the National Academy of Sciences of the United States of America 97, 274-278 (2000); Hacein-Bey-Abina, S., et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, HB, et al. Lancet 364, 2181-2187 (2004)]. In this case, the edited cells have a selective advantage and can be amplified through expansion, even if the edited cells are in small numbers, resulting in a therapeutic benefit to the patient. In contrast, editing of other hematopoietic diseases, such as chronic granulomatous disease (CGD), does not alter the fitness of edited hematopoietic progenitor cells and may increase the therapeutic modification threshold. CGD is caused by mutations in genes that code for phagocyte oxidase proteins that are normally used by neutrophils to generate reactive oxygen species that kill pathogens [Mukherjee, S. & Thrasher, AJGene 525, 174-181 (2013)].Since the dysfunction of these genes does not affect the fitness or development of hematopoietic progenitors, but only the ability of mature hematopoietic cell types to fight infection, there is likely to be no preferential expansion of edited cells in this disease. Indeed, no selective advantage has been observed for gene-corrected CGD cells in gene therapy trials, making long-term cell engraftment difficult [Malech, HL, et al. Proceedings of the National Academy of Sciences of the United States of America 94, 12133-12138 (1997); Kang, HJ, et al. Molecular therapy: the journal of the American Society of Gene Therapy 19, 2092-2101 (2011)]. Thus, significantly higher levels of editing may be required to treat diseases such as CGD, where editing confers an intermediate fitness advantage, compared to diseases where editing confers a fitness increase to the target cells. If editing imposes a fitness disadvantage, such as in the case of the restoration function for tumor suppressor genes in cancer cells, modified cells may be outcompeted by their diseased counterparts, resulting in low therapeutic benefit relative to the editing rate. This latter class of disease may be particularly difficult to treat with genome editing therapy. X-linked chronic granulomatous disease (CGD) is an inherited disorder of host defense caused by the lack or reduction of phagocyte NADPH oxidase activity. From this disclosure and the knowledge in the art, the skilled artisan can use the CRISPR-Cas9 system (e.g., with a suitable HDR template that delivers the coding sequence of phagocyte NADPH oxidase) to target and correct the mutation (lack or reduction of phagocyte NADPH oxidase activity); specifically, sgRNA can target the mutation that causes CGD (deficiency of phagocyte NADPH oxidase), and HDR can provide the coding of appropriate phagocyte NADPH oxidase expression.

[0066] In addition to cell fitness, the amount of gene product required to treat the disease also influences the minimum level of therapeutic genome editing that must be achieved to reverse symptoms. Hemophilia B is one disease where small changes in gene product levels can lead to large changes in clinical outcome. The disease is caused by mutations in the gene encoding factor IX, a protein normally secreted into the blood by the liver (factor IX functions as a component of the coagulation cascade). The clinical severity of hemophilia B is related to the amount of factor IX activity. Severe disease is associated with less than 1% of normal activity, while milder forms of the disease are associated with factor IX activity greater than 1% [Kaushansky, K. & Williams, WJ Williams hematology, (McGraw-Hill Medical, New York, 2010); Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)]. This suggests that editing a small proportion of hepatocytes to restore factor IX expression could have a major impact on clinical outcome. Studies using ZFNs to correct a mouse model of hemophilia B shortly after birth have demonstrated that a correction of 3-7% was sufficient to reverse disease symptoms, providing preclinical evidence for this hypothesis [Li, H., et al. Nature 475, 217-221 (2011)].

[0067] Disorders in which small changes in gene product levels can affect clinical outcomes and diseases in which edited cells have a fitness advantage are ideal targets for genome editing therapies because the therapeutic modification threshold is low enough to allow high response rates given current technology.

[0068] Targeting these diseases has currently led to successful editing therapies in preclinical and Phase I clinical trials (see table below). Extending these promising results to diseases with an intermediate fitness advantage for the edited cells, or where large amounts of gene product are required for treatment, will require improvements in DSB repair pathway engineering and nuclease delivery. The table below shows examples of the application of genome editing to therapeutic models.

[0069] [Table 1]

[0070] One embodiment comprises contacting a hematopoietic stem cell carrying a hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or hereditary tyrosinemia mutation with an sgRNA targeting a genomic locus of interest for hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or hereditary tyrosinemia (e.g., in Li, Genovese or Yin) and a Cas9 protein; advantageously, contacting with a suitable HDR template to correct the mutation.

[0071] Efficiency of DSB repair pathways NHEJ and HDR DSB repair activity varies greatly depending on cell type and cell state. NHEJ is not highly regulated by the cell cycle and is efficient across all cell types, allowing high levels of gene disruption in accessible target cell populations. In contrast, HDR operates primarily during the S / G2 phase and is therefore restricted to actively dividing cells, limiting therapies requiring precise genome modification to mitotic cells [Ciccia, A. & Elledge, SJ Molecular cell 40, 179-204 (2010); Chapman, JR, et al. Molecular cell 47, 497-510 (2012)].

[0072] The efficiency of correction by HDR can be controlled by the epigenetic state or sequence of the target locus, or the specific repair template configuration used (single-stranded vs. double-stranded, long vs. short homology arms) [Hacein-Bey-Abina, S., et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, HB, et al. Lancet 364, 2181-2187 (2004); Beumer, KJ, et al. G3 (2013)]. The relative activity of NHEJ and HDR machinery in the target cell can also affect gene correction efficiency, as these pathways can compete for the resolution of DSBs [Beumer, KJ, et al. Proceedings of the National Academy of Sciences of the United States of America 105, 19821-19826 (2008)]. HDR also poses delivery challenges not seen with NHEJ strategies, as it requires co-delivery of nuclease and repair template. In practice, these limitations have led to low levels of HDR in therapeutically relevant cell types so far. Clinical translation has therefore focused primarily on NHEJ strategies for disease treatment, although proof-of-concept preclinical HDR treatments have now been reported for mouse models of hemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475, 217-221 (2011); Yin, H., et al. Nature biotechnology 32, 551-553 (2014)].

[0073] Cell and tissue targeting Any given genome editing application may involve a combination of proteins, small RNA molecules, and / or repair templates, making the delivery of these multiple moieties a substantial challenge compared to small molecule therapeutics. Two main strategies have been developed for the delivery of genome editing tools: ex vivo and in vivo. In ex vivo therapy, diseased cells are removed from the body, edited, and then transplanted back into the patient (Figure 14, top panel). Ex vivo editing has the advantage that the target cell population is well defined and it is possible to specify the specific dosage of therapeutic molecules to be delivered to the cells. The latter consideration may be particularly important when off-target modifications are a concern, as such mutations can be reduced by titrating the amount of nuclease (Hsu et al., 2013). Another advantage of ex vivo approaches is that they can typically achieve high editing rates, as efficient delivery systems for proteins and nucleic acids to cells in culture have been developed for research and gene therapy applications.

[0074] However, ex vivo approaches have two major drawbacks that limit their application to a small number of diseases. First, the target cells must have the ability to survive manipulation outside the body. For many tissues, such as the brain, culturing cells outside the body is a major challenge because the cells either do not survive or lose the properties necessary for their function in vivo. Thus, ex vivo therapy is generally limited to tissues that have adult stem cell populations that are amenable to ex vivo culture and manipulation, such as the hematopoietic system. Second, cultured cells often engraft poorly upon reintroduction into the patient, reducing the efficacy of the treatment. However, engraftment can be enhanced by ablative conditioning regimens that deplete host cells before transplantation, which is clinically feasible but poses significant risks to the patient [Bunn, HF & Aster, J. Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)].

[0075] In vivo genome editing involves the targeted delivery of an editing system to a cell type in its native tissue (Figure 14, bottom panel). In vivo editing allows for the treatment of diseases where the diseased cell population is not amenable to ex vivo manipulation. Furthermore, because nucleases are delivered to cells in situ, treatment of multiple tissues and cell types is possible. Perhaps due to these properties, in vivo therapy is applicable to a wider range of diseases than ex vivo therapy.

[0076] To date, in vivo editing has been largely achieved by the use of viral vectors with defined tissue-specific tropism. Such vectors are currently limited in terms of cargo carrying capacity and tropism, limiting this therapy to organ systems that are efficient for transduction with clinically useful vectors, such as the liver, muscle and eye [Kotterman, MA & Schaffer, DV Nature reviews. Genetics 15, 445-451 (2014); Nguyen, TH & Ferry, N. Gene therapy 11 Suppl 1, S76-84 (2004); Boye, SE, et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 509-519 (2013)].

[0077] A major potential barrier to in vivo delivery is the immune response that may arise in response to the large amounts of virus required for therapy, but this phenomenon is not unique to genome editing and is seen in other virus-based gene therapies [Bessis, N., et al. Gene therapy 11 Suppl 1, S10-17 (2004)]. It is also possible that peptides of the editing nuclease itself may be presented on MHC class I molecules to stimulate an immune response, but there is little evidence to support this happening at the preclinical level. Another major challenge of this therapy is to control the distribution in vivo, and thus the dosage, of the genome-editing nuclease, which leads to off-target mutation profiles that can be difficult to predict.

[0078] Successful examples of genome editing therapeutic strategies Ex vivo editing therapy Long-standing clinical insights into the purification, culture and transplantation of hematopoietic cells have led to diseases affecting the blood system, such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome and sickle cell anemia, being the focus of ex vivo editing therapies. Another reason for the focus on hematopoietic cells is that relatively efficient delivery systems already exist, thanks to previous efforts attempting to design gene therapies for blood disorders. Despite these advantages, cell engraftment efficiency upon transplantation is often low, so this therapy is necessarily applied to diseases where the edited cells have a fitness advantage and thus a small number of engrafted edited cells can be expanded to treat the disease.

[0079] Fanconi Anemia: Mutations in at least 15 genes (FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ / BACH1 / BRIP1, FANCL / PHF9 / POG, FANCM, FANCN / PALB2, FANCO / Rad51C, and FANCP / SLX4 / BTBD12) can cause Fanconi Anemia. Proteins produced by these genes are involved in a cellular process known as the FA pathway. The FA pathway is turned on (activated) when DNA damage blocks the process of making new copies of DNA, called DNA replication. The FA pathway sends specific proteins to the area of ​​damage, which triggers DNA repair to begin so DNA replication can continue. The FA pathway is particularly responsive to a specific type of DNA damage known as interstrand crosslinks (ICLs). ICL occurs when two DNA building blocks (nucleotides) on opposite strands of DNA abnormally bind or link together, thereby halting the process of DNA replication. ICL can be caused by the accumulation of toxic substances produced in the body or by treatment with certain cancer therapy drugs. Eight proteins associated with Fanconi anemia group together to form a complex known as the FA core complex. The FA core complex activates two proteins called FANCD2 and FANCI. When these two proteins are activated, DNA repair proteins are brought to the area of ​​the ICL so that the crosslinks can be removed and DNA replication can continue. FA core complex. More specifically, the FA core complex is a nuclear multiprotein complex consisting of FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, which functions as an E3 ubiquitin ligase and mediates the activation of the ID complex, a heterodimer composed of FANCD2 and FANCI. Once monoubiquitinated, the FA core complex interacts with classical tumor suppressors downstream of the FA pathway, including FANCD1 / BRCA2, FANCN / PALB2, FANCJ / BRIP1, and FANCO / Rad51C, thereby contributing to DNA repair by homologous recombination (HR).Eighty to ninety percent of FA cases result from mutations in one of three genes, FANCA, FANCC, and FANCG. These genes provide instructions for the production of components of the FA core complex. Mutations in such genes related to the FA core complex can render the complex nonfunctional and disrupt the entire FA pathway. As a result, DNA damage is not repaired efficiently and ICLs accumulate over time. Geiselhart, "Review Article,Disrupted Signaling through the Fanconi Anemia Pathway Leads to Dysfunctional Hematopoietic Stem Cell Biology:Underlying Mechanisms and Potential Therapeutic Strategies," Anemia Volume 2012 (2012), Article ID 265790, http: / / dx.doi.org / 10.1155 / 2012 / 265790, discussed FA and animal studies involving intrafemoral injection of lentivirus encoding the FANCC gene that led to correction of HSCs in vivo. In light of this disclosure and knowledge in the art, a CRISPR-Cas9 system can be used that targets one or more mutations associated with FA, for example, a CRISPR-Cas9 system having one or more sgRNAs and one or more HDR templates that target one or more of the mutations in FANCA, FANCC, or FANCG that give rise to FA and provide corrected expression of one or more of FANCA, FANCC, or FANCG, respectively.

[0080] One such disease is HIV, where infection confers a fitness disadvantage on CD4+ T cells.

[0081] The rationale for genome editing for HIV treatment comes from the observation that individuals homozygous for a loss-of-function mutation in CCR5, a cellular coreceptor for the virus, are highly resistant to infection and otherwise healthy, suggesting that mimicking this mutation with genome editing could be a safe and effective treatment strategy [Liu, R., et al. Cell 86, 367-377 (1996)]. This idea was clinically validated when HIV-infected patients received allogeneic bone marrow transplants from donors homozygous for loss-of-function CCR5 mutations, resulting in restoration of undetectable levels of HIV and normal CD4 T cell counts [Hutter, G., et al. The New England journal of medicine 360, 692-698 (2009)]. Because of cost and potential graft-versus-host disease, bone marrow transplants are not a realistic treatment strategy for many HIV patients, but HIV treatments that alter the patient's own T cells are feasible.

[0082] Early studies using ZFN and NHEJ to knock out CCR5 in humanized mouse models of HIV showed that transfer of CCR5-edited CD4 T cells improved viral load and CD4 T cell counts [Perez, EE, et al. Nature biotechnology 26, 808-816 (2008)]. Importantly, these models also showed that HIV infection led to selection of CCR5-null cells, suggesting that editing may confer a fitness advantage such that a small number of edited cells may be therapeutic.

[0083] Following this and other promising preclinical studies, genome editing therapy to knock out CCR5 in patient T cells is now being tested in humans [Holt, N., et al. Nature biotechnology 28, 839-847 (2010); Li, L., et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013)]. In a recent Phase I clinical trial, CD4+ T cells from HIV patients were removed, edited with ZFNs designed to knock out the CCR5 gene, and transferred back into the patient in an autologous transplant [Tebas, P., et al. The New England journal of medicine 370, 901-910 (2014)]. Initial results from this trial suggest that ZFN-mediated genome editing of the CCR5 locus is safe, however, the short follow-up time does not allow a complete understanding of the risks and efficacy of the treatment.

[0084] In recent years, ex vivo editing therapy has been expanded to include gene correction strategies. The barrier of ex vivo HDR has been overcome in a recent paper by Genovese and coworkers, who achieved gene correction of a mutant IL2RG gene in hematopoietic stem cells (HSCs) from a patient suffering from SCID-X1 [Genovese, P., et al. Nature 510, 235-240 (2014)]. Genovese et.al. achieved gene correction in HSCs using a multimodality strategy. First, HSCs were transduced with an integration-deficient lentivirus containing an HDR template encoding the therapeutic cDNA of IL2RG. After transduction, cells were electroporated with mRNA encoding a ZFN that targets a mutational hotspot in IL2RG to stimulate HDR-based gene correction. To increase the HDR rate, culture conditions were optimized with small molecules to promote HSC division. With optimized culture conditions, nucleases, and HDR templates, gene-corrected HSCs from SCID-X1 patients were obtained in culture at therapeutically relevant rates. HSCs from unaffected individuals undergoing the same gene correction procedure were able to maintain long-term hematopoiesis in mice, the gold standard for HSC function. HSCs have the capacity to give rise to all hematopoietic cell types and can be autologously transplanted, making them an extremely useful cell population for any hematopoietic genetic disorder [Weissman, IL & Shizuru, JA Blood 112, 3543-3553 (2008)]. Gene-corrected HSCs could in principle be used to treat a wide range of genetic blood disorders, making this an exciting breakthrough for therapeutic genome editing.

[0085] In vivo editing therapy In vivo editing therapies face similar challenges as ex vivo strategies and are also limited by a paucity of efficient delivery systems. Inefficient modification of targeted loci is exacerbated by inefficient delivery, making it particularly difficult to treat tissues that lack a robust delivery platform. However, there are already some exciting preclinical therapeutic successes in organ systems where delivery is efficient.

[0086] The first successful in vivo editing therapy was demonstrated in a mouse model of hemophilia B [Li, H., et al. Nature 475, 217-221 (2011)]. As mentioned above, hemophilia B is an X-linked recessive genetic disease caused by loss-of-function mutations in the gene encoding factor IX, a key component of the coagulation cascade. The disease can be significantly transformed into a milder form when factor IX activity is restored to more than 1% of its level in severely affected individuals, as clinical complications are generally ameliorated when such patients are prophylactically infused with recombinant factor IX from an early age to achieve such levels [Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)]. Thus, only low levels of HDR gene correction may be required to change a patient's clinical outcome. In addition, factor IX is synthesized and secreted by the liver, an organ that can be efficiently transduced by viral vectors encoding the editing system. Based on the knowledge in the art and the teachings of this disclosure, one of skill in the art can correct HSCs using the CRISPR-Cas9 system (e.g., containing a suitable HDR template that delivers the coding sequence for factor IX) to target and correct the mutation (an X-linked recessive genetic disease caused by a loss-of-function mutation in the gene encoding factor IX) for hemophilia B; specifically, the sgRNA can target the mutation that gives rise to hemophilia B, and HDR can result in the coding of appropriate factor IX expression.

[0087] Using hepatotropic adeno-associated virus (AAV) serotypes encoding ZFNs and corrected HDR templates, up to 7% gene correction of a mutant humanized factor IX gene was achieved in mouse liver [Li, H., et al. Nature 475, 217-221 (2011)], improving clot formation kinetics, a measure of coagulation cascade function, and demonstrating for the first time that in vivo editing therapy is not only feasible but also effective.

[0088] Building on this work, other groups have recently used CRISPR-Cas9 in vivo genome editing in the liver to successfully create mutations that treat a mouse model of hereditary tyrosinemia and protect against cardiovascular disease. These two different applications demonstrate the versatility of this approach to disorders involving liver dysfunction [Yin, H., et al. Nature biotechnology 32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492 (2014)]. Application of in vivo editing to other organ systems is necessary to prove the broad applicability of this strategy. Efforts are currently underway to optimize both viral and non-viral vectors to expand the range of disorders that can be treated with this therapy [Kotterman, MA & Schaffer, DV Nature reviews. Genetics 15, 445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555 (2014)].

[0089] Specificity of editing nucleases The specificity of genome editing tools is one of the major safety concerns for clinical application. Genetic modifications are permanent, and harmful off-target mutations can create cells with oncogenic potential and other undesirable side effects. Moreover, oncogenic mutations caused by off-target editing can lead to the expansion of edited cells, so even low off-target mutagenesis levels can have serious consequences.

[0090] Two problems remain unsolved: the evaluation and reduction of off-target effects. Numerous studies have attempted to evaluate the targeting specificity of ZFNs, TALENs, and Cas9 nucleases. A limited number of studies have characterized the specificity of ZFNs [Pattanayak, V., et al. Nature methods 8, 765-770 (2011); Gabriel, R., et al. Nature biotechnology 29, 816-823 (2011)] and TALENs [Guilinger, JP, et al. Nature methods 11, 429-435 (2014)], but only highlight the challenge of detecting ZFN and TALEN off-target activity. Notably, these two independent studies attempt to characterize the off-target profile of the same pair of CCR5-targeting ZFNs, but report distinct, non-overlapping off-target sites, highlighting the challenges involved in analyzing nuclease specificity.

[0091] Many studies have attempted to evaluate the specificity of Cas9, in part because the mechanism of Cas9 RNA-guided DNA targeting is simple and it is remarkably easy to generate hypotheses about possible off-target mechanisms based on Watson-Crick base pairing rules. Early bacterial [Sapranauskas, R., et al. Nucleic acids research 39, 9275-9282 (2011)], biochemical [Jinek, M., et al. Science 337, 816-821 (2012); Gasiunas, G., et al. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012)], and mammalian [Cong, L., et al. Science 339, 819-823 (2013)] experiments suggested that the 8-12 bp 3' seed region of the guide sequence may be sensitive to single-base mismatches, but further studies have shown that this rule of thumb is not necessarily accurate, especially in the presence of high concentrations of Cas9 and guide RNA [Fu, Y., et al. Nature biotechnology 31,822-826(2013);Cho,SW,et al.Genome research 24,132-141(2014);Hsu,PD,et al.Nature biotechnology 31,827-832(2013);Mali,P.,et al.Nature biotechnology 31,833-838(2013);Pattanayak,V.,et al.Nature biotechnology 31,839-843(2013)]. Many of these studies were performed in cell lines and examined Cas9-mediated mutagenesis at genomic sites with high homology levels to the on-target sequence, and not surprisingly, some off-target sites showing high homology were found to be significantly mutated by the nuclease. However, the range of possible off-target sites evaluated in these studies was limited to computationally predicted sites.Recently, whole genome sequencing of Cas9-edited cell lines revealed a low incidence of off-target mutations, suggesting that Cas9-mediated genome editing may be specific [Veres, A., et al. Cell stem cell 15, 27-30 (2014)]. Despite these studies, unbiased assessment of genome-wide off-targeting using more advanced methods, such as direct capture of DSBs [Crosetto, N., et al. Nature methods 10, 361-365 (2013)] and techniques that can potentially detect larger structural perturbations (i.e., translocations) imposed by nuclease treatment, is still urgently needed and must be attempted to understand the true mutagenesis risk imposed by programmable nucleases. It is worth noting that off-target effects may be cell type specific; for example, off-target effects in transformed cell lines with dysregulated DSB repair pathways may overestimate the off-target effects that may be observed in primary healthy cells.

[0092] To reduce the frequency of off-target effects, many groups have been rapidly improving the targeting specificity of Cas9. For example, transforming Cas9 into a single-stranded DNA nickase that functions as a forced heterodimer dramatically reduces off-target indel formation at computationally predicted off-target sites [Mali, P., et al. Nature biotechnology 31, 833-838 (2013); Ran, FA, et al. Cell 154, 1380-1389 (2013)]. In addition, guide RNA based on fusion between catalytically inactive Cas9 and FokI nuclease domain as well as RNA-guided truncation of FokI nuclease can also achieve improved levels of targeting specificity [Fu, Y., et al. Nature biotechnology 32, 279-284 (2014); Guilinger, JP, et al. Nature biotechnology 32, 577-582 (2014); Tsai, SQ, et al. Nature biotechnology 32, 569-576 (2014)]. These and future improved nuclease strategies are considered for therapeutic applications whenever and wherever.

[0093] Crispr-Cas Systems and Compositions for Therapeutic Applications, E.g., Genome Editing In general, in addition to the discussion of CRISPR-Cas systems or CRISPR systems throughout this specification, CRISPR-Cas systems or CRISPR systems are used in the documents described herein, such as WO 2014 / 093622 (PCT / US2013 / 074667), and collectively refer to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, and may include sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or "RNAs" as that term is used herein (e.g., RNAs that guide Cas9, such as CRISPR RNA, and transactivating (tracr)RNA, or single guide RNA (sgRNA) (chimeric RNA), or other sequences and transcripts from a CRISPR locus. In general, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also called a protospacer in the context of endogenous CRISPR systems). In the context of the formation of a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. A target sequence can include any polynucleotide, for example, a DNA polynucleotide or an RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, direct repeats can be identified in silico by searching for repeat motifs that meet any or all of the following criteria: 1. found within a 2Kb window of genomic sequence adjacent to a type II CRISPR locus; 2. spans 20-50 bp; 3. are spaced 20-50 bp apart. In some embodiments, two of these criteria may be used, for example 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria may be used.In some embodiments, it will be preferred that in the CRISPR complex, the tracr sequence has one or more hairpins and is 30 or more nucleotides long, 40 or more nucleotides long, or 50 or more nucleotides long; the guide sequence is 10-30 nucleotides long, and the CRISPR / Cas enzyme is a type II Cas9 enzyme. In embodiments of the present invention, the terms guide sequence and guide RNA are used interchangeably in the above-mentioned documents, such as WO 2014 / 093622 (PCT / US2013 / 074667). In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, or more, or greater than about 50%, greater than about 60%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97.5%, greater than about 99%, or more. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such suitable algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).In some embodiments, the guide sequence is about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 75 or more, or more than about 5, more than about 10, more than about 11, more than about 12, more than about 13, more than about 14, more than about 15 ... More than about 16, more than about 17, more than about 18, more than about 19, more than about 20, more than about 21, more than about 22, more than about 23, more than about 24, more than about 25, more than about 26, more than about 27, more than about 28, more than about 29, more than about 30, more than about 35, more than about 40, more than about 45, more than about 50, more than about 75, or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, less than about 12, or less nucleotides in length. Preferably, the guide sequence is 10-30 nucleotides in length. The ability of the guide sequence to induce sequence-specific binding of the CRISPR complex to a target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, can be introduced into a host cell having a corresponding target sequence, for example, by transfection with a vector encoding the components of the CRISPR sequence, and then evaluation of preferential cleavage within the target sequence can be performed, for example, by the Surveyor assay described herein. Similarly, cleavage of a target polynucleotide sequence can be evaluated in a test tube by providing components of a CRISPR complex including the target sequence, the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test sequence reaction and the control guide sequence reaction. Other assays are possible and will occur to those skilled in the art. The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of the cell.Exemplary target sequences include sequences that are unique in the target genome. For example, in the case of S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything; W is A or T) has a single occurrence in the genome. A unique target sequence in the genome can include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In the case of S. thermophilus CRISPR1 Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; W is A or T), which has a single occurrence in the genome. A unique target sequence in the genome can include a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; W is A or T), which has a single occurrence in the genome. For S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in the genome can include S. pyogenes Cas9 of the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.In each of these sequences, "M" can be A, G, T, or C and does not need to be considered when considering a sequence unique. In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. In some embodiments, about 75%, about 50%, about 40%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or less than about 75%, about 50%, about 40%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or less than about 1% of the nucleotides of the guide sequence are involved in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry, University of Vienna, using a centroid structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1):23-24; and P. A. Carr and G. M. Church, 2009, Nature Biotechnology 27(12):1151-62).

[0094] In general, the tracr mate sequence includes any sequence that has sufficient complementarity with the tracr sequence to promote one or more of: (1) excision of the guide sequence adjacent to the tracr mate sequence in cells that contain the corresponding tracr sequence; and (2) formation of a CRISPR complex at the target sequence that contains the tracr mate sequence that hybridizes to the tracr sequence. In general, the degree of complementarity is about the optimal alignment of the tracr mate sequence and the tracr sequence along the shorter length of these sequences. The optimal alignment can be determined by any suitable alignment algorithm, and can further take into account secondary structures, such as self-complementarity within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter length of these sequences when optimally aligned is about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97.5%, about 99% or more, or greater than about 25%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, greater than about 97.5%, greater than about 99% or more. In some embodiments, the tracr sequence is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, about 50, or more nucleotides in length, or more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 11, more than about 12, more than about 13, more than about 14, more than about 15, more than about 16, more than about 17, more than about 18, more than about 19, more than about 20, more than about 25, more than about 30, more than about 40, more than about 50, or more nucleotides in length. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between these sequences forms a transcript having a secondary structure, e.g., a hairpin.In one embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has two, three, four, or five hairpins. In a further embodiment of the invention, the transcript has up to five hairpins. In the hairpin structure, the portion of the sequence 5' of the last "N" upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop corresponds to the tracr sequence. Further non-limiting examples of single polynucleotides comprising guide sequences, tracr mate sequences, and tracr sequences are as follows (listed 5' to 3'), in which the "N" in the sequence represents the bases of the guide sequence, the first block of lowercase letters represents the tracr mate sequence, the second block of lowercase letters represents the tracr sequence, and the final polyT sequence represents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtttcgttatttaaTTTTTT;(2)NNNNNNNNNNNNNNNNNNNNg tttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT;(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataag gcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT;(4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT;(5)NNNNN and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT. In some embodiments, sequences (1)-(3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4)-(6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript that contains the tracr mate sequence.

[0095] In some embodiments, candidate tracrRNAs can be predicted later by sequences that meet any or all of the following criteria: 1. repeat-inducing sequence homology (search for motifs in Geneious with mismatches up to 18 bp); 2. presence of a putative Rho-dependent transcription terminator in the transcription direction; and 3. stable hairpin secondary structure between the tracrRNA and the direct repeat. In some embodiments, two of these criteria can be used, e.g., 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used.

[0096] In some embodiments, the chimeric synthetic guide RNA (sgRNA) design may include at least a 12 bp duplex structure between the direct repeats and the tracrRNA.

[0097] It is important to control the concentration of the delivered CRISPR enzyme mRNA and guide RNA to minimize toxicity and off-target effects. The optimal concentration of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in cells or non-human eukaryotic animal models and analyzing the degree of modification at potential off-target genomic loci using deep sequencing. For example, for a guide sequence targeting 5'-GAGTCCGAGCAGAAGAAGAA-3' in the EMX1 gene of the human genome, deep sequencing can be used to evaluate the level of modification at the following two off-target loci: 1:5'-GAGTCCTAGCAGGAGAAGAA-3' and 2:5'-GAGTCTAAGCAGAAGAAGAA-3'. The concentration that produces the lowest level of off-target modification and the highest level of on-target modification should be selected for in vivo delivery. Alternatively, to minimize the level of toxicity and off-target effects, CRISPR enzyme nickase mRNA (e.g., S.pyogenes Cas9 with D10A mutation) can be delivered with a pair of guide RNAs that target the desired site. The two guide RNAs need to be spaced apart as follows: Guide sequences and strategies that minimize toxicity and off-target effects can be similar to those in WO2014 / 093622 (PCT / US2013 / 074667).

[0098] CRISPR system is advantageously delivered from type II CRISPR system.In some embodiments, one or more elements of CRISPR system are derived from a certain organism that contains endogenous CRISPR system, for example, Streptococcus pyogenes.In a preferred embodiment of the present invention, CRISPR system is type II CRISPR system, and Cas enzyme is Cas9 that catalyzes DNA cleavage. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof.

[0099] In some embodiments, the unmodified CRISPR enzyme, e.g., Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme induces cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme induces cleavage of one or both strands within about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, about 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme loses the ability to cleave one or both strands of the target polynucleotide that contains the target sequence. For example, an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). Other examples of mutations that turn Cas9 into a nickase include, but are not limited to, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III, or HNH domains) can be mutated to create a mutant Cas9 that has substantially lost all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that has substantially lost all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to have substantially lost all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about 25% or less, about 10% or less, about 5% or less, about 1% or less, about 0.1% or less, about 0.01% or less, or less than the DNA cleavage activity of the non-mutated form of the enzyme; an example would be when the DNA cleavage activity of the mutated form is zero, or negligible compared to the non-mutated form.If the enzyme is not SpCas9, the mutations can be made at some or all of the residues corresponding to positions 10, 762, 840, 854, 863, and / or 986 of SpCas9 (e.g., can be confirmed by standard sequence comparison tools). In particular, some or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A, and / or D986A; conservative substitutions of any of the substituted amino acids are also contemplated. The same (or conservative) substitutions of these mutations at the corresponding positions in other Cas9s are also preferred. D10 and H840 in SpCas9 are particularly preferred. However, in other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred. For example, in Sa Cas9, a mutation at N580, e.g., N580A, is advantageous. Orthologues of SpCas9 can be used in the practice of the invention. The Cas enzyme may refer to a general class of enzymes that share homology with the largest nucleases with multiple nuclease domains from type II CRISPR systems, and thus may be identified as Cas9. Most preferably, the Cas9 enzyme is or is derived from spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9). "StCas9" refers to wild-type Cas9 from S. thermophilus, the protein sequence of which is present in the SwissProt database under the accession number G3ECR1. Similarly, S. pyogenes Cas9 or spCas9 is also deposited in the SwissProt database under the accession number Q99ZW2. By derived, we mean that the derived enzyme is largely based on the wild-type enzyme in that it has a high degree of sequence homology with the wild-type enzyme, but has been mutated (modified) in any of the ways described herein. It should be understood that the terms Cas and CRISPR enzyme are generally used interchangeably herein unless expressly stated.As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it should be understood that the present invention includes many more Cas9s from other species of microorganisms, such as SpCas9, SaCa9, and St1Cas9. The enzymatic action of Cas9 from Streptococcus pyogenes or any related Cas9 performs double-stranded cleavage at the sequence of the target site that hybridizes to the 20 nucleotides of the guide sequence, and the target sequence has a protospacer adjacent motif (PAM) sequence (examples include NGG / NRG or PAM, which can be determined as described herein) following the 20 nucleotides. The CRISPR activity of Cas9 for site-specific DNA recognition and cleavage is determined by the guide sequence, the tracr sequence that partially hybridizes to the guide sequence, and the PAM sequence. Further aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15;37(1):7. The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, and two non-coding RNA elements, tracrRNA, and a characteristic array of repetitive sequences (direct repeats) interspaced with short lengths of non-repetitive sequences (spacers, each about 30 bp). In this system, targeted DNA double-strand breaks (DSBs) are made in four successive steps. First, two non-coding RNAs, the pre-crRNA array, and the tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the direct repeats of the pre-crRNA, and the hybridized pre-crRNA is then processed into mature crRNAs containing individual spacer sequences.Third, the mature crRNA:tracrRNA complex guides Cas9 to the DNA target consisting of the protospacer and the corresponding PAM by heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of the target DNA upstream of the PAM to generate a DSB within the protospacer. A pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs) is also encompassed by the term "tracr-mate sequence". In certain embodiments, Cas9 can be constitutively present, inducibly present, conditionally present, administered, or delivered. Cas9 optimization can be used to develop new functions that can facilitate function or create chimeric Cas9 proteins. And Cas9 can be used as a general DNA binding protein.

[0100] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). Without wishing to be bound by theory, the tracr sequence may comprise or consist of all or a portion of the wild-type tracr sequence (e.g., about 20, about 26, about 32, about 45, about 48, about 54, about 63, about 67, about 85, or more, or more than about 20, more than about 26, more than about 32, more than about 45, more than about 48, more than about 54, more than about 63, more than about 67, more than about 85, or more nucleotides of the wild-type tracr sequence) and may also form part of a CRISPR complex, e.g., by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence.

[0101] An example of a codon-optimized sequence, in this case, is a sequence optimized for expression in a eukaryote, e.g., a human (i.e., optimized for expression in a human), or another eukaryote, animal, or mammal described herein; see, for example, the SaCas9 human codon-optimized sequence in WO 2014 / 093622 (PCT / US2013 / 074667). While this is preferred, it is understood that other examples are possible and that codon optimization for other host species other than humans, or for specific organisms, is also known. In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in a specific cell, e.g., a eukaryotic cell. The eukaryotic cell may be or may be derived from a specific organism, e.g., a mammal, including but not limited to a human, or a non-human eukaryote, animal, or mammal described herein, e.g., a mouse, rat, rabbit, dog, livestock, or a non-human mammal or primate. In some embodiments, processes that alter the genetic identity of a human germline and / or alter the genetic identity of an animal without any substantial medical benefit to the human or animal, and likely to cause suffering to the human or animal, and animals resulting from such processes may also be eliminated. In general, codon optimization refers to the process of modifying a nucleic acid sequence to facilitate expression in a host cell of interest by replacing at least one codon (e.g., about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 50, or more, or more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 10, more than about 15, more than about 20, more than about 25, more than about 50, or more) of the native sequence with a more frequently or most frequently used codon used in the genes of that host cell, while maintaining the native amino acid sequence. Different species show a particular bias towards certain codons for certain amino acids.Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is believed to depend, among other things, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, for example, Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) of a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0102] In some embodiments, the vector encodes a CRISPR enzyme that includes one or more nuclear localization sequences (NLSs), e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, or more NLSs. In some embodiments, the CRISPR enzyme has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more NLSs at or near the amino terminus, or more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, or more NLSs at or near the carboxy terminus. , about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more NLSs, or combinations thereof (e.g., 0 or at least 1 or more NLSs at the amino terminus and 0 or 1 or more NLSs at the carboxy terminus). When more than one NLS is present, each can be selected independently from the other such that a single NLS can be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the present invention, the CRISPR enzyme comprises up to 6 NLSs. In some embodiments, an NLS is considered to be near the N-terminus or C-terminus if the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus or C-terminus.Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV; an NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the IBB domain from importin alpha having the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; the fibroid T protein having the sequences VSRKRPRP and PPKKARED; the human p53 sequence POPKKKPL; the mouse c-abl The NLS sequence includes the sequence SALIKKKKKMAP of IV; the sequence DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL of hepatitis virus delta antigen; the sequence REKKKFLKRR of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. In general, one or more NLSs are strong enough to accumulate detectable amounts of CRISPR enzyme in the nucleus of eukaryotic cells. In general, the strength of the nuclear localization activity can result from the number of NLSs in the CRISPR enzyme, the specific NLS used, or a combination of these factors. The detection of accumulation in the nucleus can be carried out by any suitable technique. For example, a detectable marker can be fused to the CRISPR enzyme, which allows the location in the cell to be visualized, for example, in combination with a means for detecting the location of the nucleus (for example, a nuclear-specific stain, for example, DAPI). Cell nuclei can also be isolated from cells and their contents can then be analyzed by any suitable process for detecting proteins, for example immunohistochemical analysis, Western blot, or enzyme activity assays.Accumulation in the nucleus can also be determined indirectly, for example, by assaying for the effect of CRISPR complex formation (e.g., assaying for DNA cleavage or mutation at the target sequence, or assaying for altered gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity) compared to a control not exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking one or more NLS.

[0103] Aspects of the present invention relate to a reduction in expression of a gene product, or a template polynucleotide further introduced into a DNA molecule encoding the gene product, or an intervening sequence that is precisely cleaved by allowing the reannealing and joining of two 5' overhangs, or the activity or function of the gene product being altered, or an increase in expression of the gene product. In one embodiment of the present invention, the gene product is a protein. Only sgRNA pairs that form 5' overhangs with less than 8 bp overlap between guide sequences (offsets greater than -8 bp) were able to mediate the occurrence of detectable indels. Importantly, each guide used in these assays was able to efficiently induce indels when paired with wild-type Cas9, suggesting that the relative position of the guide pair is the most important parameter in predicting double nicking activity. Since Cas9n and Cas9H840A nick opposite strands of DNA, replacement of Cas9n with Cas9H840A using a given sgRNA pair should be the reverse of the overhang type; however, no indels are observed to occur, as with Cas9H840A, suggesting that Cas9H840A is a CRISPR enzyme that is substantially devoid of total DNA cleavage activity (this is when the DNA cleavage activity of the mutant enzyme is less than about 25%, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, less than about 0.01%, or less than that of the DNA cleavage activity of the non-mutated form of the enzyme; thus, an example would be when the DNA cleavage activity of the mutant form is zero or negligible compared to the non-mutated form, e.g., no indels are observed to occur, as with Cas9H840A in eukaryotic systems as opposed to biochemical or prokaryotic systems). However, a pair of sgRNAs that form a 5' overhang with Cas9n should in principle form a corresponding 3' overhang and double nicking instead. Thus, a pair of sgRNAs that results in the formation of a 3' overhang with Cas9n can be used with another mutant Cas9 to form a 5' overhang and double nicking. Thus, in some embodiments, a recombination template is also provided. The recombination template can be a component of another vector that is included in a separate vector or provided as a separate polynucleotide, as described herein.In some embodiments, the recombination template is designed to serve as a template for homologous recombination within or near the target sequence that is nicked or cut by the CRISPR enzyme, for example, as part of the CRISPR complex. The template polynucleotide can be of any suitable length, for example, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 150, about 200, about 500, about 1000, or more, or more than about 10, more than about 15, more than about 20, more than about 25, more than about 50, more than about 75, more than about 100, more than about 150, more than about 200, more than about 500, more than about 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide that includes the target sequence. When optimally aligned, a template polynucleotide can overlap one or more nucleotides (e.g., about 1, about 5, about 10, about 15, about 20, or more, or more than about 1, about 5, about 10, about 15, about 20, or more nucleotides) of the target sequence. In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 1000, about 5000, about 10000, or more nucleotides of the target sequence.

[0104] In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into the host cell such that the expression of the elements of the CRISPR system induces the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements in separate vectors. Alternatively, the RNA of the CRISPR system can be delivered to a transgenic Cas9 animal or mammal, for example, an animal or mammal that expresses Cas9 constitutively, inducibly, or conditionally; or an animal or mammal that expresses Cas9 by other methods, for example, by prior administration of one or more vectors that encode Cas9 and express Cas9 in vivo, or that have cells that contain Cas9. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in this first vector. The elements of the CRISPR system combined in a single vector can be in any suitable orientation, for example, one element can be placed 5' (upstream of the second element) or 3' (downstream of the second element) relative to the second element. The coding sequence of one element can be placed on the same or opposite strand of the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding a CRISPR enzyme and one or more of a guide sequence, a tracr mate sequence (optionally operably linked to a guide sequence), and a tracr sequence integrated within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.Delivery vehicles, vectors, particles, nanoparticles, formulations, and components thereof for expression of one or more elements of the CRISPR system are used as described in the above-mentioned documents, for example, WO 2014 / 093622 (PCT / US2013 / 074667). In some embodiments, the vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning" sites). In some embodiments, one or more insertion sites (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and, optionally, an insertion site downstream of a regulatory element operably linked to the tracr mate sequence, such that upon expression after insertion of the guide sequence into the insertion site, the guide sequence directs sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, the vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different, corresponding target sequences in a cell. For example, a single vector can contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or more, or more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 15, more than about 20, or more guide sequences.In some embodiments, vectors comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more such guide sequences can be provided and optionally delivered to cells. In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, e.g., a Cas protein. The CRISPR enzyme, or CRISPR enzyme mRNA, or CRISPR guide RNA or RNA can be delivered separately; and advantageously, at least one of these is delivered by a nanoparticle complex. The CRISPR enzyme mRNA can be delivered before the guide RNA to allow time for the CRISPR enzyme to be expressed. The CRISPR enzyme mRNA may be administered 1-12 hours (preferably about 2-6 hours) prior to administration of the guide RNA. Alternatively, the CRISPR enzyme mRNA and the guide RNA can be administered together. Advantageously, a second booster dose of guide RNA can be administered 1-12 hours (preferably about 2-6 hours) after the first administration of the CRISPR enzyme mRNA + guide RNA. Additional administration of CRISPR enzyme mRNA and / or guide RNA may be useful to achieve the most efficient level of genome modification.

[0105] In one aspect, the present invention provides a method for using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the present invention has a wide variety of utilities, including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in multiple cell types. Thus, the CRISPR complex of the present invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex includes a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in a target polynucleotide. The guide sequence is linked to a tracr mate sequence, which hybridizes to a tracr sequence. In one embodiment, the present invention provides a method for cleaving a target polynucleotide. The method includes modifying a target polynucleotide with a CRISPR complex that binds to and cleaves the target polynucleotide. Typically, when the CRISPR complex of the present invention is introduced into a cell, it forms a break (e.g., a single-strand or double-strand break) in the genomic sequence. For example, this method can be used to break a disease gene in the cell. The break formed by the CRISPR complex can be repaired by a repair process, such as the error-prone non-homologous end joining (NHEJ) pathway or high-fidelity homology-directed repair (HDR). During these repair processes, an exogenous polynucleotide template can be introduced into the genomic sequence. In some methods, the genomic sequence is modified using the HDR process. For example, an exogenous polynucleotide template is introduced into the cell, which includes the sequence to be integrated adjacent to the upstream sequence and the downstream sequence. The upstream sequence and the downstream sequence share sequence similarity with both sides of the integration site in the chromosome. If desired, the donor nucleotide can be DNA, such as a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear fragment of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle, such as a liposome or a poloxamer.The exogenous polynucleotide template comprises the sequence to be integrated (e.g., a mutant gene). The sequence for integration may be a sequence endogenous to the cell or may be a sequence exogenous. Examples of sequences to be integrated include polynucleotides encoding proteins or non-coding RNA (e.g., microRNA). Thus, the sequence for integration may be operably linked to one or more appropriate control sequences. Alternatively, the sequence to be integrated may provide a control function. The upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the genomic sequence upstream of the target site for integration. Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the target site for integration. The upstream and downstream sequences in the exogenous polynucleotide template may have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target genomic sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with the target genome sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or about 100% sequence identity with the target genome sequence. The upstream sequence or downstream sequence may comprise about 20 bp to about 2500 bp, for example, about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, or about 2500 bp. In some methods, exemplary upstream or downstream sequences have from about 200 bp to about 2000 bp, from about 600 bp to about 1000 bp, or particularly from 700 bp to about 1000 bp. In some methods, the exogenous polynucleotide template can further comprise a marker.Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selection markers. The exogenous polynucleotide template of the present invention can be produced using recombinant techniques (see, for example, Sambrook et al., 2001, and Ausubel et al., 1996). In the method of modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double-stranded break is introduced into the genome sequence by a CRISPR complex, and the break is repaired by homologous recombination of the template so that the exogenous polynucleotide template is integrated into the genome. The presence of the double-stranded break promotes the integration of the template. In another embodiment, the present invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. The method includes increasing or decreasing the expression of the target polynucleotide by using a CRISPR enzyme that binds to the target polynucleotide. In some methods, the target polynucleotide can be inactivated to modify its expression in the cell. For example, when a CRISPR complex binds to a target sequence in a cell, the target polynucleotide is inactivated, such that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild-type sequence. For example, a protein or microRNA coding sequence can be inactivated to prevent the transcription of the protein or microRN or pre-microRNA. In some methods, a control sequence can be inactivated to prevent the control sequence from functioning as a control sequence. The term "control sequence" as used herein refers to any nucleic acid sequence that provides for the transcription, translation, or accessibility of a nucleic acid sequence. Examples of control sequences include promoters, transcription terminators, and enhancers. The target polynucleotide of a CRISPR complex can be any polynucleotide that is endogenous or exogenous to a eukaryotic cell. For example, the target polynucleotide can be a polynucleotide that is present in the nucleus of a eukaryotic cell.A target polynucleotide may be a sequence that codes for a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-associated genes or polynucleotides. Examples of target polynucleotides include disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that gives rise to a transcription or translation product at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to non-disease control tissues or cells. A disease-associated gene may be a gene that becomes expressed at an abnormally high level; a disease-associated gene may be a gene that becomes expressed at an abnormally low level, and this altered expression correlates with the onset and / or progression of the disease. A disease-associated gene also refers to a gene that has a mutation or genetic variation that is directly involved in the pathogenesis of the disease or is in linkage disequilibrium with a gene that is involved in the pathogenesis of the disease. A transcription or translation product may be known or unknown, and may be at a normal or abnormal level. The target polynucleotide of the CRISPR complex can be any polynucleotide, endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide present in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence that codes for a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Without wishing to be bound by theory, it is believed that the target sequence must be associated with a PAM (protospacer adjacent motif); i.e., a short sequence that is recognized by the CRISPR complex. The exact sequence and length requirements for the PAM vary depending on the CRISPR enzyme used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). Exemplary PAM sequences are provided in the Examples section below, and one of skill in the art would be able to identify additional PAM sequences for use with a given CRISPR enzyme.In some embodiments, the method comprises a step of allowing a CRISPR complex to bind to a target polynucleotide, resulting in cleavage of the target polynucleotide, thereby modifying the target polynucleotide, the CRISPR complex comprising a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in the target polynucleotide, the guide sequence being linked to a tracr mate sequence, the tracr mate sequence being hybridized to a tracr sequence. In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises a step of allowing a CRISPR complex to bind to a polynucleotide, the binding increasing or decreasing expression of the polynucleotide; the CRISPR complex comprising a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in the target polynucleotide, the guide sequence being linked to a tracr mate sequence, the tracr mate sequence being hybridized to a tracr sequence. Similar considerations and provisos apply to the method of modifying a target polynucleotide as described above. In fact, these sampling, culturing and reintroduction options apply to all aspects of the invention. In one aspect, the invention provides a method for modifying a target polynucleotide in a eukaryotic cell, which can be performed in vivo, ex vivo or in vitro. In some embodiments, the method includes sampling a cell or cell population from a human or non-human animal, and modifying the one or more cells. The culturing can be performed at all stages ex vivo. The one or more cells can even be reintroduced into a non-human animal or plant. For the reintroduced cells, it is particularly preferred that the cells are stem cells.

[0106] Indeed, in any aspect of the invention, a CRISPR complex can comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence, which can be linked to a tracr mate sequence, which can hybridize to the tracr sequence.

[0107] The present invention relates to the engineering and optimization of systems, methods and compositions used for controlling gene expression involved in sequence targeting, such as genome perturbation or genome editing, associated with CRISPR-Cas system and its components.In an advantageous embodiment, the Cas enzyme is Cas9.The advantage of the method of the present invention is that the CRISPR system minimizes or avoids off-target binding and its side effects.This is achieved by using a system that is arranged to have a high degree of sequence specificity for target DNA.

[0108] self-inactivation system Once the intended change has been introduced, such as by editing the intended copy of the gene in the genome of the cell, there is no need to continue CRISRP / Cas9 expression in the cell any longer. In fact, continued expression may be undesirable in some cases, such as when off-target effects occur at unintended genomic sites. Thus, timed expression may be useful. Inducible expression provides one approach, but applicants have also engineered a self-inactivating CRISPR-Cas9 system that relies on the use of a non-coding guide target sequence within the CRISPR vector itself. Thus, after expression begins, the CRISPR system may effect its own destruction, but may have time to edit the genomic copy of the target gene before the destruction is complete (the target gene requires at most two edits for a typical point mutation in a diploid cell). Simply put, a self-inactivating CRISPR-Cas system comprises an additional RNA (i.e., guide RNA) that targets either the coding sequence of the CRISPR enzyme itself or targets one or more non-coding guide target sequences that are complementary to a unique sequence present in one or more of the following: (a) within a promoter driving expression of a non-coding RNA element; (b) within a promoter driving expression of a Cas9 gene; (c) within 100 bp of the ATG translation start codon in the Cas9 coding sequence; (d) within the inverted terminal repeats (iTRs) of a viral delivery vector, for example in the AAV genome.

[0109] HDR Efficiency Although the amount of genome modification required in the target cell population to produce a therapeutic effect varies depending on the disease, the efficacy of many editing therapies is improved by increasing the editing rate. As mentioned above, the editing rate is controlled by the activity of DSB repair pathways and the delivery efficiency to the target cells. Therefore, improving either one of these factors is likely to improve the efficacy of editing therapies.

[0110] Attempts to increase the activity rate of DSB repair pathways have generally focused on HDR, as cell cycle regulation and the challenges of delivering HDR templates with nucleases make strategies using this pathway less efficient than NHEJ. Currently, cell cycle regulation is somewhat circumvented for slow-cycling cell types by stimulating mitosis with pharmacological agents ex vivo [Kormann, MS, et al. Nature biotechnology 29, 154-157 (2011)]. However, true postmitotic cells appear to be unsuitable for such manipulation, limiting the applicability of this strategy. Attempts have been made to circumvent the need for HDR entirely by directly ligating a DNA template containing a therapeutic transgene to the targeted DSB. Such ligation events have been observed, but at rates too low to be therapeutically useful [Ran, FA, et al. Cell 154, 1380-1389 (2013); Orlando, SJ, et al. Nucleic acids research 38, e152 (2010)]. Perhaps dramatically new approaches are needed to improve HDR efficiency and increase the therapeutic efficacy of strategies that require precise genome modification.

[0111] Genome editing offers interesting opportunities to tackle a number of incurable diseases. Nevertheless, this technology is still in its early stages and requires multiple iterations to systematically optimize its efficacy, safety, and specificity. In addition, despite the enormous excitement surrounding genome editing, strategic planning and rigorous yet feasible regulatory processes are needed to ensure the successful development of this class of potentially life-changing medicines.

[0112] Service, including a summary of the service Various nucleic acid or protein delivery methods can be used to introduce genome editing nucleases into target cells ex vivo or in vivo. Depending on the delivery method selected, nucleases can be expressed transiently or permanently in target cells. Delivery systems must be carefully selected, considering that nucleases may exhibit off-target cleavage activity or induce immune responses. For ex vivo applications, such as editing hematopoietic stem cells, electroporation can be used to achieve transient nuclease expression by delivery of DNA-based nuclease expression vectors, mRNA, or protein. Both integration-competent and integration-deficient lentiviral vectors have also been successfully used to drive nuclease expression. However, integration of lentiviral vectors may be less desirable, as it drives constitutive expression and may lead to more off-target activity. In addition, all three nuclease platforms have also been demonstrated to be amenable to modification such that proteins can be delivered directly into cells, either by engineered cell permeability or chemical conjugation [Guilinger, JP, et al. Nature methods 11, 429-435 (2014); Zuris, JA, et al. Nature biotechnology (2014); Gaj, T., et al. Nature methods 9, 805-807 (2012)].

[0113] For in vivo applications, the most promising delivery system is viral vectors, especially adeno-associated virus (AAV) vectors, which have recently been approved for clinical use [Wirth, T., et al. Gene 525, 162-169 (2013)]. AAVs come in many serotypes and have been shown to have high delivery efficacy to various tissue types, including eye, brain, liver, and muscle [Samulski, RJ & Muzyczka, N. Annual Review of Virology 1, 427-451 (2014)]. However, AAV vectors have a relatively small packaging capacity, which poses some challenges for nuclease delivery. ZFNs are relatively small and a dimeric ZFN pair can be packaged into a single AAV, whereas a dimeric TALEN pair is much larger and would likely have to be packaged into two separate AAV vectors. As for Cas9, short orthologs could be packaged into a single AAV along with the guide RNA. To date, AAV-mediated nuclease expression has been demonstrated to be successful in several tissue types, including liver and brain [Li, H., et al. Nature 475, 217-221 (2011); Swiech, L., et al. Nature biotechnology (2014)].

[0114] Despite the potential of AAV-mediated in vivo nuclease expression, there are several challenges that require further development. First, AAV-mediated nuclease expression is often constitutive, and it would be more desirable to be able to block nuclease expression after successful genome editing events in target cells. Second, patients who have already been naturally exposed to AAV are likely to have developed immunity to certain serotypes. Therefore, AAV may not be a suitable delivery vehicle for these patients. To overcome these challenges faced with viral vectors, nanoparticle-based and lipid-based in vivo mRNA or protein delivery systems may provide attractive alternatives [Zuris, JA, et al. Nature biotechnology (2014); Kormann, MS, et al. Nature biotechnology 29, 154-157 (2011)].

[0115] Using this disclosure and knowledge in the art, a CRISPR-Cas system, or a component thereof, or a nucleic acid molecule thereof (including, for example, an HDR template), or a nucleic acid molecule encoding or providing a component thereof, may be delivered by the delivery systems described generally and in detail herein.

[0116] Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, e.g., Cas9, and / or any of the RNAs, e.g., guide RNAs, can be delivered using any suitable vector, e.g., plasmid or viral vector, e.g., adeno-associated virus (AAV), lentivirus, adenovirus, or other types of viral vectors, or combinations thereof. Cas9 and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vector. In some embodiments, the vector, e.g., plasmid or viral vector, is delivered to the tissue of interest, e.g., by intramuscular injection, otherwise delivery is intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery can be a single dose or multiple doses. Those skilled in the art will appreciate that the actual dose delivered herein can vary widely depending on a variety of factors, e.g., the choice of vector, the target cell, organism, or tissue, the general health of the subject to be treated, the degree of transformation / modification desired, the route of administration, the mode of administration, the type of transformation / modification desired, etc.

[0117] Such dosages may further comprise, for example, carriers (such as water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharma- ceutically acceptable carriers (such as phosphate buffered saline), pharma- ceutically acceptable excipients, and / or other compounds known in the art. The dosages may further comprise one or more pharma- ceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts, such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling substances, flavorings, coloring agents, microspheres, polymers, suspending agents, and the like, may also be present therein. In addition, one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc., especially if the dosage form is in a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin, and combinations thereof. A thorough discussion of pharma- ceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0118] In one embodiment herein, delivery is by adenovirus, and the delivery is at least 1×10 5 In one embodiment herein, the dose is preferably at least about 1×10 particles (also called particle units, pu) of adenoviral vector. 6 particles (e.g., about 1 × 10 6 ~1×10 12 particles), more preferably at least about 1×10 7particles, more preferably at least about 1×10 8 particles (e.g., about 1 × 10 8 ~1×10 11 of particles or about 1×10 8 ~1×10 12 particles), and most preferably at least about 1×10 0 particles (e.g., about 1 × 10 9 ~1×10 10 of particles or about 1×10 9 ~1×10 12 particles), or even at least about 1×10 10 particles (e.g., about 1 × 10 10 ~1×10 12 Alternatively, the dose is about 1×10 14 Particles of less than about 1×10 13 Particles less than or equal to about 1×10 12 Particles less than or equal to about 1×10 11 Particles of less than about 1×10 10 Particles below 1×10 9 The dose may therefore be, for example, about 1×10 6 particle unit (pu), approximately 2 × 10 6 pu, approx. 4×10 6 pu, about 1×10 7 pu, approx. 2×10 7 pu, approx. 4×10 7 pu, about 1×10 8 pu, approx. 2×10 8 pu, approx. 4×10 8 pu, about 1×10 9 pu, approx. 2×10 9 pu, approx. 4×10 9 pu, about 1×10 10 pu, approx. 2×10 10 pu, approx. 4×10 10 pu, about 1×10 11 pu, about 2×10 11 pu, approx. 4×10 11 pu, about 1×10 12 pu, about 2×10 12 pu, or approximately 4 x 10 12The adenovirus vector may comprise a single dose of the adenovirus vector, including the adenovirus vector of pu.See, for example, adenovirus vectors in U.S. Patent No. 8,454,972 B2, granted to Nabel et al. on June 4, 2013, which is incorporated herein by reference; for dosage, see lines 36-58 of paragraph 29 thereof.In one embodiment herein, the adenovirus is delivered by multiple doses.

[0119] In one embodiment herein, delivery is by AAV. The therapeutically effective amount of AAV for in vivo delivery to humans is about 1×10 10 ~Approx. 1×10 10 The dosage is considered to be in the range of about 20 to about 50 ml of saline containing 1000 functional AAV / ml solution. The dosage can be adjusted to balance the therapeutic benefit against any side effects. In one embodiment herein, the dose of AAV is generally about 1×10 5 ~1×10 50 AAV genome, approximately 1 x 10 8 ~1×10 20 AAV genome, approximately 1 x 10 10 ~Approx. 1×10 16 genome, or approximately 1 × 10 11 ~Approx. 1×10 16 The concentration of AAV genome ranges from 1 × 10 to 1 × 10. 13 The AAV genome may be of about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. Other effective dosages can be readily established by one of skill in the art through routine testing to establish a dose-response curve. See, for example, U.S. Patent No. 8,404,658 B2, issued to Hajjar et al. on March 26, 2013, at paragraph 27, lines 45-60.

[0120] In one embodiment herein, delivery is by plasmid. In such a plasmid composition, the dosage should be sufficient for the plasmid to elicit a response. For example, a suitable amount of plasmid DNA in a plasmid composition may be about 0.1 to about 2 mg, or about 1 μg to about 10 μg for a 70 kg person. A plasmid of the present invention generally comprises: (i) a promoter; (ii) a sequence encoding a CRISPR enzyme operably linked to the promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmid may also encode an RNA component of the CRISPR complex, although one or more of these may alternatively be encoded on a different vector.

[0121] The dosages herein are based on an average 70 kg person. The frequency of administration is within the scope of a medical or veterinary practitioner (e.g., physician, veterinarian) or person skilled in the art. Also, it should be noted that mice used in experiments are typically about 20 g, and that experiments on mice can be scaled up to a 70 kg person.

[0122] In some embodiments, the RNA molecules of the invention are delivered in liposome or lipofection formulations, etc., and can be prepared by methods well known to those of skill in the art, such as those described in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference. In particular, improved and improved delivery systems aimed at the delivery of siRNA to mammalian cells have been developed (see, e.g., Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003, 327:761-766; Lewis et al., Nat. Gen. 2002, 32:107-108, and Simeoni et al., NAR 2003, 31, 11:2717-2724) and can be applied to the present invention. siRNAs have recently been used successfully to suppress gene expression in primates (see, eg, Tolentino et al., Retina 24(4):660, which may also be applied to the present invention).

[0123] In fact, delivery of RNA is a useful method of in vivo delivery. It is possible to use liposomes or nanoparticles to deliver Cas9 and gRNA (and, for example, HR repair template) into cells. Thus, delivery of CRISPR enzymes, such as Cas9, and / or delivery of the RNA of the present invention can be carried out in RNA form by microvesicles, liposomes, or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposome particles for in vivo delivery. Liposomal transfection reagents, such as Life Technologies' Lipofectamine and other commercially available reagents, can effectively deliver RNA molecules to the liver.

[0124] Means for delivery of RNA also preferably include nanoparticle delivery of RNA (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010) or exosome delivery (Schroeder, A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641). Indeed, exosomes should be particularly useful for delivery of siRNa, a system that has some similarity to the CRISPR system. For example, El-Andaloussi S, et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo." Nat Protoc. 2012 Dec;7(12):2112-26. doi:10.1038 / nprot.2012.131. Epub 2012 Nov 15.) describes how exosomes are promising tools for drug delivery across various biological barriers and can be used to deliver siRNA in vtiro and in vivo. In this approach, targeted exosomes are generated by transfection of an expression vector containing an exosomal protein fused to a peptide ligand. Exosomes are then purified and characterized from transfected cell supernatants, and RNA is then introduced into the exosomes. Delivery or administration according to the present invention, particularly but not limited to the brain, can be performed using exosomes.Vitamin E (α-tocopherol) can be conjugated to CRISPR Cas and delivered to the brain with high density lipoprotein (HDL) in a manner similar to that used to deliver short interfering RNA (siRNA) to the brain, for example, by Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)). Mice were infused with osmotic minipumps (Model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE / HDL and connected to a Brain Infusion Kit 3 (Alzet). Brain infusion cannulas were placed approximately 0.5 mm behind the bregma at the midline for infusion into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could produce a similar degree of target reduction by the same ICV infusion method. Similar doses of CRISPR Cas conjugated to α-tocopherol co-administered with HDL to target the brain may be contemplated in humans in the present invention, for example, about 3 nmol to about 3 μmol of CRISPR Cas to target the brain. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describe a method of lentivirus-mediated delivery of short hairpin RNA targeting PKCγ for in vivo gene silencing in the spinal cord of rats. Zou et al. 9 Approximately 10 μl of recombinant lentivirus with a titer of 1×10 transducing units (TU) / ml was administered via an intrathecal catheter. Similar amounts of CRISPR Cas expressed in brain-targeting lentiviral vectors can be contemplated in humans in the present invention, for example, 1×10 9 Approximately 10-50 ml of CRISPR Cas targeted to the brain with lentivirus having a titer of 1000 transducing units (TU) / ml can be contemplated.

[0125] With regard to localized delivery to the brain, this can be accomplished in a variety of ways. For example, substances can be delivered into the striatum, for example, by injection. Injection can be performed stereotactically via a craniotomy.

[0126] In some aspects, the present invention provides a method comprising delivering one or more polynucleotides, such as, or one or more vectors as described herein, one or more transcripts thereof, and / or transcribed therefrom or proteins, to a host cell. In some aspects, the present invention further provides a cell produced by such a method, and an animal comprising or produced therefrom. In some embodiments, a CRISPR enzyme combined with (and optionally complexed with) a guide sequence is delivered to the cell. Conventional viral and non-viral based gene transfer methods can be used for the introduction of nucleic acids in mammalian cells or target tissues.

[0127] Using such methods, nucleic acid encoding the components of CRISPR system can be administered to cells in culture or to cells in a host organism.Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acid, and nucleic acid complexed with delivery vehicle, such as liposome.Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genome after delivery to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and See Immunology Doerfler and Boehm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994). Non-viral methods of delivery of nucleic acids include lipofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424; WO 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); see U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). Delivery of nucleic acids using RNA or DNA virus-based systems utilizes a highly evolved process of targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro, and optionally the modified cells can be administered to patients (ex vivo). Conventional virus-based systems can include retrovirus, lentivirus, adenovirus, adeno-associated and herpes simplex virus vectors for gene transfer. Retrovirus, lentivirus and adeno-associated virus gene transfer methods allow integration into the host genome, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.The tropism of retroviruses can be altered by the incorporation of foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system can therefore depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats that have the packaging capacity for up to 6-10 kb of foreign sequences. A minimal cis-acting LTR is sufficient for vector replication and packaging, which in turn can be used to integrate therapeutic genes into target cells, resulting in persistent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral vector particles are contemplated (see, e.g., U.S. Patent Application Publication No. 20120164118, assigned to Fred Hutchinson Cancer Research Center). Cocal virus is a genus of Vesiculovirus, the causative agent of vesicular stomatitis in mammals.Cocalvirus was originally isolated from ticks in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)) and has been identified infecting insects, cattle, and horses in Trinidad, Brazil, and Argentina. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common in people living in rural areas, where the virus is endemic and laboratory-acquired; infection in humans usually results in influenza-like symptoms. Cocalvirus envelope glycoprotein shares 71.5% identity with VSV-G Indiana at the amino acid level, and phylogenetic comparison of vesiculovirus envelope genes indicates that, among vesiculoviruses, cocalvirus is most closely related to VSV-G Indiana, although it is serologically distinct. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006 (1984). Cocarbecyclovirus envelope pseudotyped retroviral vector particles can include, for example, lentivirus, alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, and epsilonretrovirus vector particles that can include Gag, Pol, and / or one or more accessory proteins of a retrovirus and a cocarbecyclovirus envelope protein. Within certain aspects of these embodiments, the Gag, Pol, and accessory proteins are lentivirus and / or gammaretrovirus. For applications where transient expression is preferred, an adenovirus-based system can be used. Adenovirus-based vectors can exhibit extremely high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors, which can be produced in large quantities in a relatively simple system.Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, e.g., in in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). The construction of recombinant AAV vectors has been described in numerous publications, e.g., U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 5:3251-3260 (1985); al.,Mol.Cell.Biol.4:2072-2081(1984);Hermonat&Muzyczka,PNAS 81:6466-6470(1984);and Samulski et al., J. Virol. 63:03822-3828 (1989). Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and Ψ2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually made by creating cell lines that package nucleic acid vectors into viral particles. The vectors typically contain the minimum viral sequences required for packaging and subsequent integration into the host, with other viral sequences being replaced by expression cassettes for the polynucleotides to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have ITR sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line that contains a helper plasmid that encodes the other AAV genes, namely rep and cap, but lacks ITR sequences.This cell line can also be infected with adenovirus as a helper. The helper virus promotes the replication of AAV vectors and the expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in large amounts because it lacks ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Therefore, AAV is considered an ideal candidate for use as a transduction vector. Such AAV transduction vectors can contain sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helper functions provided in trans. Recombinant AAV (rAAV). can be used to deliver exogenous genes to cells of various lineages. In these vectors, the AAV cap and / or rep genes are deleted from the viral genome and replaced with a DNA segment of choice. Current AAV vectors can accommodate insert DNA of up to 4300 bases. There are several ways to generate rAAV, and the present invention provides rAAV and methods for preparing rAAV. For example, one or more plasmids containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells. In addition, a second or additional helper plasmid is co-transfected into these cells to provide the AAV rep and / or cap genes essential for the replication and packaging of the recombinant viral construct. Under these conditions, the AAV rep and / or cap proteins act in trans to stimulate the replication and packaging of the rAAV construct. The rAAV is harvested 2-3 days after transfection. Traditionally, rAAV is harvested from the cells along with adenovirus. Contaminating adenovirus is then inactivated by heat treatment. In the present invention, rAAV is advantageously recovered from the cell supernatant rather than from the cells themselves.Thus, in a first aspect, the invention provides for preparing an rAAV, and in addition to the above, the rAAV may be prepared by a method comprising or consisting essentially of: infecting a susceptible cell with an rAAV containing exogenous DNA, including expression DNA, and a helper virus (e.g., a poxvirus, such as adenovirus, herpesvirus, vaccinia virus, etc.), where the rAAV lacks a functional cap and / or rep (and the helper virus (e.g., a poxvirus, such as adenovirus, herpesvirus, vaccinia virus, etc.) provides the cap and / or rev functions that the rAAV lacks); or infecting a susceptible cell with an rAAV containing exogenous DNA, including expression DNA, where the recombinant lacks a functional cap and / or rep. or infecting a susceptible cell with an rAAV containing foreign DNA, including expression DNA, wherein the recombinant is lacking a functional cap and / or rep, and the cell is providing the cap and / or rep functions that the recombinant is lacking; or transfecting a susceptible cell with an AAV lacking a functional cap and / or rep, and a plasmid for inserting foreign DNA into the recombinant and for providing the rep and / or cap functions, such that transfection results in a rAAV containing foreign DNA, including expression DNA, that is lacking a functional cap and / or rep. The rAAV may be derived from AAV as described herein, and may advantageously be rAAV1, rAAV2, AAV5 or rAAV with a capsid, or a hybrid that may include AAV1, AAV2, AAV5 or any combination thereof.The AAV of the rAAV can be selected with respect to the cells that the rAAV targets; for example, for targeting brain or neuronal cells, AAV serotypes 1, 2, 5 or hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof can be selected; and for targeting cardiac tissue, AAV4 can be selected. In addition to 293 cells, other cells that can be used in the practice of the invention and the relative infectivity of certain AAV serotypes in vitro for those cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) are as follows:

[0128] [Table 2]

[0129] The present invention relates to an exogenous nucleic acid molecule encoding a CRISPR (clustered regularly interspaced short palindromic repeats) system, e.g., a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (a putative nuclease or helicase protein), e.g., Cas9, and a terminator, and two or more, advantageously up to the packaging size limit of the vector, e.g., five cassettes in total (including the first cassette) comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette is roughly: promoter-gRNA1-terminator, promoter-gRNA2-terminator... promoter-gRNA(N) -terminator (where N is the number of inserts that is the upper limit of the packaging size limit of the vector)), or two or more individual rAAVs, each comprising one or more cassettes of a CRISPR system, e.g., a first rAAV comprises a first cassette comprising or essentially consisting of a promoter, a nucleic acid molecule encoding a Cas, e.g. Cas9, and a terminator, and a second rAAV comprises a plurality of four cassettes comprising or essentially consisting of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette is generally represented as promoter-gRNA1-terminator, promoter-gRNA2-terminator...The present invention provides an rAAV comprising a promoter-gRNA(N)-terminator, where N is the number of inserts that is the upper limit of the packaging size limit of the vector. Since rAAV is a DNA virus, the nucleic acid molecule in the AAV or rAAV discussion herein is preferably DNA. The promoter is preferably the human synapsin I promoter (hSyn) in some embodiments. Further methods of delivering nucleic acids to cells are known to those skilled in the art. See, for example, US Patent Publication No. 20030087817, which is incorporated herein by reference. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected as it naturally occurs in the subject. In some embodiments, the cell to be transfected is taken from the subject. In some embodiments, the cell is derived from a cell taken from the subject, such as a cell line. A wide range of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts;10.1 Mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, B EAS-2B, bEnd.3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (e.g., the American Type Culture Collection (ATCC) (Manassus, Va.). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain one or more vector-derived sequences. In some embodiments, cells transiently transfected with components of the CRISPR system described herein (e.g., by transient transfection of one or more vectors or transfection with RNA) and modified through the activity of the CRISPR complex are used to establish new cell lines that include cells that contain the modifications but lack any other exogenous sequences. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used in the evaluation of one or more test compounds.

[0130] Increasing NHEJ or HR efficiency also aids in delivery. NHEJ efficiency is preferably increased by co-expression of terminal processing enzymes, such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4): 787-797). HR efficiency is preferably increased by transient inhibition of NHEJ machinery, such as Ku70 and Ku86. HR efficiency can also be increased by co-expression of prokaryotic or eukaryotic homologous recombination enzymes, such as RecBCD, RecA.

[0131] General Packaging and Promoters A method for packaging a Cas9-encoding nucleic acid molecule, e.g., DNA, into a vector, e.g., a viral vector, to mediate genome modification in vivo includes: Achieving NHEJ-mediated gene knockout: Single viral vector: Vectors containing two or more expression cassettes: Promoter-Cas9 coding nucleic acid molecule-Terminator: Promoter-gRNA1-Terminator: Promoter-gRNA2-Terminator: Promoter-gRNA(N)-Terminator: (up to the size limit of the vector): Dual viral vectors: Vector 1, containing one expression cassette driving the expression of Cas9: Promoter-Cas9 coding nucleic acid molecule-Terminator: Vector 2, comprising one or more expression cassettes driving the expression of one or more guide RNAs: Promoter-gRNA1-Terminator: Promoter-gRNA(N)-Terminator: (up to the size limit of the vector): Mediates homology-dependent repair: In addition to the single and dual viral vector approaches described above, an additional vector is used to deliver the homology-dependent repair template.

[0132] Promoters used to drive expression of the Cas9-encoding nucleic acid molecule may include: AAV ITRs may serve as promoters: This is advantageous in that no additional promoter elements (which may take up space in the vector) are required. The additional space freed up can be used to drive expression of additional elements (such as gRNAs). Also, since ITR activity is relatively weak, it can be used to mitigate potential toxicity from overexpression of Cas9. For ubiquitous expression, promoters can be used: CMV, CAG, CBh, PGK, SV40, and Ferritin heavy or light chain, etc. For expression in the brain or other CNS, the following promoters can be used: SynapsinI for any neuron, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For expression in the liver, the albumin promoter can be used. For lung expression, SP-B can be used. In the case of endothelial cells, ICAM can be used. In the case of hematopoietic cells, IFNβ or CD45 can be used. For osteoblasts, OG-2 can be used.

[0133] Promoters used to drive the guide RNA may include: Pol III promoters, e.g., U6 or H1 Use of a Pol II promoter and an intron cassette to express the gRNA.

[0134] Adeno-associated virus (AAV) Cas9 and one or more guide RNAs can be delivered using adeno-associated virus (AAV), lentivirus, adenovirus, or other types of plasmid or viral vectors, particularly formulations and doses from, for example, U.S. Pat. Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV), and 5,846,946 (formulations, doses for DNA plasmids), as well as from clinical trials and publications on clinical trials for lentivirus, AAV, and adenovirus. For example, in the case of AAV, the administration route, formulation, and dose can be similar to U.S. Pat. No. 8,454,972 and clinical trials on AAV. In the case of adenovirus, the administration route, formulation, and dose can be similar to U.S. Pat. No. 8,404,658 and clinical trials on adenovirus. In the case of plasmid delivery, the administration route, formulation, and dose can be similar to U.S. Pat. No. 5,846,946 and clinical studies on plasmids. Dosages can be based on or extrapolated to an average 70 kg person (e.g., adult human male) and can be adjusted for different weights and species of patients, subjects, and mammals. The frequency of administration is within the domain of the medical or veterinary practitioner (e.g., physician, veterinarian) and will depend on the usual factors including age, sex, general health, other conditions of the patient or subject, and the specific condition or symptom being addressed. The viral vector can be injected into the tissue of interest. For cell type specific genome modification, expression of Cas9 can be driven by a cell type specific promoter. For example, liver specific expression can use the albumin promoter, and neuron specific expression (e.g., when targeting CNS disorders) can use the Synapsin I promoter. With regard to in vivo delivery, AAV is advantageous over other viral vectors for a few reasons: low toxicity (which can be due to purification methods that do not require ultracentrifugation of cell particles that can activate an immune response);

[0135] AAV does not integrate into the host genome and is therefore unlikely to cause insertional mutagenesis.

[0136] AAV has a packaging limit of 4.5Kb or 4.75Kb. This means that Cas9 and the promoter and transcription terminator must all fit into the same viral vector. Constructs larger than 4.5Kb or 4.75Kb will greatly reduce virus production. SpCas9 is quite large, with the gene itself exceeding 4.1Kb, making it difficult to pack into AAV. Thus, embodiments of the present invention include utilizing a relatively short homologue of Cas9. For example:

[0137] [Table 3]

[0138] Therefore, these species are generally preferred Cas9 species.

[0139] For AAV, the AAV can be AAV1, AAV2, AAV5, or any combination thereof. AAV can be selected for the AAV for the cells to be targeted; for example, when targeting brain or neuronal cells, AAV serotypes 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected; when targeting cardiac tissue, AAV4 can be selected. AAV8 is useful for delivery to the liver. The promoters and vectors herein are individually preferred. A table of specific AAV serotypes for these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

[0140] [Table 4]

[0141] Lentivirus Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and postmitotic cells. The best known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.

[0142] Lentiviruses will be preferred as follows: After cloning of pCasES10 (containing the lentiviral transfer plasmid backbone), low passage (p=5) HEK293FT were seeded to 50% confluence in T-75 flasks the day before transfection in antibiotic-free DMEM supplemented with 10% fetal bovine serum. After 20 hours, the medium was replaced with OptiMEM (serum-free) medium, and transfection was performed 4 hours later. Cells were transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotyped), and 7.5 μg of psPAX2 (gag / pol / rev / tat). Transfection was performed in 4 mL of OptiMEM containing cationic lipid delivery agent (50 uL of Lipofectamine 2000 and 100 ul of Plus reagent). After 6 hours, the medium was changed to antibiotic-free DMED containing 10% fetal bovine serum. Although these methods use serum in cell culture, serum-free methods are preferred.

[0143] Lentivirus can be purified as follows: After 48 hours, viral supernatant was harvested. The supernatant was first cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. The supernatant was then ultracentrifuged at 24,000 rpm for 2 hours. The viral pellet was resuspended in 50 μl of DMEM overnight at 4° C. It was then aliquoted and flash frozen at −80° C.

[0144] In another embodiment, minimal non-primate lentiviral vectors based on Equine Infectious Anemia Virus (EIAV) are also contemplated, particularly for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8:275-285). In another embodiment, RetinoStat®, an Equine Infectious Anemia Virus-based lentiviral gene therapy vector expressing the angiogenesis inhibitor proteins endostatin and angiostatin, delivered by subretinal injection for the treatment of web form age-related macular degeneration, is also contemplated (e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), which can be modified for the CRISPR-Cas system of the present invention.

[0145] In another embodiment, a self-inactivating lentiviral vector containing an siRNA targeting a common exon shared by HIV tat / rev, a nuclear-localizing TAR decoy, and an anti-CCR5 specific hammerhead ribozyme (e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and / or adapted for the CRISPR-Cas system of the present invention. A minimum of 2.5×106 CD34+ cells per kg of patient body weight can be harvested and pre-stimulated for 16-20 hours at a concentration of 2×106 cells / ml in X-VIVO15 medium (Lonza) containing 2 μmol / L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml), and thrombopoietin (10 ng / ml) (CellGenix). Pre-stimulated cells can be transduced with lentiviruses in quintuple infections on fibronectin-coated 75 cm tissue culture flasks (25 mg / cm) (RetroNectin, Takara Bio Inc.) for 16-24 h.

[0146] Lentiviral vectors have been disclosed in the treatment of Parkinson's disease, see, e.g., US Patent Publication No. 20120295960 and US Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed in the treatment of eye diseases, see, e.g., US Patent Publication Nos. 20060281180, 20090007284, 20110117189; US Patent No. 20090017543; US Patent No. 20070054961, US Patent No. 20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., U.S. Patent Application Publication Nos. 20110293571; 20110293571, 20040013648, 20070025970, 20090111106, and U.S. Patent No. 7,259,015.

[0147] RNA delivery Delivery of RNA: CRISPR enzymes, such as Cas9 and / or any of the RNAs, such as guide RNAs, can also be delivered in the form of RNA. Cas9 mRNA can be produced using in vitro transcription. For example, Cas9 mRNA can be synthesized using a PCR cassette that contains the following elements: T7_promoter-kozak sequence (GCCACC)-Cas9-3'UTR from β-globin-polyA tail (a series of 120 or more adenines). This cassette can be used for transcription by T7 polymerase. Guide RNA can also be transcribed using in vitro transcription from a cassette that contains a T7_promoter-GG-guide RNA sequence.

[0148] To facilitate expression and reduce potential toxicity, the CRISPR enzyme coding sequence and / or guide RNA can be modified to include one or more modified nucleotides, for example, with a pseudo-U or a 5-methyl-C.

[0149] mRNA delivery methods currently show particular promise for delivery to the liver.

[0150] While most clinical studies on RNA delivery have focused on RNAi or antisense, these systems can be adapted to deliver RNA for the practice of the present invention. The following references on RNAi etc. should be read:

[0151] Particulate delivery systems and / or formulations: Several types of particulate delivery systems and / or formulations are known to be useful for a wide range of biomedical applications. In general, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to their diameter. Coarse particles encompass the range of 2,500-10,000 nanometers. Fine particles are 100-2,500 nanometers in size. Ultrafine particles, or nanoparticles, are generally 1-100 nanometers in size. This 100 nm limit is based on the fact that the novel properties that distinguish particles from bulk materials typically occur at a critical length scale below 100 nm.

[0152] As used herein, a particle delivery system / formulation is defined as any biological delivery system / formulation that includes a particle according to the present invention. A particle according to the present invention is any entity that has a maximum dimension (e.g., diameter) of less than 100 microns (μm). In some embodiments, a particle according to the present invention has a maximum dimension of less than 10 μm. In some embodiments, a particle according to the present invention has a maximum dimension of less than 2000 nanometers (nm). In some embodiments, a particle according to the present invention has a maximum dimension of less than 1000 nanometers (nm). In some embodiments, a particle according to the present invention has a maximum dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, a particle according to the present invention has a maximum dimension (e.g., diameter) of 500 nm or less. In some embodiments, a particle according to the present invention has a maximum dimension (e.g., diameter) of 250 nm or less. In some embodiments, a particle according to the present invention has a maximum dimension (e.g., diameter) of 200 nm or less. In some embodiments, a particle according to the present invention has a maximum dimension (e.g., diameter) of 150 nm or less. In some embodiments, the particles of the invention have a maximum dimension (e.g., diameter) of 100 nm or less. Smaller particles, such as particles having a maximum dimension of 50 nm or less, are used in some embodiments of the invention. In some embodiments, the particles of the invention have a maximum dimension in the range of 25 nm to 200 nm.

[0153] Characterization of particles (including, for example, characterizing morphology, dimensions, etc.) is performed using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry, and nuclear magnetic resonance (NMR). Characterization (sizing) may be performed on the native particles (i.e., before loading) or after loading with cargo (cargo, as used herein, refers to, for example, one or more components of the CRISPR-Cas system, such as the CRISPR enzyme or mRNA or guide RNA, or any combination thereof, and may include additional carriers and / or excipients) to provide particles of optimal size for delivery to any in vitro, ex vivo, and / or in vivo application of the invention. In certain preferred embodiments, the characterization of particle size (e.g., diameter) is based on measurement using dynamic laser scattering (DLS).Regarding particles, their preparation and use methods and their measurement, reference is made to US Patent No. 8,709,843; US Patent No. 6,007,845; US Patent No. 5,855,913; US Patent No. 5,985,309; US Patent No. 5,543,158; and the publication by James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014, doi:10.1038 / nnano.2014.84.

[0154] Particulate delivery systems within the scope of the present invention may be provided in any form, including, but not limited to, solid, semi-solid, emulsion, or colloidal particles. Thus, any delivery system described herein may be provided as a particulate delivery system within the scope of the present invention, including, but not limited to, for example, lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns.

[0155] Nanoparticles The CRISPR enzyme mRNA and guide RNA can be delivered simultaneously using nanoparticles or lipid envelopes.

[0156] For example, Su X, Fricke J, Kavanagh DG, Irvine DJ ("In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles" Mol Pharm. 2011 Jun 6;8(3):774-87. doi:10.1021 / mp100390w. Epub 2011 Apr 1) describe biodegradable core-shell nanoparticles with a poly(β-amino ester) (PBAE) core covered by a phospholipid bilayer shell. These were developed for delivery of mRNA in vivo. The pH-responsive PBAE component was selected to promote endosomal disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. They are therefore preferred for delivery of RNA of the present invention.

[0157] In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are contemplated, which can be applied to oral, intravenous, and nasal peptide delivery, all of which are delivered to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs, are also contemplated. Molecular envelope technology involves engineered polymer envelopes that are delivered to the disease site protected (e.g., Mazza, M. et al. ACS Nano, 2013.7(2):1016-1026; Siew, A., et al. Mol Pharm, 2012.9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012.9(6):1764-74; Garrett, NL, et al. J Biophotonics, 2012.5(5-6):458-68; Garrett, NL, et al. J Raman Spect, 2012.43(5):681-688; Ahmad, S., et al. J Royal Soc Interface 2010.7:S423-33; Uchegbu, IF Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006.7(12):3452-9, and Uchegbu, IF, et al. Int J Pharm, 2001.224:185-199). A dose of about 5 mg / kg is contemplated, either in a single dose or multiple doses depending on the target tissue.

[0158] In one embodiment, nanoparticles developed in Dan Anderson's lab at MIT that can deliver RNA to cancer cells to stop tumor growth can be used and / or adapted to the CRISPR Cas system of the present invention. In particular, Anderson's lab has developed a fully automated combination system for the synthesis, purification, characterization, and formulation of new biomaterials and nanoformulations. For example, Alabi et al.,Proc Natl Acad Sci US A.2013 Aug 6;110(32):12881-6;Zhang et al.,Adv Mater.2013 Sep 6;25(33):4641-5;Jiang et al.,Nano Lett.2013 Mar 13;13(3):1059-64;Karagiannis et al. al., ACS Nano.2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano.2012 Aug 28;6(8):6922-9, and Lee et al., Nat Nanotechnol.2012 Jun 3;7(6):389-93.

[0159] US Patent Publication No. 20110293703 relates to lipid compounds that are also particularly useful for the administration of polynucleotides, which can be applied to the delivery of the CRISPR Cas system of the present invention. In one aspect, the amino alcohol lipid compounds are combined with the agent to be delivered to a cell or subject to form microparticles, nanoparticles, liposomes, or micelles. The agent to be delivered by the particles, liposomes, or micelles can be in gas, liquid, or solid form, and the agent can be a polynucleotide, protein, peptide, or small molecule. The amino alcohol lipid compounds can be combined with other amino alcohol lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles can then be combined with optional pharmaceutical excipients to form pharmaceutical compositions.

[0160] US20110293703 also provides a method for preparing an amino alcohol lipid compound. One or more equivalents of an amine are reacted with one or more equivalents of an epoxide-terminated compound under suitable conditions to form the amino alcohol lipid compound of the present invention. In certain embodiments, all amino groups of the amine are sufficiently reacted with the epoxide-terminated compound to form a tertiary amine. In other embodiments, not all amino groups of the amine are sufficiently reacted with the epoxide-terminated compound to form a tertiary amine, thus forming a primary or secondary amine in the amino alcohol lipid compound. These primary or secondary amines can remain as is or can be reacted with another electrophile, for example, a different epoxide-terminated compound. As will be appreciated by those skilled in the art, reacting an amine with less than an excess of an epoxide-terminated compound will result in a number of different amino alcohol lipid compounds with different numbers of tails. Certain amines can be fully functionalized with two epoxide-derived compound tails, while other molecules are not fully functionalized with epoxide-derived compound tails. For example, a diamine or polyamine may contain one, two, three, or four epoxide-derived compound tails that are separated from various amino moieties of the molecule to form primary, secondary, and tertiary amines. In certain embodiments, not all amino groups are fully functionalized. In certain embodiments, two of the same type of epoxide-terminated compounds are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of the amino alcohol lipid compounds may be performed with or without a solvent, and the synthesis may be performed at elevated temperatures of 30-100°C, preferably about 50-90°C. The prepared amino alcohol lipid compounds may be optionally purified. For example, a mixture of amino alcohol lipid compounds may be purified to obtain amino alcohol lipid compounds having a specific number of epoxide-derived compound tails. Or, the mixture may be purified to obtain a specific stereoisomer or positional isomer. The amino alcohol lipid compounds may also be alkylated and / or acylated with alkyl halides (e.g., methyl iodide) or other alkylating agents.

[0161] US20110293703 also provides libraries of amino alcohol lipid compounds prepared by the methods of the invention. These amino alcohol lipid compounds can be prepared and / or screened using high throughput techniques including liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, the amino alcohol lipid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into cells.

[0162] US Patent Publication No. 20130302401 relates to a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization. The PBAAs of the present invention can be used in biotechnology and medical applications as coatings (e.g., thin or multi-thin coatings for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cell encapsulation agents. When used as surface coatings, these PBAAs caused different levels of inflammation both in vitro and in vivo, depending on their chemical structure. The wide chemical diversity of this class of materials allowed the identification of polymer coatings that inhibit macrophage activity in vitro. Furthermore, these coatings reduce inflammatory cell recruitment and attenuate fibrosis following subcutaneous injection of carboxylated polystyrene microparticles. These polymers can be used to form polyelectrolyte complex capsules for cell encapsulation. The present invention may also have many other biological applications, such as, for example, antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Publication No. 20130302401 can be applied to the CRISPR Cas system of the present invention.

[0163] In another embodiment, lipid nanoparticles (LNPs) are also contemplated. Anti-transthyretin short interfering RNA has been delivered to humans encapsulated within lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29), and such systems can be adapted and applied to the CRISPR Cas system of the present invention. Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated. Agents that reduce the risk of injection-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine. Multiple doses of about 0.3 mg / kg every 4 weeks for a total of five doses are also contemplated.

[0164] LNP has been shown to be highly effective in delivering siRNA to the liver (see, for example, Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470), and thus delivery of RNA encoding CRISPR Cas to the liver is contemplated. Approximately four doses of 6 mg / kg of LNP every two weeks may be contemplated. Tabernero et al. demonstrated that tumor regression was observed after the first two cycles of 0.7 mg / kg LNP administration, and by the end of six cycles, the patient achieved a partial response, including complete regression of lymph node metastases and substantial shrinkage of liver tumors. A complete response was obtained after 40 doses in this patient, who remained in remission and completed treatment after 26 months of administration. Two patients with extrahepatic sites of disease involving kidney, lung, and lymph nodes and RCC who had progressed after prior treatment with a VEGF pathway inhibitor had stable disease at all sites for approximately 8 to 12 months, and a patient with PNET and hepatic metastases continued on the extension study for 18 months (36 doses) with stable disease.

[0165] However, changes in LNPs must be considered. Cationic lipids are combined with negatively charged lipids to induce a monolayer structure that promotes intracellular delivery. Charged LNPs are rapidly removed from circulation after intravenous injection, so ionic cationic lipids with pKa values ​​below 7 have been developed (see, for example, Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). Negatively charged polymers, such as RNA, can be introduced into LNPs at low pH values ​​(e.g., pH 4), and the ionic lipids can exhibit positive charges. However, at physiological pH values, LNPs exhibit low surface charges that are compatible with long circulation times. We focused on four ionic cationic lipids, namely, 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). These lipid-containing LNP siRNA systems have been shown to exhibit significantly different gene silencing properties in hepatocytes in vivo, with different potencies according to the series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP utilizing the Factor VII gene silencing model (see, for example, Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, Dec.2011). In particular, for formulations containing DLinKC2-DMA, a dose of 1 μg / ml of LNP or CRISPR Cas RNA within or associated with this LNP may be contemplated.

[0166] For preparation of LNPs and CRISPR Cas encapsulation, the method of Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011 can be used and / or adapted from this document. The cationic lipids, 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-O-[2''-(methoxypolyethylene glycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(ω-methoxy-poly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DOMG), were obtained from Tekmira. Cholesterol can be purchased from Sigma (St Louis, MO). Specific CRISPR Cas RNAs can be encapsulated in LNPs containing cationic lipids DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (40:10:40:10 molar ratio):DSPC:CHOL:PEGS-DMG or PEG-c-DOMG). Optionally, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) can be encapsulated to assess cellular uptake, intracellular delivery, and biodistribution. Encapsulation can be performed by dissolving a lipid mixture consisting of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanol to a final lipid concentration of 10 mmol / l. This lipid solution in ethanol can be added dropwise to 50 mmol / l citrate, pH 4.0, to form multivesicles, to a final concentration of 30% ethanol (vol / vol).Large unilamellar vesicles can be formed after passing the multilamellar vesicles through a double 80 nm Nuclepore polycarbonate filter using an extruder (Northern Lipids, Vancouver, Canada). Encapsulation can be achieved by adding RNA dissolved at 2 mg / ml in 50 mmol / l citrate, pH 4.0, containing 30% ethanol (vol / vol) dropwise to the extruded preformed large unilamellar vesicles and incubating at 31° C. for 30 min with constant mixing to a final RNA / lipid weight ratio of 0.06 / 1 wt / wt. Removal of ethanol and neutralization of the formulation buffer was performed by dialysis against phosphate buffered saline (PBS), pH 7.4, using a Spectra / Por 2 regenerated cellulose dialysis membrane for 16 h. Size distribution of nanoparticles can be determined by dynamic light scattering using a NICOMP 370 particle sizer (Nicomp Particle Sizing, Santa Barbara, CA) in vesicle / intensity mode and Gaussian fitting. The particle size of all three LNP systems can be about 70 nm in diameter. RNA encapsulation efficiency can be determined by removing free RNA from samples collected before and after dialysis using VivaPureD MiniH columns (Sartorius Stedim Biotech). Encapsulated RNA can be extracted from eluted nanoparticles and quantified at 260 nm. RNA to lipid ratio was determined by measuring cholesterol content in vesicles using cholesterol enzyme assay from Wako Chemicals USA (Richmond, VA). In relation to the present discussion of LNP and PEG lipids, PEGylated liposomes or LNPs are also suitable for delivery of CRISPR-Cas systems or their components.

[0167] For the preparation of large LNPs, Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011 can be used and / or adapted. A lipid premix solution (total lipid concentration of 20.4 mg / ml) can be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol in a molar ratio of 50:10:38.5. Sodium acetate can be added to the lipid premix in a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids can then be hydrated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol / l, pH 3.0) under vigorous stirring, which results in the spontaneous formation of liposomes in aqueous buffer containing 35% ethanol. The liposome solution can be incubated at 37° C. to allow for a time-dependent increase in particle size. Aliquots can be removed at various times during incubation to assess the change in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size has been achieved, an aqueous PEG-lipid solution (stock = 10 mg / ml PEG-DMG in 35% (vol / vol) ethanol) can be added to the liposome mixture to a final PEG molar concentration of 3.5% of total lipid. Upon addition of the PEG-lipid, the liposomes should effectively stop growing further in size. RNA can then be added to the empty liposomes at a ratio of RNA to total lipid of approximately 1:10 (wt:wt) and then incubated for 30 minutes at 37° C. to form the loaded LNPs. The mixture can then be dialyzed overnight in PBS and filtered through a 0.45-μm syringe filter.

[0168] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means of delivering the CRISPR-Cas system to the intended target. Significant data indicates that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs based on nucleic acid-functionalized gold nanoparticles are useful.

[0169] References that may be used in connection with the teachings of this specification include: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257; Hao et al., Small. 2011 7:3158-3162; Zhang et al., ACS Nano. 2011 5:6962-6970; Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391; Young et al., Nano Lett. 2012 12:3867-71; Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80; Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013), and Mirkin, et al., Small, 10:186-192.

[0170] Self-assembled nanoparticles containing RNA can be formed using polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG). This system has been used, for example, as a means to deliver siRNA to target tumor neovasculature expressing integrins and suppress expression of vascular endothelial growth factor receptor-2 (VEGF R2), thereby achieving tumor vascularization (see, for example, Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to provide a net molar excess of ionized nitrogen (polymer) to phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between the cationic polymer and nucleic acid result in the formation of polyplexes with a mean particle size distribution of approximately 100 nm, and are therefore referred to herein as nanoplexes. A dose of approximately 100-200 mg of CRISPR Cas is contemplated for delivery in Schiffelers et al.'s self-assembled nanoparticles.

[0171] The nanoplexes of Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39) can also be applied to the present invention. The nanoplexes of Bartlett et al. are prepared by mixing equal amounts of aqueous solutions of cationic polymer and nucleic acid, and adding a net molar excess of ionized nitrogen (polymer) to phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between the cationic polymer and nucleic acid result in the formation of polyplexes with an average particle size distribution of approximately 100 nm, and are therefore referred to herein as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized as follows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered from Macrocyclics (Dallas, TX). It was added to a microcentrifuge tube with an amine-modified RNA sense strand in carbonate buffer (pH 9) and a 100-fold molar excess of DOTA-NHSester. The contents were allowed to react with stirring at room temperature for 4 hours. The DOTA-RNA sense strand conjugate was ethanol precipitated, resuspended in water, and annealed to the unmodified antisense strand to obtain DOTA-siRNA. All liquids were treated with Chelex-100 (Bio-Rad, Hercules, CA) to remove traces of metal contamination. Tf-targeted and non-targeted siRNA nanoparticles can be formed using cyclodextrin-containing polycations. Typically, nanoparticles were formed in water with a charge ratio of 3 (±) and an siRNA concentration of 0.5 g / liter. 1% of the adamantane-PEG molecules on the surface of the targeted nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (wt / vol) glucose carrier solution for injection.

[0172] Davis et al. (Nature, Vol 464, 15 April 2010) will conduct an RNA clinical trial using a targeted nanoparticle delivery system (clinical trial registration number NCT00689065). Patients with solid tumors that are refractory to standard cancer therapy will receive the targeted nanoparticles via a 30-minute intravenous infusion on days 1, 3, 8, and 10 of a 21-day cycle. The nanoparticles consist of a synthetic delivery system that includes: (1) a linear cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF) targeting ligand that is presented on the outer surface of the nanoparticle for binding to the TF receptor (TFR) on the surface of cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG) used to improve the stability of the nanoparticles in biological fluids), and (4) an siRNA designed to suppress the expression of RRM2 (the sequence used in the clinic has already been designated siR2B+5). TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anticancer target. These nanoparticles (clinical version designated CALAA-01) have been shown to be well tolerated in multiple-dose studies in non-human primates. Although one patient with chronic myeloid leukemia has received siRNA via liposomal delivery, Davis et al.'s clinical trial is the first human study to use a targeted delivery system to deliver siRNA systemically to treat patients with solid tumors. To confirm that a targeted delivery system could effectively deliver functional siRNA to human tumors, Davis et al. administered siRNA to three patients comprising three different dosing cohorts: one with metastatic melanoma, each at 18, 24, and 30 mg / m2, respectively. 2 Biopsies from patients A, B, and C were examined, who received a dose of CALAA-01 of 0.01 mg / kg / day. Similar doses may be contemplated for the CRISPR Cas system of the present invention. Delivery of the present invention may be achieved with nanoparticles that include linear cyclodextrin-based polymers (CDPs), human transferrin protein (TF) targeting ligands presented on the outer surface of the nanoparticles for binding to TF receptors (TFRs) on the surface of cancer cells, and / or hydrophilic polymers (e.g., polyethylene glycol (PEG) used to improve nanoparticle stability in biological fluids).

[0173] In the context of the present invention, it is preferred that one or more components of the CRISPR complex, such as the CRISPR enzyme or mRNA or guide RNA or sgRNA or HDR template, if present, can be delivered using one or more particles or nanoparticles or lipid envelopes. Other delivery systems or vectors can be used in conjunction with the nanoparticle aspects of the present invention.

[0174] In general, a "nanoparticle" is any particle having a diameter of less than 1000 nm. In certain preferred embodiments, the nanoparticles of the present invention have a maximum dimension (e.g., diameter) of less than 500 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 25 nm to 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension of less than 100 nm. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 35 nm to 60 nm.

[0175] Nanoparticles encompassed by the present invention can be provided in various forms, such as solid nanoparticles (e.g., metals, e.g., silver, gold, iron, titanium), non-metallic, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can be prepared. Nanoparticles formed from semiconductor materials can also be labeled quantum dots if they are small enough (typically less than 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or contrast agents, and can be adapted for similar purposes of the present invention.

[0176] Semi-solid and soft nanoparticles may be produced and are within the scope of the present invention. The prototype semi-solid nanoparticle is the liposome. Various types of liposomal nanoparticles are currently in clinical use as delivery systems for anticancer drugs and vaccines. Nanoparticles that are half hydrophilic and half hydrophobic, called Janus particles, are particularly effective in stabilizing emulsions. These nanoparticles can self-assemble at the water / oil interface and function as solid surfactants.

[0177] No. 8,709,843, incorporated herein by reference, provides a drug delivery system for targeted delivery of particles containing therapeutic agents to tissues, cells, and intracellular compartments. The present invention provides targeted particles comprising surfactants, hydrophilic polymers, or polymers conjugated to lipids. No. 6,007,845, incorporated herein by reference, provides particles having a multiblock copolymer core formed by covalently bonding a multifunctional compound to one or more hydrophobic polymers and one or more hydrophilic polymers, and comprising a biologically active material. No. 5,855,913, incorporated herein by reference, provides a particulate composition having aerodynamically light particles with a tap density of less than 0.4 g / cm3 and an average diameter of 5 μm to 30 μm, and comprising a surfactant on the surface thereof, for drug delivery to the pulmonary system. U.S. Patent No. 5,985,309, incorporated herein by reference, provides particles comprising hydrophilic or hydrophobic complexes of surfactants and / or positively or negatively charged therapeutic or diagnostic agents with oppositely charged molecules for delivery to the pulmonary system. U.S. Patent No. 5,543,158, incorporated herein by reference, provides biodegradable injectable nanoparticles having a biologically active material on the surface and a biodegradable solid core comprising poly(alkylene glycol) moieties. International Publication No. WO2012135025 (also published as U.S. Patent Application Publication No. 20120251560), incorporated herein by reference, describes conjugated polyethyleneimine (PEI) polymers and conjugated aza macrocycles (collectively referred to as "conjugated lipomers" or "lipomers"). In certain embodiments, it can be envisioned that such methods and materials described herein, e.g., conjugated lipomers, can be used in conjunction with CRISPR-Cas systems to achieve genomic perturbations in vivo, ex vivo, and in vitro to regulate gene expression, including regulating the expression of proteins.

[0178] In one embodiment, the nanoparticles can be an epoxide-modified lipid polymer, advantageously 7C1 (see, for example, James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi:10.1038 / nnano.2014.84). C71 was synthesized by reacting C15 epoxide-terminated lipid with PEI600 in a 14:1 molar ratio, and formulated with C14PEG2000 into nanoparticles (diameter 35-60 nm) that are stable in PBS solution for at least 40 days. Epoxide-modified lipid-polymers can be utilized to deliver the CRISPR-Cas system of the invention to pulmonary, cardiovascular, or renal cells, although one skilled in the art could adapt the system for delivery to other target organs. Doses of about 0.05 to about 0.6 mg / kg are contemplated. Administration over several days or weeks with a total dose of about 2 mg / kg is also contemplated.

[0179] Exosomes Exosomes are endogenous nano-vesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used autologous dendritic cells to generate exosomes. Targeting to the brain was achieved by engineering dendritic cells to express Lamp2b, an exosomal membrane protein, fused to a neuron-specific RVG peptide. Purified exosomes were loaded with exogenous RNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate the knockdown, and no nonspecific uptake into other tissues was observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by potent mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target for Alzheimer's disease.

[0180] To obtain a pool of immunologically inert exosomes, Alvarez-Erviti et al. harvested bone marrow from inbred C57BL / 6 mice with the same major histocompatibility complex (MHC) haplotype. Because immature dendritic cells produce large amounts of exosomes that do not contain T cell activators, such as MHC-II and CD86, Alvarez-Erviti et al. selected dendritic cells with granulocyte / macrophage colony-stimulating factor (GM-CSF) for 7 days. The next day, exosomes were purified from the culture supernatant using a well-established ultracentrifugation protocol. The resulting exosomes were physically homogenous, with a size distribution peaking at a diameter of 80 nm as determined by nanoparticle tracking analysis (NTA) and electron microscopy. Alvarez-Erviti et al. 6 Between 6 and 12 μg (measured based on protein concentration) of exosomes were obtained per cell.

[0181] Next, Alvarez-Erviti et al. investigated the possibility of introducing exogenous cargo into the modified exosomes using an electroporation protocol adapted for nanoscale applications. Since electroporation of membrane particles at the nanometer scale is not well characterized, non-specific Cy5-labeled RNA was used for empirical optimization of the electroporation protocol. The amount of encapsulated RNA was analyzed after ultracentrifugation and lysis of the exosomes. Electroporation at 400V and 125μF provided the greatest retention of RNA and was therefore used for all subsequent experiments.

[0182] Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNA encapsulated in 150 μg RVG exosomes to normal C57BL / 6 mice and compared the knockdown efficiency to four controls: untreated mice, mice injected with RVG exosomes alone, mice injected with BACE1 siRNA complexed with an in vivo cationic liposome reagent, and mice injected with BACE1 siRNA complexed to RVG-9R, an RVG peptide conjugated to 9D-arginine that electrostatically binds to the siRNA. Cortical tissue samples were analyzed 3 days after administration and significant protein knockdown was observed in both siRNA-RVG-9R and siRNARVG exosome treated mice (45%, P<0.05 vs. 62%, P<0.01), resulting from a significant reduction in BACE1 mRNA levels (66% [+ or -] 15%, P<0.001 and 61% [+ or -] 13%, P<0.01, respectively). Furthermore, applicants demonstrated a significant reduction in the levels of total [β]-amyloid 1-42 (55%, P<0.05), the main component of amyloid plaques in Alzheimer's disease pathology, in RVG-exosome treated animals. The observed reduction was greater than the reduction in β-amyloid 1-40 demonstrated in normal mice following intracerebroventricular injection of BCAE1 inhibitors. Alvarez-Erviti et al. performed 5' rapid amplification of cDNA ends (RACE) of BCAE1 cleavage products and provided evidence of RNAi-mediated knockdown by siRNA.

[0183] Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomes induced immune responses in vivo by assessing serum concentrations of IL-6, IP-10, TNFα, and IFN-α. Following exosome treatment, non-significant changes in all cytokines were recorded, as were siRNA transfection reagent treatments in contrast to siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirming the immunologically inert profile of exosome treatment. Given that exosomes encapsulate only 20% of the siRNA, RVG exosome delivery appears to be more efficient than RVG-9R delivery, as comparable mRNA knockdown and greater protein knockdown were achieved with 1 / 5th the siRNA without a corresponding level of immune stimulation. This experiment demonstrates the therapeutic potential of RVG exosome technology, which may be suitable for long-term silencing of genes associated with neurodegenerative diseases. The exosome delivery system of Alvarez-Erviti et al. can be applied to the delivery of the CRISPR-Cas system of the present invention to therapeutic targets, particularly neurodegenerative diseases. In the present invention, a dose of about 100-1000 mg of CRISPR Cas encapsulated in about 100-1000 mg of RVG exosomes can be contemplated.

[0184] El-Andaloussi et al. (Nature Protocols 7, 2112-2126 (2012)) disclose how exosomes derived from cultured cells can be used to deliver RNA in vitro and in vivo. The protocol first describes the generation of targeted exosomes by transfection of an expression vector containing an exosomal protein fused to a peptide ligand. Next, El-Andaloussi et al. describe methods for purifying and characterizing exosomes from the supernatant of transfected cells. Next, El-Andaloussi et al. detail the key steps of introducing RNA into exosomes. Finally, El-Andaloussi et al. outline how exosomes can be used to efficiently deliver RNA to mouse brains in vitro and in vivo. Examples of expected results are also provided in which exosome-mediated RNA delivery is evaluated by functional assays and imaging. The entire protocol takes approximately three weeks. Delivery or administration according to the present invention can be performed using exosomes produced from autologous dendritic cells. From the teachings herein, this can be utilized in the practice of the present invention.

[0185] In another embodiment, plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomes are nano-sized vesicles (30-90 nm in size) produced by many cell types, including dendritic cells (DCs), B cells, T cells, mast cells, epithelial cells, and tumor cells. These vesicles are formed by inward budding of late endosomes and are then released into the extracellular environment upon fusion with the plasma membrane. Since exosomes naturally transport RNA between cells, this property may be useful in gene therapy, and this disclosure may be utilized in the practice of the present invention.

[0186] Exosomes from plasma can be prepared by separating the plasma by centrifugation of buffy coat at 900g for 20 min, collecting the cell supernatant, removing the cells by centrifugation at 300g for 10 min, and centrifuging at 16500g for 30 min, followed by filtration through a 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at 120000g for 70 min. Chemical transfection of siRNA into exosomes is performed according to the manufacturer's instructions for the RNAi Human / Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to 100 ml of PBS to a final concentration of 2 mmol / ml. After addition of HiPerFect transfection reagent, the mixture is incubated at RT for 10 min. Exosomes are re-isolated using aldehyde / sulfate latex beads to remove excess micelles. Chemical transfection of CRISPR Cas into exosomes can be performed similarly to siRNA. Exosomes can be cultured with monocytes and lymphocytes isolated from peripheral blood of healthy donors.Therefore, it can be envisaged that exosomes containing CRISPR Cas can be introduced into monocytes and lymphocytes and then reintroduced into humans.Therefore, delivery or administration according to the present invention can be carried out using plasma exosomes.

[0187] Liposomes Delivery or administration according to the present invention can be carried out with liposomes. Liposomes are spherical vesicular structures composed of a single or multi-membrane lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have attracted considerable attention as drug delivery carriers because they are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes to the blood-brain barrier (BBB) ​​(see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 46967 (for reference)). Liposomes can be formed from several different types of lipids; however, phospholipids are most commonly used to form liposomes as drug carriers. Liposome formation occurs spontaneously when a lipid membrane is mixed with an aqueous solution, but can also be promoted by applying force in the form of shaking by using a homogenizer, sonicator, or extruder (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 (for reference)).

[0188] Some other additives can be added to liposomes to modify their structure and properties. For example, either cholesterol or sphingomyelin can be added to the liposome mixture to stabilize the liposome structure and prevent the leakage of cargo inside the liposome. In addition, liposomes have been prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate, and their average vesicle size has been adjusted to about 50-100 nm (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 (for reference)). The liposome formulation may be mainly composed of natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside. Because the formulation is prepared only from phospholipids, the liposome formulation faces a number of challenges, one of which is instability in plasma. Several attempts have been made to overcome these challenges, especially the treatment of lipid membranes. One of these attempts has focused on the treatment of cholesterol. The addition of cholesterol to conventional formulations reduces the rapid release of encapsulated bioactive compounds into the plasma, or 1,2-dioleoyl-sn-glycero-3-phosphaethanolamine (DOPE) enhances stability (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 (for reference)).

[0189] In certain advantageous embodiments, Trojan Horse liposomes (also known as molecular Trojan Horses) are desirable, and protocols can be found at http: / / cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long. These particles can deliver transgenes throughout the brain after vascular injection. It is believed, without limitation, that neutral lipid particles with specific antibodies conjugated to their surface can cross the blood-brain barrier by endocytosis. Applicants hypothesize that Trojan Horse liposomes can be utilized to deliver the CRISPR family of nucleases to the brain by vascular injection, thus enabling whole-brain transgenic animals without the need for fetal manipulation. In vivo administration of about 1-5 g of DNA or RNA in liposomes can be contemplated.

[0190] In another embodiment, the CRISPR Cas system or its components can be administered in liposomes, such as stable nucleic acid lipid particles (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injection of about 1 mg / kg / day, 3 mg / kg / day, or 5 mg / kg / day of the specific CRISPR Cas targeted in SNALP is contemplated. Daily treatment can be performed for about 3 days, followed by weekly administration for 5 weeks. In another embodiment, the specific CRISPR Cas encapsulated in SNA...

Claims

1. A composition comprising a CRISPR-Cas system for use in treating a genetic eye disease, comprising: The composition comprises a plurality of adeno-associated virus (AAV) vectors, each of which comprises a CRISPR-Cas system; The CRISPR-Cas system comprises: (A) a polynucleotide encoding a CRISPR-Cas system RNA comprising: (a) a guide sequence capable of hybridizing to a target sequence associated with a genetic eye disease; (b) a tracr mate sequence; and (c) a tracr sequence; and (B) a polynucleotide encoding Cas9; Including, the composition is formulated for topical administration to an eye of a subject; when administered to the eye of a subject, the CRISPR-Cas system is capable of forming a CRISPR-Cas complex comprising Cas9 and a CRISPR-Cas system RNA; the genetic eye disease is a retinal disease, and the composition is formulated for subretinal administration and administered via subretinal injection. composition.

2. A composition for use according to claim 1, comprising The retinal disease is an inherited retinal disease. composition.

3. A composition for use according to claim 1 or 2, comprising The Cas9 is a nuclease that induces a cleavage of both strands at the target sequence, or the Cas9 is a nickase that induces a cleavage of a single strand at the target sequence. composition.

4. A composition for use according to any one of claims 1 to 3, comprising In use, the Cas9 forms a complex with the CRISPR-Cas system RNA. composition.

5. A composition for use according to claim 4, comprising In use, 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. composition.

6. A composition for use according to any one of claims 1 to 5, comprising The hereditary eye disease is Leber Congenital Amauroscopy, Usher Syndrome, Stargardt disease, Sorsby Dystrophy, retinitis pigmentosa, or age-related macular degeneration; composition.

7. A composition for use according to any one of claims 1 to 6, comprising The CRISPR-Cas system RNA is a chimeric RNA, and (a), (b) and (c) are arranged in a 5' to 3' direction; composition.

8. A composition for use according to any one of claims 1 to 7, comprising The CRISPR-Cas system is a multiplexed system containing multiple guide sequences and a single tracr sequence; composition.

9. A composition for use according to any one of claims 1 to 8, comprising The Cas9 comprises one or more nuclear localization sequences (NLS); composition.

10. A composition for use according to any one of claims 1 to 9, comprising The Cas9 comprises one or more mutations. composition.

11. A composition for use according to any one of claims 1 to 10, comprising the Cas9 comprises one or more mutations in a catalytic domain; The Cas9 is fused to a heterologous functional domain; composition.

12. A composition for use according to any one of claims 1 to 11, comprising Further comprising a recombinant template, composition.

13. A composition for use according to any one of claims 1 to 12, comprising Formulated for single dose administration; composition.

14. A composition for use according to any one of claims 1 to 13, comprising The subject is a mammal or a human subject. composition.

15. A composition for use according to any one of claims 1 to 14, comprising The CRISPR-Cas system comprises two or more guide sequences. composition.

16. A composition for use according to any one of claims 1 to 15, comprising The Cas9 is Staphylococcus aureus Cas9 (SaCas9); composition.

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