Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

Abstract

To provide alternative and robust systems and techniques for nucleic sequence targeting with a wide array of applications.SOLUTION: The problem is solved by a CRISPR complex comprising a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within a target polynucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE AND INCORPORATION BY REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Applications Nos. 61 / 736,527, filed December 12, 2012; 61 / 748,427, filed January 2, 2013; 61 / 758,468, filed January 30, 2013; 61 / 769,046, filed February 25, 2013; 61 / 791,409 and 61 / 802,174, filed March 15, 2013; and 61 / 802,174, filed March 28, 2013. This application claims the benefit of and priority to US Patent No. 61 / 814,263, filed April 20, 2013; US Patent No. 61 / 819,803, filed May 6, 2013; US Patent No. 61 / 828,130, filed May 28, 2013; US Patent Nos. 61 / 835,931 and 61 / 836,123, filed June 17, 2013, and US Patent No. 61 / 847,537, filed July 17, 2013. Also, U.S. Provisional Patent Application Nos. 61 / 799,800, filed March 15, 2013; 61 / 835,931, 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 836,080, and 61 / 835,973, filed June 17, 2013. See also US Patent 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.

[0002] The above applications, and all documents cited in those applications or during their prosecution ("application cited documents"), and all documents cited or referenced in those application cited documents, as well as all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this invention. More specifically, all references are incorporated by reference to the same extent as if each individual document were individually and specifically 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 in the control of gene expression, including sequence targeting, e.g., genomic perturbation or gene editing, involving Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its components.

[0004] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support under an NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health. The U.S. government has certain rights in this invention. [Background technology]

[0005] Recent advances in genome sequencing technology and analytical methods have significantly accelerated the ability to classify and map genetic factors related to a wide range of biological functions and diseases.Accurate genome targeting technology is needed to enable the systematic reverse engineering of causal gene mutations by enabling the selective perturbation of individual genetic elements, and to advance synthetic biology, biotechnology and pharmaceutical applications.Genome editing technology, such as designer zinc finger, transcription activator-like effector (TALE), or homing meganuclease, can be used to produce targeted genome perturbations, but there is still a need for new genome engineering technology that is inexpensive, easy to set up, scalable, and easy to target multiple locations in eukaryotic genomes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 97 / 03211 Brochure Summary of the Invention [Problem to be solved by the invention]

[0007] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences, but can program a single Cas enzyme with a short RNA molecule to recognize specific DNA targets.The addition of the CRISPR-Cas system to the repertoire of genome sequencing technologies and analytical methods significantly simplifies methodology and accelerates the ability to classify and map genetic factors related to a wide range of biological functions and diseases.In order to effectively utilize the CRISPR-Cas system in genome editing without adverse effects, it is important to understand the aspects of engineering, optimization, and cell type / tissue / organ-specific delivery of these genome engineering tools, which are an embodiment of the claimed invention. [Means for solving the problem]

[0008] There is an urgent need for alternative and robust systems and techniques for targeting nucleic acid sequences with wide applicability.The embodiments of the present invention meet this need and provide related advantages.An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes with target sequence in target polynucleotide.The guide sequence binds to tracr mate sequence, and then the tracr mate sequence hybridizes with tracr sequence.

[0009] In one aspect, the present invention provides the method of using one or more elements of CRISPR-Cas system.The CRISPR complex of the present invention provides an effective means for modifying target polynucleotide.The CRISPR complex of the present invention has a wide range of uses, including modifying (for example, deleting, inserting, translocating, inactivating, activating) target polynucleotide in a wide variety of cell types in various tissues and organs.Therefore, the CRISPR complex of the present invention has a wide range of applications, for example, in gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis and prognosis.

[0010] Embodiments of the invention relate to Cas9 enzymes and nucleic acid molecules encoding same, and chimeric Cas9 enzymes with improved targeting specificity in CRISPR-Cas9 systems having guide RNAs with optimal activity that are shorter in length than wild-type Cas9 enzymes, as well as methods of improving the target specificity of a Cas9 enzyme or methods of designing a CRISPR-Cas9 system, comprising designing or preparing a guide RNA with optimal activity and / or selecting or preparing a Cas9 enzyme that is smaller in size or length than wild-type Cas9 (which allows for more advancement in packaging of the nucleic acid encoding it into a delivery vector due to less coding of it in the delivery vector compared to wild-type Cas9), and / or generating a chimeric Cas9 enzyme.

[0011] Also provided is the use of the subject sequences, vectors, enzymes or systems in medicine, and in gene or genome editing.

[0012] In further embodiments of the present invention, the Cas9 enzyme may contain one or more mutations and may be used as a general-purpose DNA-binding protein, with or without fusion to a functional domain. The mutations may be artificially introduced or may be gain-of-function or loss-of-function mutations. Mutations may include, but are not limited to, mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively. Additional mutations have been characterized. In one embodiment of the present invention, the transcriptional activation domain may be VP64. In another embodiment of the present invention, the transcriptional repressor domain may be KRAB or SID4X. Other embodiments of the present invention relate to mutant Cas9 enzymes fused to domains including, but not limited to, transcriptional activators, repressors, recombinases, transposases, histone remodelers, demethylases, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains, or chemical-inducible / regulatory domains.

[0013] In further embodiments, the present invention provides methods for generating mutant tracrRNAs and direct repeat sequences or mutant chimeric guide sequences that enhance the performance of these RNAs in cells. Aspects of the invention also provide for selection of said sequences.

[0014] The present invention also provides a method for simplifying the cloning and delivery of components of CRISPR complexes.In a preferred embodiment of the present invention, a suitable promoter, such as U6 promoter, is amplified together with DNA oligos and added to guide RNA.The resulting PCR product can then be transfected into cells to drive the expression of guide RNA.The present invention also relates to guide RNA that is transcribed in vitro or ordered from a synthesis company and directly transfected.

[0015] In one aspect, the present invention provides a method for improving activity by using a more active polymerase. In a preferred embodiment, expression of a guide RNA under the control of a T7 promoter is driven by expression of a T7 polymerase in the cell. In an advantageous embodiment, the cell is a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a human cell. In a more preferred embodiment, the human cell is a patient-specific cell.

[0016] In one embodiment, the present invention provides a method for reducing the toxicity of Cas enzymes. In a specific embodiment, the Cas enzyme is any Cas9 as described herein, for example, any naturally occurring bacterial Cas9, and any chimera, mutant, homolog, or ortholog. In a preferred embodiment, Cas9 is delivered to cells in the form of mRNA. This allows for transient expression of the enzyme, thereby reducing toxicity. In another preferred embodiment, the present invention also provides a method for expressing Cas9 under the control of an inducible promoter, and a construct used therein.

[0017] In another aspect, the present invention provides methods for improving the in vivo application of CRISPR-Cas systems. In a preferred embodiment, the Cas enzyme is wild-type Cas9 or any of the modified forms described herein, including any naturally occurring bacterial Cas9 and any chimera, mutant, homolog, or ortholog. An advantageous aspect of the present invention provides for the selection of a Cas9 homolog that is easily packaged into a viral vector for delivery. Cas9 orthologs typically share the general organization of three to four 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 refer to the guide sequence.

[0018] The catalytic residues of the 5' RuvC domain are identified by homology comparison of the Cas9 of interest with other Cas9 orthologs (from the S. pyogenes Type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscillanovicida Type II CRISPR locus), and the conserved Asp residue (D10) is mutated to alanine to convert the Cas9 into a complementary strand nicking enzyme. Similarly, the conserved His and Asn residues in the HNH domain are mutated to alanine to convert the Cas9 into a non-complementary strand nicking enzyme. In some embodiments, both sets of mutations can be made to convert the Cas9 into a non-cleaving enzyme.

[0019] In some embodiments, the CRISPR enzyme is a type I or type III CRISPR enzyme, preferably a type II CRISPR enzyme. The type II CRISPR enzyme can be any Cas enzyme. A preferred Cas enzyme can be identified as Cas9, which can refer to a general class of enzymes that share homology with the largest nucleases with multiple nuclease domains in the type II CRISPR system. Most preferably, the Cas9 enzyme is derived 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 that it has a high degree of sequence homology with the wild-type enzyme, but it has been mutated (modified) in some way as described herein.

[0020] It will be understood that the terms Cas and CRISPR enzyme are generally used interchangeably herein unless otherwise clear. As noted above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus of Streptococcus pyogenes. However, it will be understood that the present invention encompasses many more Cas9s from other microbial species, such as SpCas9, SaCas9, and St1Cas9. Further examples are provided herein. Those skilled in the art will be able to determine appropriate corresponding residues in Cas9 enzymes other than SpCas9 by comparison of relevant amino acid sequences. Thus, when a particular amino acid substitution is referenced using SpCas9 numbering, the present disclosure is intended to encompass corresponding modifications in other Cas9 enzymes, unless the context makes clear that this is not intended to refer to other Cas9 enzymes.

[0021] In this case, an example of a codon-optimized sequence that is human-optimized (i.e., optimized for expression in humans) is provided herein (see SaCas9 human codon-optimized sequence). While this is preferred, it will be understood that other examples are possible, and codon optimization for host species is known.

[0022] In further embodiments, the present invention provides methods for enhancing Cas9 function by creating chimeric Cas9 proteins. Chimeric Cas9 proteins may be novel Cas9s containing fragments derived from two or more naturally occurring Cas9s. These methods may involve fusing the N-terminal fragment of one Cas9 homolog to the C-terminal fragment of another Cas9 homolog. These methods also allow for the selection of novel properties exhibited by chimeric Cas9 proteins.

[0023] In the present methods, where the organism is an animal or a plant, it will be understood that the modification may be performed ex vivo or in vitro, for example in cell culture, and optionally not in vivo. In other embodiments, the modification may be performed in vivo.

[0024] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: A)-I. CRISPR-Cas system chimeric RNA (chiRNA) 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 a CRISPR enzyme containing at least one nuclear localization sequence [(a), (b), and (c) are arranged in a 5' to 3' orientation; 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 a CRISPR enzyme complexed with (1) a guide sequence that hybridizes to the target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence, and the polynucleotide sequence encoding the CRISPR enzyme 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 CRISPR enzymes, and III. Polynucleotide sequences containing the tracr sequence [Once 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 a CRISPR enzyme 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 the CRISPR enzyme is DNA or RNA. The present invention provides a method for modifying an organism or non-human organism by manipulating a target sequence at a genomic locus of interest, comprising delivering a non-naturally occurring or engineered composition comprising:

[0025] Some or all of the polynucleotide sequence encoding the CRISPR enzyme, the guide sequence, the tracr mate sequence, or the tracr sequence may be RNA. The polynucleotide encoding the sequence encoding the CRISPR enzyme, the guide sequence, the tracr mate sequence, or the tracr sequence may be RNA and may be delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns.

[0026] When a polynucleotide is referenced that is RNA and is said to "comprise" a feature such as a tracr mate sequence, it will be understood that the RNA sequence includes the feature. When a polynucleotide is DNA and is said to include a feature such as a tracr mate sequence, 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 referenced DNA or RNA sequence is or can be translated (first transcribed, in the case of DNA).

[0027] Accordingly, in certain embodiments, the present invention provides methods for modifying an organism, including a human, e.g., a mammal or a non-human mammal or organism, by manipulating a target sequence in a genomic locus of interest comprising delivering a non-naturally occurring or engineered composition comprising a viral or plasmid vector system comprising one or more viral or plasmid vectors operably encoding the composition for expression, wherein the composition comprises: (A) I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, the first regulatory element being operably linked to a polynucleotide sequence comprising (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence; and II. an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally, as in some embodiments, the at least one or more nuclear localization sequences may not comprise an NLS). a second regulatory element operably linked to a sequence of (a), (b), and (c) arranged in a 5' to 3' orientation, wherein components I and II are on the same or different vectors of the system, wherein, when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and the CRISPR complex comprises (1) the guide sequence that hybridizes to the target sequence, and (2) the tracr mate sequence that hybridizes to the tracr sequence; or (B) a non-naturally occurring or engineered composition comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) a first regulatory element operably linked to at least one or more tracr mate sequences; II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme; and III.A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising a third regulatory element operably linked to a tracr sequence, wherein components I, II, and III are on the same or different vectors of the system, wherein, when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and 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. In some embodiments, components I, II, and III are on the same vector. In other embodiments, components I and II are on the same vector while component III is on a separate vector. In other embodiments, components I and III are on the same vector while component II is on a separate vector. In other embodiments, components II and III are on the same vector while component I is on a separate vector. In other embodiments, each of components I, II, and III is on a different vector. The present invention also provides viral or plasmid vector systems as described herein.

[0028] Preferably, the vector is a viral vector, such as a lentivirus or baculovirus, or preferably an adenovirus / adeno-associated virus vector, although other delivery means are known and provided (e.g., yeast systems, microvesicles, gene guns / vectors coupled to gold nanoparticles, etc.). In some embodiments, one or more of the viral or plasmid vectors may be delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns.

[0029] By manipulation of a target sequence, Applicants also mean epigenetic manipulation of the target sequence, which may be manipulation of the chromatin state of the target sequence by altering 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 promoting three-dimensional folding, etc.

[0030] When reference is made to a method of modifying an organism or mammal, including a human, or a non-human mammal or organism by manipulating 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 from that organism. In the case of humans, for example, applicants particularly contemplate single cells or cell populations, which may preferably be modified 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, in vivo embodiments are of course also contemplated.

[0031] In certain embodiments, the present invention provides a method of treating or inhibiting a pathology caused by an abnormality in a target sequence in a genomic locus of interest in a subject (e.g., a mammal or a human) or non-human subject (e.g., a mammal) in need thereof, the pathology caused by an abnormality in a target sequence in a genomic locus of interest, the method comprising modifying the subject or non-human subject by manipulating the target sequence, wherein the pathology is susceptible to treatment or inhibition by manipulation of the target sequence, comprising providing treatment comprising delivering a non-naturally occurring or engineered composition comprising an AAV or lentiviral vector system comprising one or more AAV or lentiviral vectors operably encoding the composition for expression, wherein the target sequence is manipulated by the composition upon expression, the composition comprising: (A) a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence operably linked to a polynucleotide sequence comprising: (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, 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, as in some embodiments, the at least one or more nuclear localization sequences may not comprise an NLS), wherein (a), (b), and (c) are arranged in a 5' to 3' orientation, and components I and II are on the same or different vectors of the system, such that, upon transcription, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs CRISPR to the target sequence. a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) a first regulatory element operably linked to at least one or more tracr mate sequences; II. (b) a CRISPR complex comprising a CRISPR enzyme complexed with (1) a guide sequence that hybridizes to the target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence; or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) a first regulatory element operably linked to at least one or more tracr mate sequences;A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising: a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme; and a third regulatory element operably linked to a III.tracr sequence, wherein components I, II, and III are on the same or different vectors of the system, wherein, upon transcription, a 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 a CRISPR enzyme complexed with (1) the guide sequence hybridizing to the target sequence, and (2) the tracr mate sequence hybridizing to the tracr sequence. In some embodiments, components I, II, and III are on the same vector. In other embodiments, components I and II are on the same vector while component III is on a separate vector. In other embodiments, components I and III are on the same vector while component II is on a separate vector. In other embodiments, components II and III are on the same vector while component I is on a separate vector. In other embodiments, components I, II, and III are each on a different vector. The present invention also provides viral (e.g., AAV or lentiviral) vector systems as described herein, and can be part of vector systems as described herein.

[0032] Some methods of the present invention may include inducing expression. In some methods of the present invention, the organism or subject is a eukaryote (including mammals, including humans) or a non-human eukaryote or a non-human animal or a non-human mammal. In some embodiments, the organism or subject is a non-human animal, and may be an arthropod, such as an insect, or a nematode. In some methods of the present invention, the organism or subject is a plant. In some methods of the present invention, the organism or subject is a mammal or a non-human mammal. The non-human mammal may be, for example, a rodent (preferably a mouse or a rat), an ungulate, or a primate. In some methods of the present invention, the organism or subject is algae, including microalgae, or a fungus. In some methods of the present invention, the viral vector is an AAV or lentivirus, and may be part of a vector system as described herein. In some methods of the present invention, the CRISPR enzyme is Cas9. In some methods of the present invention, expression of the guide sequence is under the control of a T7 promoter and is driven by expression of T7 polymerase.

[0033] In some embodiments, the invention encompasses methods of delivering a CRISPR enzyme, the methods comprising delivering mRNA encoding the CRISPR enzyme to a cell. In some of these methods, the CRISPR enzyme is Cas9.

[0034] The present invention also provides a method for preparing the vector system of the present invention, particularly the viral vector system as described herein. In some embodiments, the present invention encompasses a method for preparing the AAV of the present invention, which method comprises transfecting an AAV-infected cell with one or more plasmids containing or essentially consisting of one or more nucleic acid molecules encoding AAV, and providing AAVrep and / or cap, which are essential for AAV replication and packaging. In some embodiments, the AAVrep and / or cap, which are essential for AAV replication and packaging, are provided by transfecting the cell with one or more helper plasmids or one or more helper viruses. In some embodiments, the helper virus is a poxvirus, adenovirus, herpesvirus, or baculovirus. In some embodiments, the poxvirus is a vaccinia virus. In some embodiments, the cell is a mammalian cell. And in some embodiments, the cell is an insect cell, and the helper virus is a baculovirus. In other embodiments, the virus is a lentivirus.

[0035] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f.sp. lycopersici, which causes tomato wilt, attacks only tomatoes, and F. oxysporum f. dianthii and Puccinia graminis f.sp. tritici attack only wheat. Plants have pre-existing and induced defenses to resist many pathogens. Mutation and recombination events between plant generations, particularly as pathogens replicate at higher frequencies than plants, result in genetic variability that leads to susceptibility. Non-host resistance can also exist in plants, for example, when the host and pathogen are incompatible. Horizontal resistance, e.g., partial resistance to all pathogen strains, typically controlled by many genes, and vertical resistance, e.g., complete resistance to some pathogen strains but not others, typically controlled by a few genes, may also exist. At the gene-for-gene level, plants and pathogens coevolve, with genetic changes in one balancing out changes in the other. Thus, breeders use natural variability to combine the most useful genes for yield, quality, uniformity, cold tolerance, and resistance. Sources of resistance genes include native or exotic varieties, landraces, wild plant relatives, and induced mutations (e.g., treatment of plant material with mutagens). The present invention provides plant breeders with a novel means of inducing mutations. Thus, by analyzing the genomes of sources of resistance genes and using the present invention in varieties with desired characteristics or traits, those skilled in the art can induce the production of resistance genes with greater precision than previous mutagens, thereby accelerating and improving plant breeding programs.

[0036] The present invention further encompasses compositions of the invention or CRISPR enzymes thereof (and / or mRNAs encoding the CRISPR enzymes) for use in medicine or therapy. In some embodiments, the present invention encompasses compositions of the invention or CRISPR enzymes thereof (and / or mRNAs encoding the CRISPR enzymes) for use in methods of the present invention. In some embodiments, the present invention provides use of compositions of the invention or CRISPR enzymes thereof (and / or mRNAs encoding the CRISPR enzymes) in ex vivo gene or genome editing. In particular embodiments, the present invention encompasses use of compositions of the invention or CRISPR enzymes thereof (and / or mRNAs encoding the CRISPR enzymes) in the manufacture of medicines for ex vivo gene or genome editing or in methods of the present invention. In some embodiments, the present invention encompasses compositions of the invention or CRISPR enzymes thereof (and / or mRNAs encoding the CRISPR enzymes) in which the 3' end of the target sequence is flanked by a 5'-motif-containing PAM (protospacer adjacent motif) sequence, 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), respectively, as described below.

[0037] It will be understood that SpCas9 or SaCas9 is derived from or derived from Streptococcus pyogenes (S. pyogenes) or Staphylococcus aureus (S. aureus) Cas9.

[0038] Aspects of the invention include improving the specificity of gene targeting mediated by CRISPR enzymes, such as Cas9, and reducing the likelihood of off-target modifications by CRISPR enzymes, such as Cas9. The invention in some embodiments includes a method of modifying an organism or non-human organism by minimizing off-target modifications through manipulation of first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in a cell, comprising delivering a non-naturally occurring or engineered composition comprising: I. A first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, the first polynucleotide sequence comprising: (a) a first guide sequence capable of hybridizing to a first target sequence; (b) the first tracr mate sequence, and (c) the first tracr sequence; II. A second CRISPR-Cas system chiRNA polynucleotide sequence, wherein the second polynucleotide sequence comprises: (a) a second guide sequence capable of hybridizing to a second target sequence; (b) a second tracr mate sequence, and (c) a second tracr sequence, and III. A polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations, wherein (a), (b), and (c) are arranged in a 5' to 3' orientation, such that, 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 a first and second CRISPR complex to the first and second target sequences, respectively, wherein the first CRISPR complex comprises (1) a first guide sequence that hybridizes to the first target sequence, and (2) a first tracr mate sequence that hybridizes to the first tracr sequence. a first CRISPR complex comprising a CRISPR enzyme complexed with a target sequence, and a second CRISPR complex comprising a CRISPR enzyme complexed with (1) a second guide sequence that hybridizes to a second target sequence, and (2) a second tracr mate sequence that hybridizes to a second tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of a DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence to induce a double-stranded break, thus minimizing off-target modifications and modifying an organism or non-human organism.

[0039] In some methods of the present invention, the polynucleotide sequence encoding a CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences, or the first and second tracr sequences are partly or entirely RNA. In a further embodiment of the present invention, the polynucleotide encoding the sequence encoding a 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. 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 can be contained in a vector system comprising one or more vectors. In a preferred embodiment of the present invention, the CRISPR enzyme is a Cas9 enzyme, for example, SpCas9. In an embodiment 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. In a highly preferred embodiment, the CRISPR enzyme has 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.

[0040] In a preferred method of the invention, a first guide sequence directs cleavage of one strand of a DNA duplex near a first target sequence, and a second guide sequence directs 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.

[0041] The invention in some embodiments encompasses methods of modifying an organism or non-human organism by minimizing off-target modifications through manipulation of first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in a cell, comprising delivering a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising: I. A first regulatory element operably linked to: (a) a first guide sequence capable of hybridizing to a first target sequence, and (b) at least one tracr mate sequence; II. A second regulatory element operably linked to: (a) a second guide sequence capable of hybridizing to a second target sequence, and (b) at least one tracr mate sequence; 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; wherein components I, II, III, and IV are 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 hybridizing to the first target sequence, and (2) the tracr mate sequence hybridizing to the tracr sequence, and the second CRISPR complex comprises ( The CRISPR enzyme comprises a CRISPR enzyme complexed with (1) a second guide sequence that hybridizes to a second target sequence, and (2) a tracr mate sequence that hybridizes to the tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of a DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence to induce a double-stranded break, thus modifying an organism or non-human organism by minimizing off-target modifications.

[0042] The present invention also provides a vector system as described herein. The system can include one, two, three, or four different vectors. Thus, components I, II, III, and IV can be on one, two, three, or four different vectors, and any combination of possible component locations is contemplated herein, such as: components I, II, III, and IV can be on the same vector; components I, II, III, and IV can each be on a different vector; components I, II, III, and IV can all be on two or three different vectors, and any combination of locations is contemplated, etc.

[0043] 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, the one or more mutations being selected from the group consisting of D10A, E762A, H840A, N854A, N863A, and D986A. In a highly preferred embodiment, the CRISPR enzyme has 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 further embodiment of the present invention, one or more viral vectors are delivered by liposome, nanoparticle, exosome, microvesicle, or gene gun.

[0044] In a preferred method of the invention, a first guide sequence directs cleavage of one strand of a DNA duplex near a first target sequence, and a second guide sequence directs 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.

[0045] The invention in some embodiments encompasses methods of modifying a genomic locus of interest by minimizing off-target modifications by introducing into a cell that contains and expresses a double-stranded DNA molecule encoding a gene product of interest an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand, respectively, of the DNA molecule, whereby the guide RNA targets the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, thereby altering expression of the gene product; and wherein the Cas protein and the two guide RNAs do not occur together in nature.

[0046] In a preferred method of the present invention, a Cas protein nicks each of the first and second strands of a DNA molecule encoding a gene product, thereby creating a 5' overhang. In an embodiment of the present 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 present invention, the 5' overhang is at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.

[0047]

[0013] Embodiments of the present invention also encompass guide RNAs comprising a tracr mate sequence and a guide sequence fused to the tracr sequence. In aspects of the present invention, the Cas protein is codon-optimized for expression in eukaryotic cells, preferably mammalian or human cells. In further embodiments of the present invention, the Cas protein is a type II CRISPR-Cas protein, such as a Cas9 protein. In highly preferred embodiments, the Cas protein is a Cas9 protein, such as SpCas9. In aspects of the present invention, the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A, and D986A. In highly preferred embodiments, the Cas protein has a D10A mutation.

[0048] Aspects of the invention relate to reducing the expression of a gene product, or introducing a template polynucleotide into a DNA molecule encoding the gene product, or precisely excising an intervening sequence by reannealing and ligating two 5' overhangs, or altering the activity or function of a gene product, or increasing the expression of a gene product. In one embodiment of the invention, the gene product is a protein.

[0049] The present invention also encompasses an engineered, non-naturally occurring CRISPR-Cas system 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 double-stranded DNA molecule encoding a gene product in a cell, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, thereby altering expression of the gene product; and wherein the Cas protein and the two guide RNAs do not occur together in nature.

[0050] In an embodiment of the invention, the guide RNA may comprise a tracr mate sequence and a guide sequence fused to the tracr 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 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. 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. In a highly preferred embodiment, the Cas protein has a D10A mutation.

[0051] Aspects of the invention relate to reducing the expression of a gene product, or introducing a template polynucleotide into a DNA molecule encoding the gene product, or precisely excising an intervening sequence by reannealing and ligating two 5' overhangs, or altering the activity or function of a gene product, or increasing the expression of a gene product. In one embodiment of the invention, the gene product is a protein.

[0052] The present invention also encompasses engineered, non-naturally occurring vector systems comprising one or more vectors comprising: 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 encoding a gene product; b) a second regulatory element operably linked to the Cas protein; wherein components (a) and (b) are on the same or different vectors of the system, whereby the guide RNA targets a DNA molecule encoding a gene product, and the Cas protein nicks each of the first and second strands of the DNA molecule encoding the gene product, thereby altering expression of the gene product; and the Cas protein and the two guide RNAs do not naturally occur together.

[0053] In an embodiment of the invention, the guide RNA may comprise a tracr mate sequence and a guide sequence fused to the tracr 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 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. 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. In a highly preferred embodiment, the Cas protein has a D10A mutation.

[0054] Aspects of the present invention relate to reducing the expression of a gene product, or introducing a template polynucleotide into a DNA molecule encoding the gene product, or precisely excising an intervening sequence by reannealing and ligating two 5' overhangs, or altering the activity or function of a gene product, or increasing the expression of a gene product. In one embodiment of the present invention, the gene product is a protein. In a preferred embodiment of the present 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 a gene gun.

[0055] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide, the guide sequence in turn being bound to a tracr mate sequence hybridized to a tracr sequence. In some embodiments, the cleavage comprises cleaving one or both strands at the location of the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved 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 in 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 method further comprises delivering one or more vectors to the eukaryotic cell, 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. In some embodiments, the vectors are delivered into the eukaryotic cell in a subject. In some embodiments, the modification is performed in the eukaryotic cell in cell culture. In some embodiments, the method further comprises isolating the eukaryotic cell from a subject before the modification. In some embodiments, the method further comprises returning the eukaryotic cell and / or cells derived therefrom to the subject.

[0056] In one aspect, the present invention provides a method for modifying the expression of polynucleotide in eukaryotic cells.In some embodiments, the method comprises: allowing CRISPR complex to bind to polynucleotide, whereby said binding causes the expression of said polynucleotide to increase or decrease; said CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence in said polynucleotide, and said guide sequence is then linked to a tracr mate sequence that hybridizes to a tracr sequence.In some embodiments, the method further comprises delivering one or more vectors into said eukaryotic cell, and said one or more vectors drive the expression of one or more of CRISPR enzyme, the guide sequence that is linked to a tracr mate sequence, and the tracr sequence.

[0057] In one aspect, the present invention provides a method for generating a model eukaryotic cell containing 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 includes (a) introducing one or more vectors into a eukaryotic cell, 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; and (b) binding a CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide within the disease gene, where the CRISPR complex comprises (1) a guide sequence hybridized to a target sequence within the target polynucleotide, and (2) a CRISPR enzyme complexed with a tracr mate sequence hybridized to a tracr sequence, thereby generating a model eukaryotic cell containing a mutated disease gene. In some embodiments, the cleavage includes cleavage of one or both strands at the location of the target sequence by the CRISPR enzyme. In some embodiments, the cleavage results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing the cleaved 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 in 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.

[0058] In one aspect, the present invention provides a method for selecting one or more prokaryotic cells by introducing one or more mutations into a gene in one or more prokaryotic cells, the method comprising: introducing one or more vectors into the prokaryotic cells (the one or more vectors drive expression of one or more of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template; the editing template comprises one or more mutations that prevent CRISPR enzyme cleavage); allowing the editing template to homologously recombine with a target polynucleotide in the cell to be selected; and allowing a CRISPR complex to bind to the target polynucleotide to cause cleavage of the target polynucleotide within the gene (the CRISPR complex comprises (1) a guide sequence hybridized to a target sequence within the target polynucleotide, and (2) a tracr mate sequence hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death), thereby enabling selection of one or more prokaryotic cells into which one or more mutations have been introduced. In a preferred embodiment, the CRISPR enzyme is Cas9. In another embodiment of the invention, the cells to be selected may be eukaryotic cells. This embodiment of the invention allows for the selection of defined cells without requiring a selectable marker or a two-step process that may include a counterselection system.

[0059] In one aspect, the present invention provides a method for modifying target polynucleotide in eukaryotic cells.In some embodiments, the method comprises allowing a CRISPR complex to bind to target polynucleotide, causing the cleavage of said target polynucleotide, thereby modifying said target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence in said target polynucleotide, and said guide sequence binds to a tracr mate sequence, and the tracr mate sequence then hybridizes to the tracr sequence.

[0060] In another embodiment, the present invention provides a method for modifying expression of a polynucleotide in a eukaryotic cell, the method comprising increasing or decreasing expression of a target polynucleotide using a CRISPR complex that binds to the polynucleotide.

[0061] If desired, to achieve the expression modification in cells, one or more vectors comprising tracr sequence, guide sequence linked to tracr mate sequence, and sequence encoding CRISPR enzyme are delivered to cells.In some methods, one or more vectors comprise the regulatory element operably linked to the enzyme coding sequence encoding the CRISPR enzyme, comprising nuclear localization sequence; and the regulatory element operably linked to tracr mate sequence and one or more insertion sites for inserting guide sequence upstream of tracr mate sequence.When guide sequence is expressed, it directs the sequence-specific binding of CRISPR complex to target sequence in cells.Typically, CRISPR complex comprises (1) guide sequence hybridized to target sequence, and (2) CRISPR enzyme complexed with tracr mate sequence hybridized to tracr sequence.

[0062] In some methods, target polynucleotide can be inactivated to achieve the expression modification in cell.For example, when CRISPR complex is bound to target sequence in cell, target polynucleotide is inactivated so that sequence is not transcribed, or encoded protein is not produced, or sequence does not function as wild-type sequence functions.For example, protein or microRNA coding sequence can be inactivated so that protein is not produced.

[0063] In certain embodiments, the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A, or D986A, and / or the one or more mutations are in the RuvC1 or HNH domain of the CRISPR enzyme, or other forms of mutations as discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, and 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 enzyme further comprises a functional domain. 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 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.

[0064] In some embodiments, the CRISPR enzyme is a type I or type III CRISPR enzyme, but preferably a type II CRISPR enzyme. The type II CRISPR enzyme can be any Cas enzyme. The Cas enzyme can be identified as Cas9, which can refer to a general class of enzymes that share homology with the largest nucleases with multiple nuclease domains in the type II CRISPR system. Most preferably, the Cas9 enzyme is derived 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 that it has a high degree of sequence homology with the wild-type enzyme, but it has been mutated (modified) in some way as described herein.

[0065] It will be understood that the terms Cas and CRISPR enzyme are generally used interchangeably herein unless otherwise clear. As noted above, many of the residue numbering used herein refers to the Cas9 enzyme from the Type II CRISPR locus of Streptococcus pyogenes. However, it will be understood that the invention encompasses many more Cas9s from other microbial species, such as SpCas9, SaCa9, and St1Cas9.

[0066] In this case, an example of a codon-optimized sequence that is human-optimized (i.e., optimized for expression in humans) is provided herein (see SaCas9 human codon-optimized sequence). While this is preferred, it will be understood that other examples are possible, and codon optimization for host species is known.

[0067] Preferably, delivery is in the form of a vector, which may be a viral vector such as a lentivirus or baculovirus, or preferably an adenovirus / adeno-associated virus vector, but other delivery means are known and provided (such as a yeast system, microvesicles, gene gun / vector combined with gold nanoparticles, etc.).Vector not only refers to a viral system or yeast system (for example, where the nucleic acid of interest may be operably linked to and under the control of a promoter (such as to provide RNA processed in the end) in terms of expression), but also refers to the direct delivery of nucleic acid to a host cell.In the methods herein, the vector may be a viral vector, preferably AAV, but other viral vectors such as lentivirus, as discussed herein, may also be used.For example, baculovirus can be used for expression in insect cells.These insect cells can then be useful for producing large quantities of additional vectors, such as AAV or lentivirus vectors, suitable for delivery of the present invention.Also contemplated is a method for delivering the present CRISPR enzyme, which includes delivering mRNA encoding the CRISPR enzyme into cells. It will be understood that in certain embodiments the CRISPR enzyme is truncated, and / or comprises fewer than 1000 amino acids or fewer than 4000 amino acids, and / or is a nuclease or nickase, and / or is codon optimized, and / or comprises one or more mutations, and / or comprises a chimeric CRISPR enzyme, and / or comprises other optional elements as discussed herein. AAV and lentiviral vectors are preferred.

[0068] In certain embodiments, the 3' end of the target sequence is adjacent to or follows a PAM suitable for a CRISPR enzyme, typically Cas and particularly Cas9.

[0069] For example, a suitable PAM is 5'-NRG or 5'-NNGRR for the SpCas9 or SaCas9 enzyme (or derived enzyme), respectively.

[0070] It will be understood that SpCas9 or SaCas9 is derived from or derived from Streptococcus pyogenes (S. pyogenes) or Staphylococcus aureus (S. aureus) Cas9.

[0071] Accordingly, it is an object of the present invention not to include within its scope any previously known product, process for making that product, or method for using that product, to which applicants reserve their rights and which hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the present invention is not intended to include within its scope any product, process for making that product, or method for using that product that does not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (35 U.S.C. § 112, first paragraph) or the European Patent Office (EPO) (Article 83 EPC), to which applicants reserve their rights and which hereby disclose a disclaimer of any previously described product, process for making that product, or method for using that product.

[0072] In this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like, may have the meaning ascribed to them in U.S. Patent Law; for example, they may mean "includes," "included," "including," and the like; and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent Law, for example, it is noted that they allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention. These and other embodiments are disclosed by, or are obvious from, the following detailed description and are encompassed thereby.

[0073] The novel features of the invention are set forth with particularity in the appended claims.

[0074] A better understanding of the features and advantages of the present invention will be obtained by reference to 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 explanation of the drawings]

[0075] [Figure 1] A schematic model of the CRISPR system is shown. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base pairs with the DNA target (blue) immediately upstream of a required 5'-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-strand break (DSB) (red triangle) approximately 3 bp upstream of the PAM. [Figure 2A] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 2B] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 2C] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 2D] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 2E] Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 2F]Exemplary CRISPR systems, possible mechanisms of action, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing nuclear localization and CRISPR activity are presented. [Figure 3A] 1 shows the results of an assessment of SpCas9 specificity for exemplary targets. [Figure 3B] 1 shows the results of an assessment of SpCas9 specificity for exemplary targets. [Figure 3C] 1 shows the results of an assessment of SpCas9 specificity for exemplary targets. [Figure 3D] 1 shows the results of an assessment of SpCas9 specificity for exemplary targets. [Figure 4A] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4B] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4C] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4D] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4E] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4F] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 4G] An exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells are presented. [Figure 5]A table of protospacer sequences is provided, and modification efficiency results for protospacer targets and corresponding PAMs designed based on exemplary S. pyogenes and S. thermophilus CRISPR systems for loci in the human and mouse genomes are summarized. Cells were transfected with Cas9 and either pre-crRNA / tracrRNA or chimeric RNA and analyzed 72 hours post-transfection. Percent indels were calculated based on Surveyor assay results from the indicated cell lines (N=3 for all protospacer targets; errors are standard error of the mean (SEM) where ND indicates not detectable using the Surveyor assay and NT indicates not tested in this study). [Figure 6A] 1 shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 6B] 1 shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 6C] 1 shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. [Figure 7] Figure 1 shows a schematic diagram of the surveyor nuclease assay for the detection of double-strand break-induced microinsertions and deletions. [Figure 8] 1 shows an exemplary bicistronic expression vector for expression of CRISPR system elements in eukaryotic cells. [Figure 9] A histogram of the distance between the adjacent S. pyogenes SF370 locus 1 PAM (NGG) (Figure 9A) and the S. thermophilus LMD9 locus 2 PAM (NNAGAAW) (Figure 9B) in the human genome; as well as the distance for each PAM in chromosomes (Chr) (Figure 9C). [Figure 10A] 1 shows an exemplary CRISPR system, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing CRISPR activity. [Figure 10B]1 shows an exemplary CRISPR system, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing CRISPR activity. [Figure 10C] 1 shows an exemplary CRISPR system, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing CRISPR activity. [Figure 10D] 1 shows an exemplary CRISPR system, exemplary adaptations for expression in eukaryotic cells, and results of studies assessing CRISPR activity. [Figure 11] 1 shows an exemplary operation of the CRISPR system for targeting genomic loci in mammalian cells. [Figure 12] 1 shows the results of Northern blot analysis of crRNA processing in mammalian cells. [Figure 13] Exemplary selection of protospacers in the human PVALB and mouse Th loci is shown. [Figure 14] 1 shows an exemplary protospacer and corresponding PAM sequence target of the S. thermophilus CRISPR system in the human EMX1 locus. [Figure 15] A table of sequences is provided for the primers and probes used in Surveyor, RFLP, genomic sequencing, and Northern blot assays. [Figure 16A] 1 shows an exemplary engineering of a CRISPR system with chimeric RNA and the results of a SURVEYOR assay for system activity in eukaryotic cells. [Figure 16B] 1 shows an exemplary engineering of a CRISPR system with chimeric RNA and the results of a SURVEYOR assay for system activity in eukaryotic cells. [Figure 16C] 1 shows an exemplary engineering of a CRISPR system with chimeric RNA and the results of a SURVEYOR assay for system activity in eukaryotic cells. [Figure 17] 1 shows a graphical representation of the results of a SURVEYOR assay for CRISPR system activity in eukaryotic cells. [Figure 18]FIG. 1 shows an exemplary visualization of several Streptococcus pyogenes (S. pyogenes) Cas9 target sites in the human genome using the UCSC genome browser. [Figure 19A] A circular representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 19B] A circular representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 19C] A circular representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 19D] A circular representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20A] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20B] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20C] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20D] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20E]A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 20F] A linear representation of a phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (approximately 1400 amino acids) and two groups of small Cas9s (approximately 1100 amino acids) is shown. [Figure 21A] Genome editing via homologous recombination. (a) Schematic of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic depicting homologous recombination (HR) at the human EMX1 locus using either a sense or antisense single-stranded oligonucleotide as the repair template. The upper red arrow indicates the sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target locus. Arrows indicate the positions of predicted fragment sizes. [Figure 21B] Genome editing via homologous recombination. (a) Schematic of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic depicting homologous recombination (HR) at the human EMX1 locus using either a sense or antisense single-stranded oligonucleotide as the repair template. The upper red arrow indicates the sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target locus. Arrows indicate the positions of predicted fragment sizes. [Figure 21C]Genome editing via homologous recombination. (a) Schematic of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic depicting homologous recombination (HR) at the human EMX1 locus using either a sense or antisense single-stranded oligonucleotide as the repair template. The upper red arrow indicates the sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target locus. Arrows indicate the positions of predicted fragment sizes. [Figure 21D] Genome editing via homologous recombination. (a) Schematic of SpCas9 nickase with a D10A mutation in the RuvC I catalytic domain. (b) Schematic depicting homologous recombination (HR) at the human EMX1 locus using either a sense or antisense single-stranded oligonucleotide as the repair template. The upper red arrow indicates the sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are shown as arrows in the right panel. (c) Sequence of the region modified by HR. d, SURVEYOR assay (n=3) for wild-type (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target locus. Arrows indicate the positions of predicted fragment sizes. [Figure 22A] 1 shows a single vector design for SpCas9. [Figure 22B] 1 shows a single vector design for SpCas9. [Figure 23] A graph depicting the length distribution of Cas9 orthologs is shown. [Figure 24A] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24B] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24C] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24D]The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24E] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24F] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24G] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24H] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24I] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24J] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24K] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24L] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 24M] The sequence where the mutation point is located within the SpCas9 gene is shown. [Figure 25A] Conditional Cas9, Rosa26 targeting vector map is shown. [Figure 25B] Constitutive Cas9, Rosa26 targeting vector map is shown. [Figure 26] A schematic representation of key elements in constitutive and conditional Cas9 constructs is shown. [Figure 27] Delivery and in vivo mouse brain Cas9 expression data are shown. [Figure 28] (A) Delivery of a GFP reporter as either DNA or mRNA into Neuro2A cells. (B) Delivery of Cas9 and chimeric RNA as RNA to the Icam2 gene results in cleavage at one of two spacers tested. (C) Delivery of Cas9 and chimeric RNA as RNA to the F7 gene results in cleavage at one of two spacers tested. [Figure 29]This demonstrates how DNA double-strand break (DSB) repair can facilitate gene editing. In the error-prone non-homologous end joining (NHEJ) pathway, the ends of DSBs are processed and rejoined together by endogenous DNA repair mechanisms, which can result in random insertion / deletion (indel) mutations at the junction site. Indel mutations occurring within the coding region of a gene can result in frameshifts and premature stop codons, resulting in gene knockout. Alternatively, the homology-directed repair (HDR) pathway can be utilized, which provides a repair template in the form of a plasmid or single-stranded oligodeoxynucleotide (ssODN) to enable high-fidelity and precise editing. [Figure 30] Figures 30A-30C show expected results for HDR in HEK and HUES9 cells. (a) Either a targeting plasmid or ssODN (sense or antisense) with homology arms can be used to edit sequences at the target genomic locus (red triangle) cleaved by Cas9. To assay the efficiency of HDR, we introduced a HindIII site (red bar) into the target locus, which was PCR-amplified with primers that anneal outside the homology region. Digestion of the PCR product with HindIII reveals the occurrence of HDR events. (b) By using ssODNs oriented in either the sense or antisense direction (s or a) relative to the locus of interest in combination with Cas9, efficient HDR-mediated editing at the target locus can be achieved. A minimum homology region of 40 bp, and preferably 90 bp, on either side of the modification is recommended (red bar). (c) Examples of the effect of ssODNs on HDR at the EMX1 locus are shown using both wild-type Cas9 and Cas9 nickase (D10A). Each ssODN contains 90 bp of homology arms flanked by 12 bp insertions of two restriction sites. [Figure 31] Figures 31A-C show strategies for repair of the cystic fibrosis ΔF508 mutation. [Figure 32]Figures 32A-B(a) shows a schematic representation of the GAA repeat expansion in FXN intron 1, and (b) shows a schematic representation of the strategy used to excise the GAA expansion region using the CRISPR / Cas system. [Figure 33] Screening for efficient SpCas9-mediated targeting of the Tet1-3 and Dnmt1, 3a, and 3b loci is shown. Surveyor assays on DNA from transfected N2A cells demonstrate efficient DNA cleavage using various gRNAs. [Figure 34] A multiplex genome targeting strategy using a two-vector system in an AAV1 / 2 delivery system is shown. Tet1-3 and Dnmt1, 3a, and 3b gRNAs are under the control of the U6 promoter. GFP-KASH is under the control of the human synapsin promoter. Restriction enzyme sites indicate a simple gRNA replacement strategy by subcloning. HA-tagged SpCas9 flanked by two nuclear localization signals (NLS) is shown. Both vectors are delivered to the brain by AAV1 / 2 viruses at a 1:1 ratio. [Figure 35] Functional validation of multiplex DNMT targeting vector #1 using the Surveyor assay is shown. To test SpCas9-mediated cleavage of DNMT gene family loci, N2A cells were co-transfected with DNMT targeting vector #1 (+) and an SpCas9-encoding vector. gRNA alone (-) served as a negative control. Cells were harvested 48 hours post-transfection for DNA purification and downstream processing. [Figure 36] Functional validation of multiplex DNMT targeting vector #2 using the Surveyor assay is shown. To test SpCas9-mediated cleavage of DNMT gene family loci, N2A cells were co-transfected with DNMT targeting vector #1 (+) and an SpCas9-encoding vector. gRNA alone (-) served as a negative control. Cells were harvested 48 hours post-transfection for DNA purification and downstream processing. [Figure 37]A schematic diagram of the short promoter and short polyA version used for HA-SpCas9 expression in vivo is shown. The size of the coding region from the L-ITR to the R-ITR is indicated on the right. [Figure 38] A schematic diagram of the short promoter and short polyA version used for in vivo HA-SaCas9 expression is shown. The size of the coding region from the L-ITR to the R-ITR is indicated on the right. [Figure 39] Expression of SpCas9 and SaCas9 in N2A cells. Representative Western blots of HA-tagged SpCas9 and SaCas9 versions under the control of various short promoters and with short polyA (spA) sequences. Tubulin is a loading control. mCherry (mCh) is a transfection control. Cells were harvested and further processed for Western blotting 48 hours after transfection. [Figure 40] This figure shows the screening of efficient SaCas9-mediated targeting of the Tet3 locus. Surveyor assays on DNA from transfected N2A cells demonstrate efficient DNA cleavage using various gRNAs with the NNGGGTPUM sequence. GFP-transfected cells and cells expressing SaCas9 alone serve as controls. [Figure 41] Expression of HA-SaCas9 in mouse brain. Animals were injected into the dentate gyrus with a virus driving expression of HA-SaCas9 under the control of the human synapsin promoter. Animals were sacrificed two weeks after surgery. The HA tag was detected using rabbit monoclonal antibody C29F4 (CellSignaling). Cell nuclei were stained blue with DAPI staining. [Figure 42] Figure 1 shows the expression of SpCas9 and SaCas9 in primary cortical neurons in culture 7 days after transduction. Representative Western blots of HA-tagged SpCas9 and SaCas9 versions under the control of various promoters and with bgh or short polyA (spA) sequences. Tubulin is a loading control. [Figure 43]Live / dead staining of primary cortical neurons 7 days after transduction with AAV1 particles carrying SpCas9 and multiple gRNA constructs containing various promoters (examples for DNMTs are shown in the last panel). AAV-transduced neurons were compared with control, non-transduced neurons. Red nuclei indicate permeabilized dead cells (second row of panels). Live cells are green (third row of panels). [Figure 44] Live / dead staining of primary cortical neurons 7 days after transduction with AAV1 particles carrying SaCas9 containing various promoters is shown. Red nuclei indicate permeabilized dead cells (second row of panels). Live cells are shown in green (third row of panels). [Figure 45] Figure 1 shows a comparison of neuronal morphology after transduction with AAV1 viruses carrying SpCas9 and gRNA multiplexes for TET and DNMT loci. Non-transduced neurons are shown as controls. [Figure 46] Functional validation of multiplex DNMT targeting vector #1 using the Surveyor assay in primary cortical neurons. To test SpCas9-mediated cleavage of DNMT gene family loci, cells were co-transduced with DNMT targeting vector #1 and SpCas9 viruses carrying various promoters. [Figure 47] This figure shows the in vivo efficiency of SpCas9 cleavage in the brain. Mice were injected with AAV1 / 2 viruses carrying gRNA multiplexes targeting DNMT family loci, along with SpCas9 viruses under the control of two different promoters: mouse Mecp2 and rat Map1b. Two weeks after injection, brain tissue was removed, and nuclei were prepared and sorted using FACS based on GFP expression driven by the synapsin promoter from the gRNA multiplex construct. After gDNA extraction, a Surveyor assay was performed. + indicates GFP-positive nuclei, and - indicates control GFP-negative nuclei from the same animal. The numbers above the gel indicate the assessed SpCas9 efficiency. [Figure 48]Purification of GFP-KASH-labeled nuclei from hippocampal neurons is shown. The outer nuclear membrane (ONM) of the nuclear membrane is tagged with a fusion of GFP and the KASH protein transmembrane domain. Strong GFP expression in the brain one week after stereotaxic surgery and AAV1 / 2 injection. Purification of nuclei from intact brains by a density gradient centrifugation step. Purified nuclei are shown. Chromatin staining with Vybrant® DyeCycle™ Ruby stain is shown in red, and GFP-labeled nuclei are shown in green. Representative FACS profiles of GFP+ and GFP- nuclei (magenta: Vybrant® DyeCycle™ Ruby stain, green: GFP). [Figure 49] SpCas9 cleavage efficiency in mouse brain is shown. Mice were injected with AAV1 / 2 viruses carrying gRNA multiplexes targeting TET family loci, along with SpCas9 viruses under the control of two different promoters: mouse Mecp2 and rat Map1b. Three weeks after injection, brain tissue was removed, and nuclei were prepared and sorted using FACS based on GFP expression driven by the synapsin promoter from the gRNA multiplex construct. After gDNA extraction, a Surveyor assay was performed. + indicates GFP-positive nuclei, and - indicates control GFP-negative nuclei from the same animal. The numbers above the gel indicate the assessed SpCas9 efficiency. [Figure 50] GFP-KASH expression in cultured cortical neurons. Neurons were transduced with AAV1 virus carrying a gRNA multimer construct targeting the TET locus. Due to the localization of the KASH domain, the strongest signal is located around the cell nucleus. [Figure 51] (Top) A list of the spacing between pairs of guide RNAs (as indicated by the placement pattern of the two PAM sequences). When used with SpCas9(D10A) nickase, only guide RNA pairs satisfying patterns 1, 2, 3, and 4 exhibited indels. (Bottom) A gel image showing that combinations of SpCas9(D10A) with guide RNA pairs satisfying patterns 1, 2, 3, and 4 resulted in the formation of indels at the target site. [Figure 52]The U6 reverse primer sequences used to create the U6 guide RNA expression cassette are listed below. Each primer must be paired with the U6 forward primer "gcactgagggcctatttcccatgattc" to create an amplicon containing U6 and the desired guide RNA. [Figure 53] A genomic sequence map from the human EMX1 locus showing the locations of the 24 patterns listed in Figure 33 is shown. [Figure 54] (Right) Gel images showing indel formation at the target site in the presence of variable 5' overhangs after cleavage by Cas9 nickase targeted with different guide RNA pairs. (Left) A table showing the gel lane numbers on the right and various parameters, including specifying the guide RNA pair used and the length of the 5' overhang present after cleavage by Cas9 nickase, is shown. [Figure 55] 5 shows a genomic sequence map from the human EMX1 locus indicating the location of various guide RNA pairs that resulted in the gel pattern of FIG. 54 (right) and are further described in Example 35. [Figure 56] Shown is HA-SpCas9 staining in the dorsal and ventral hippocampus 8 weeks after injection of viruses encoding Mecp2-HA-SpCas9 and 3x gRNA-TET together with Syn-KASH-GFP. [Figure 57] This shows that Syn_GFP-KASH expression 8 weeks after 3x gRNA virus injection is specific to neurons (NeuN-positive cells) and non-specific to glial cells (GFAP-positive). [Figure 58]Behavioral testing performed 5 weeks after CRISPR-mediated knockdown of TET and DNMT in the dentate gyrus (ventral and dorsal regions) showed increased levels of anxiety and learning impairment. A) Elevated plus maze test: time spent in the open arms. B) Open field test: time spent in the center of the arena versus the corners. C) Novel object recognition test: results were measured 3 hours after the habituation phase. D) Barnes maze: escape-finding efficiency during 3 days of training. E) Barnes maze results. F) Freezing behavior during contextual fear conditioning. G) Latency to first freezing during contextual fear conditioning. H) Trace fear conditioning results for TET KD and DNMT KD. (I) Control - Animals injected with SpCas9 virus and a GFP-KASH construct without gRNA. Animals injected with TET-SpCas9 and constructs encoding gRNAs for Tet1, Tet2, and Tet3. Animals injected with both DNMT-SpCas9 and constructs encoding gRNAs against Dnmt1, Dnmt3a, and Dnmt3b. [Figure 59] 1 shows the cleavage efficiency of the Tet locus in the brain 8 weeks after Mecp_SpCas9 virus injection compared to control animals injected with Mecp2_SpCas9 virus only. [Figure 60] 1 shows the cleavage efficiency of the Dnmt locus in the brain 8 weeks after Mecp_SpCas9 virus injection compared to control animals injected with only Mecp2_SpCas9 virus. [Figure 61] Figure 1 shows Dnmt3a staining in the brain 8 weeks after stereotaxic injection of virus encoding Mecp2_SpCas9 and a gRNA targeting the Dnmt locus. The lower panel shows a magnified image of the ROI shown in the upper panel. [Figure 62] Figure 1 shows staining of Syn_HA-SaCas9 in the dorsal hippocampus 4 weeks after virus injection. The first column shows animals injected with SaCas9 alone, the middle column shows animals injected with both SaCas9 and a gRNA directed against the TET locus, and the right column shows animals injected with a virus encoding only the gRNA. SaCas9 nuclear localization is dependent on the presence of the gRNA. [Figure 63] SpCas9 in N2a cells. A) Targeting vector and SpCas9 expression vector. B) Western blot analysis of N2a cells expressing HA-tagged SpCas9 under the control of various promoters. C) Cleavage efficiency of the Dnmt locus. D) Western blot analysis demonstrating efficient knockdown of Dnmt3a. e) Cleavage efficiency of the Tet locus. [Figure 64] Figure 1 shows SpCas9 in primary neurons. A) Schematic of the SpCas9 cloning strategy used in this study. A short promoter and short polyA are used for efficient packaging in the AAV delivery system. B) Schematic of the strategy combining multiplex targeting with nuclear membrane labeling. C) Western blot analysis showing expression of HA-tagged SpCas9 under the control of the rMap1b and mMecp2 promoters and bGH and spA signaling. D) Immunocytochemistry demonstrating co-expression of SpCas9 with GFP-KASH in primary neurons. SpCas9 under the control of the mMecp2 promoter is expressed in neurons (Map1b, NeuN), but not in astroglial cells (GFAP). [Figure 65] Figure 1 shows knockdown of Dnmt3a in primary neurons. A) Immunocytochemistry demonstrating efficient knockdown of Dnmt3a after targeting with a multiplex targeting vector and mMecp2-SpCas9. B) Quantification of Dnmt3a antibody staining in control and targeted neurons. C) Western blot analysis demonstrating reduction of Dnmt3a protein levels. D) Quantification of Western blot analysis demonstrating total knockdown of Dnmt3a protein levels by approximately 75% in mixed primary neuronal cultures (neurons and astroglia). [Figure 66]In vivo knockdown of Dnmt3a is shown. A) Cleavage efficiency of the Dnmt locus in the brain 8 weeks after Mecp_SpCas9 virus injection compared to control animals injected with Mecp2_SpCas9 virus only. B) Western blot analysis showing reduced Dnmt3a protein levels in targeted neuronal nuclei (KASH-GFP positive) compared to control nuclei (Ruby dye positive) after sorting cell nuclei using FACS. [Figure 67] Expression of SaCas9 in primary neurons. A) Size of the SaCas9 expression vector using the human synapsin promoter and bGH signal. B) Expression of SaCas9 in primary neurons (NeuN), but not in astroglia (GFAP). C) Extranuclear localization of SaCas9 in the absence of gRNA. C' Higher magnification of the SaCas9-positive neuron shown in C). D) Nuclear localization of SaCas9 in the presence of gRNA. D') Higher magnification of the SaCas9-positive neuron shown in D). E) Western blot analysis demonstrating expression of HA-tagged SaCas9 and GFP-KASH. F) Cleavage efficiency of the Dnmt locus 1 week after AAV infection. [Figure 68]Figure 1 shows gRNA-dependent nuclear localization of SaCas9. A) Confocal imaging analysis demonstrating extranuclear localization of SaCas9 in the absence of gRNA in primary neurons. B) Nuclear localization of SaCas9 in the presence of gRNA. C) Line scan analysis of confocal image A) demonstrating extranuclear localization of SaCas9 in the absence of gRNA (red, SaCas9 signal; blue, DAPI signal; green, GFP-KASH signal). D) Line scan analysis of confocal image B) demonstrating nuclear localization of SaCas9 in the presence of gRNA (red, SaCas9 signal; blue, DAPI signal; green, GFP-KASH signal). E) Subcellular localization of SaCas9 and SpCas9 in N2a cells without (-) and with (+) gRNA. The 250 kDa SaCas9 signal in the cytoplasmic fraction (tubulin-positive) indicates SaCas9 dimerization in the cytoplasm. In the presence of gRNA, SaCas9 protein shifts to the nuclear fraction (Sun2 positive). The 100 kDa SaCas9 signal indicates the gRNA-dependent formation and transport of SaCas9 homomers into the cell nucleus. In contrast, SpCas9 exists primarily as a homomer, and its nuclear localization is gRNA-independent. [Figure 69] AAV-Sa-Cas9 vectors, liver-specific AAV-Sa-Cas9 vectors, and alternative AAV-Sa-Cas9 vectors are shown. [Figure 70] Data are presented for the optimized CMV-SaCas9-NLS-U6-sgRNA vector (vector design was last submitted); new data compares N'- and C'-terminally tagged SaCas9 and demonstrates increased cleavage efficiency with C'-terminal NLS tagging. [Figure 71] SURVEYOR images showing indels created by novel Pcsk9 targets are shown. [Figure 72]To demonstrate SaCas9 specificity, genome-wide off-target sites (GWOTs) were predicted based on two criteria: they contained four or fewer mismatched bases with the intended SaCas9 target and had the minimally restrictive PAM for SaCas9, NNGRR. HEK293FT cells were transfected with either SpCas9 or SaCas9 along with their corresponding sgRNAs at the target site (EMX1:TAGGGTTAGGGGCCCCAGGC) with CGGGGT as the PAM, allowing cleavage by either SpCas9 (CGG) or SaCas9 (CGGGGT). DNA was harvested from the cells and analyzed for indels by Illumina sequencing at the on-target locus and 41 predicted off-target loci (following the protocol from Hsu et al. Nature Biotech 2013 and the data analysis pipeline developed by David Scott and Josh Weinstein). [Figure 73] This shows that SaCas9 can have greater off-target activity than SpCas9 at specific loci. DETAILED DESCRIPTION OF THE INVENTION

[0076] The drawings herein are for illustrative purposes only and are not necessarily drawn to scale.

[0077] The present invention relates to the engineering and optimization of systems, methods and compositions relating to CRISPR-Cas systems and their components for use in controlling gene expression involving sequence targeting, such as genomic perturbations or gene editing. In an advantageous embodiment, the Cas enzyme is Cas9.

[0078] 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.

[0079] Cas9 Optimization of Cas9 may be used to enhance or develop new functions, and chimeric Cas9 proteins can be created. Examples created by the applicants are provided in Example 6. Chimeric Cas9 proteins can be created by combining fragments of different Cas9 homologs. For example, two exemplary chimeric Cas9 proteins from Cas9 described herein are shown. For example, the applicants fused the N-terminus of St1Cas9 (fragments from this protein are in bold) to the C-terminus of SpCas9. Benefits of creating chimeric Cas9 include some or all of the following: reduced toxicity; improved expression in eukaryotic cells; increased specificity; reduced molecular weight of the protein, e.g., combining the smallest domains of different Cas9 homologs to create a smaller protein; and / or altered PAM sequence requirements.

[0080] Cas9 can be used as a general-purpose DNA-binding protein. For example, and as shown in Example 7, applicants used Cas9 as a general-purpose DNA-binding protein by mutating the two catalytic domains (D10 and H840) involved in cleaving both strands of the DNA target. To upregulate gene transcription at a target locus, applicants fused a transcription activation domain (VP64) to Cas9. Other transcription activation domains are known. As shown in Example 17, transcription activation is possible. Similarly, as shown in Example 17, gene repression (in this case, of the β-catenin gene) is possible using the Cas9 repressor (DNA-binding domain), which binds to the target gene sequence and thereby represses its activity.

[0081] Adeno-associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types can be used to deliver Cas9 and one or more guide RNAs, specifically using the formulations and dosages described in, for example, US Patent No. 8,454,972 (formulations, dosages for adenovirus), US Patent No. 8,404,658 (formulations, dosages for AAV) and US Patent No. 5,846,946 (formulations, dosages for DNA plasmid), and the formulations and dosages described in the clinical trials involving lentivirus, AAV and adenovirus and the literature related to such clinical trials.For example, for AAV, the administration route, formulation and dosage can be as described in US Patent No. 8,454,972 and as described in the clinical trials involving AAV.For adenovirus, the administration route, formulation and dosage can be as described in US Patent No. 8,404,658 and as described in the clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 5,846,946 and as described in clinical trials involving the plasmid. Dosages may be based on or extrapolated to an average 70 kg individual and can be adjusted for patients, subjects, or mammals of different weights and species. Dosage frequency is within the discretion of a medical or veterinary practitioner (e.g., physician, veterinarian), depending on the usual factors, including the patient's or subject's age, sex, general health, other conditions, and the specific condition or symptom being addressed.

[0082] The viral vector can be injected into the tissue of interest.For cell type-specific genome modification, Cas9 expression can be driven by cell type-specific promoter.For example, liver-specific expression can use albumin promoter, and neuron-specific expression can use synapsin I promoter.

[0083] Transgenic Animals and Plants Transgenic animals are also provided. Preferred examples include animals containing Cas9 in the sense of the Cas9-encoding polynucleotide or protein itself. Mice, rats, and rabbits are preferred. To generate transgenic mice with the constructs exemplified herein, pure linear DNA can be injected into the pronuclei of zygotes from pseudopregnant females, e.g., CB56 females. Founders can then be identified, genotyped, and backcrossed with CB57 mice. The constructs can then be cloned and optionally verified, e.g., by Sanger sequencing. Knockouts are contemplated, e.g., when one or more genes are knocked out in the model. However, knockins (alone or in combination) are also contemplated. Exemplary knockin Cas9 mice have been generated and are exemplified, but Cas9 knockins are preferred. To generate Cas9 knockin mice, the same constitutive and conditional constructs can be targeted to the Rosa26 locus as described herein (Figures 25A-B and 26). The methods of U.S. Patent Application Publication Nos. 20120017290 and 20110265198, assigned to SangamoBioSciences, Inc., for targeting the Rosa locus, can be modified to utilize the CRISPRCas system of the present invention. In another embodiment, the methods of U.S. Patent Application Publication No. 20130236946, assigned to Cellectis, for targeting the Rosa locus can also be modified to utilize the CRISPRCas system of the present invention.

[0084] Usefulness of conditional Cas9 mice: We demonstrate that a Cas9 conditional expression construct can be activated by coexpression with Cre in 293 cells. We also demonstrate that correctly targeted R1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 is followed by a P2A peptide cleavage sequence and then EGFP, we identify successful expression by observing EGFP. We demonstrate Cas9 activation in mESCs. This same concept makes conditional Cas9 mice extremely useful. We can cross these conditional Cas9 mice with mice that ubiquitously express Cre (ACTB-Cre strain) to obtain mice that express Cas9 in all cells. Delivery of chimeric RNA should be all that is required to induce genome editing in fetal or adult mice. Interestingly, when conditional Cas9 mice are bred with mice expressing Cre under the control of a tissue-specific promoter, Cas9 should be present only in tissues that also express Cre. This approach could be used to restrict genome editing to precise tissues by delivering chimeric RNA to those tissues.

[0085] As mentioned above, transgenic animals are also provided, as well as transgenic plants, particularly crops and algae.Transgenic plants can be useful in applications other than providing disease models.Such applications can include, for example, food or feed production by expressing protein, carbohydrate, nutrient or vitamin levels higher than those normally found in wild-type plants.In this regard, transgenic plants, particularly legumes and tubers, and animals, particularly mammals such as livestock (dairy cows, sheep, goats and pigs), as well as poultry and edible insects, are also preferred.

[0086] Transgenic algae or other plants, such as rapeseed, can be particularly useful, for example, in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols used in the oil or biofuel industry.

[0087] Adeno-associated virus (AAV) In terms of in vivo delivery, AAV is advantageous compared to other viral vectors for several reasons: Low toxicity (this may be due to the purification method not requiring ultracentrifugation of cellular particles that may activate the immune response) It is unlikely to cause insertional mutagenesis because it does not integrate into the host genome.

[0088] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9, a promoter, and a transcription terminator must all be packaged in the same viral vector. Constructs larger than 4.5 or 4.75 Kb can cause a significant decrease in virus production. SpCas9 is very large, with the gene itself exceeding 4.1 Kb, making it difficult to package into AAV. Therefore, embodiments of the present invention include utilizing shorter Cas9 homologs. For example:

[0089] [Table 1]

[0090] Therefore, these species are generally preferred Cas9 species. Applicants present delivery and in vivo mouse brain Cas9 expression data.

[0091] Two methods of packaging Cas9-encoding nucleic acid molecules, e.g., DNA, into viral vectors to mediate genome modification in vivo are preferred: To achieve NHEJ-mediated gene knockout: Single viral vector: Vectors containing two or more expression cassettes: Promoter-Cas9-encoding 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 to drive Cas9 expression Promoter-Cas9-encoding nucleic acid molecule-terminator Vector 2 contains another expression cassette to drive expression of one or more guide RNAs. Promoter-gRNA1-terminator Promoter-gRNA(N)-terminator (up to the size limit of the vector)

[0092] To mediate homology-directed repair. In addition to the single and dual viral vector approaches described above, additional vectors are used to deliver the homology-directed repair template.

[0093] Promoters used to drive expression of Cas9-encoding nucleic acid molecules can include: AAV ITRs can act as promoters; this is advantageous as it eliminates the need for additional promoter elements, which can take up space in the vector. The additional space freed up can be used to drive expression of additional elements (such as gRNAs). Furthermore, because ITR activity is relatively weak, it can also be used to reduce toxicity resulting from Cas9 overexpression.

[0094] For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For expression in the brain, the following promoters can be used: synapsin I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.

[0095] For expression in the liver, the albumin promoter can be used.

[0096] For expression in the lung, SP-B can be used.

[0097] ICAM can be used on endothelial cells.

[0098] For hematopoietic cells, IFNβ or CD45 can be used.

[0099] OG-2 can be used for osteoblasts.

[0100] Promoters used to drive guide RNAs include: Pol III promoters, e.g., U6 or H1 Expression of gRNA by using a Pol II promoter and intron cassette

[0101] Regarding AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof. AAV of the AAV related to the target cell can be selected; for example, AAV serotype 1, 2, 5 or hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof can be selected to target brain or nerve cells; and AAV4 can be selected to target cardiac tissue. AAV8 is useful for delivery to the liver. The above promoters and vectors are individually preferred.

[0102] RNA delivery is also a useful in vivo delivery method. Figure 27 shows delivery and in vivo mouse brain Cas9 expression data. Liposomes or nanoparticles can be used to deliver Cas9 and gRNA (and, for example, HR repair templates) to cells. Thus, delivery of CRISPR enzymes, such as Cas9, and / or delivery of the RNA of the present invention in RNA form can be via microvesicles, liposomes, or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged into liposome particles for in vivo delivery. Liposomal transfection reagents, such as LifeTechnologies' Lipofectamine and other commercially available reagents, can effectively deliver RNA molecules to the liver.

[0103] Enhancement of NHEJ or HR efficiency also aids in delivery. NHEJ efficiency is preferably enhanced by co-expressing end-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4):787-797). HR efficiency is preferably increased by transiently inhibiting NHEJ machinery such as Ku70 and Ku86. HR efficiency can also be increased by co-expressing prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD and RecA.

[0104] A variety of delivery means are described herein and further discussed in this section.

[0105] Viral delivery: CRISPR enzymes, such as Cas9, and / or any of the RNAs, such as guide RNAs, can be delivered using adeno-associated viruses (AAVs), lentiviruses, adenoviruses, or other viral vector types, or combinations thereof. Cas9 and one or more guide RNAs can be packaged into one or more viral vectors. In some embodiments, the viral vector is delivered to the target tissue, for example, by intramuscular injection, while in other cases, viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be via a single dose or multiple doses. Those skilled in the art will understand that the actual dosage delivered herein may vary significantly depending on various factors, such as the choice of vector, the target cell, organism, or tissue, the general condition of the subject, the degree of transformation / modification desired, the route and method of administration, and the type of transformation / modification desired.

[0106] Such dosages may further contain, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable carriers (e.g., phosphate-buffered saline), pharmaceutically acceptable excipients, adjuvants that enhance antigenicity, immunostimulatory compounds or molecules, and / or other compounds known in the art. Adjuvants herein may include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) to which the antigen adsorbs; or water-in-oil emulsions (MF-59, Freund's incomplete adjuvant) in which an antigen solution is emulsified in oil (sometimes containing killed mycobacteria (Freund's complete adjuvant)) to further enhance antigenicity (to inhibit antigen degradation and / or induce macrophage influx). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and, for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides. Such dosage formulations can be easily ascertained by those skilled in the art. The dosage may further contain one or more pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic acid salts, such as acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, gels or gelling materials, flavorings, coloring agents, microspheres, polymers, suspending agents, and the like, may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, and the like, may also be present, especially when the dosage form is 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.For a thorough discussion of pharmaceutically acceptable excipients, see REMINGTON'S PHERARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), incorporated herein by reference.

[0107] In certain embodiments herein, delivery is via adenovirus, which delivers at least 1 x 10 5 In certain embodiments herein, the dose is preferably at least about 1 x 10 6 particles (e.g., about 1 × 10 6 ~1×10 12 particles), more preferably at least about 1×10 7 particles, more preferably at least about 1×10 8 particles (e.g., about 1 × 10 8 ~1×10 11 particles or approximately 1 x 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 particles or approximately 1 x 10 9 ~1×10 12 particles), or even at least about 1 × 10 10 particles (e.g., about 1 × 10 10 ~1×10 12 particles) of adenoviral vector. Alternatively, the dose is about 1 x 10 14 particles or less, preferably about 1 x 10 13 particles or less, and even more preferably about 1×10 12 particles or less, and even more preferably about 1×10 11 particles or less, and most preferably about 1×10 10 particles or less (e.g., about 1 × 10 9 Thus, the dose may be, for example, about 1×10 6 Particle unit (pu), approximately 2 x 10 6 pu, approx. 4×10 6 pu, about 1×107 pu, about 2×10 7 pu, approx. 4×10 7 pu, about 1×10 8 pu, about 2×10 8 pu, approx. 4×10 8 pu, about 1×10 9 pu, about 2×10 9 pu, approx. 4×10 9 pu, about 1×10 10 pu, about 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 × 10 12 The adenovirus vector may contain a single dose of the adenovirus vector, including the adenovirus vector of pu. See, for example, the adenovirus vectors in U.S. Patent No. 8,454,972 B2 to Nabel, et al., issued June 4, 2013 (incorporated herein by reference), and the dosage amounts at column 29, lines 36-58 thereof. In some embodiments herein, the adenovirus is delivered in multiple doses.

[0108] In certain embodiments herein, delivery is via AAV. The therapeutically effective dosage for in vivo delivery of AAV to humans is about 1×10 10 ~Approx. 1×10 10 The dosage is expected to be in the range of about 20 to about 50 ml of saline containing 1×10 functional AAV / ml solution. Dosage can be adjusted to balance the therapeutic benefit versus any adverse effects. In certain embodiments herein, the AAV dose 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 range of AAV genome is approximately 1 x 1013 The AAV may be a genome-wide AAV. Such concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml of carrier solution. Other effective dosages can be readily established by those skilled in the art through routine testing to generate dose-response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., issued March 26, 2013, column 27, lines 45-60.

[0109] In some embodiments of the present disclosure, delivery is via a plasmid. In such plasmid compositions, the dosage must be a sufficient amount of plasmid to induce 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.

[0110] Dosages herein are based on an average 70 kg individual. The frequency of administration is within the discretion of a medical or veterinary practitioner (e.g., physician, veterinarian), or scientist of ordinary skill in the art.

[0111] Lentivirus Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and postmitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses envelope glycoproteins from other viruses to target a wide range of cell types.

[0112] Lentivirus can be prepared as follows. After cloning of pCasES10 (which contains the lentiviral transfer plasmid backbone), low-passage (p=5) HEK293FT cells were seeded in T-75 flasks to 50% confluence the day before transfection in DMEM containing 10% fetal bovine serum and without antibiotics. 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). Transfections were performed in 4 mL of OptiMEM containing cationic lipid delivery agents (50 μL Lipofectamine 2000 and 100 μL Plus Reagent). After 6 hours, the medium was changed to antibiotic-free DMEM containing 10% fetal bovine serum.

[0113] Lentivirus can be purified as follows. After 48 hours, the viral supernatant was collected. The supernatant was first cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. It was then spun in an ultracentrifuge at 24,000 rpm for 2 hours. The viral pellet was resuspended in 50 μl of DMEM overnight at 4°C. It was then divided into aliquots and immediately frozen at -80°C.

[0114] 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, published online November 21, 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002 / jgm.845). In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector expressing the angiogenesis inhibitor proteins endostain and angiostatin delivered by subretinal injection for the treatment of wet age-related macular degeneration, is also contemplated (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), and may be modified for use with the CRISPR-Cas system of the present invention.

[0115] In another embodiment, a self-inactivating lentiviral vector (see, e.g., DiGiusto et al. (2010) SciTransl Med 2:36ra43) carrying an siRNA targeting a common exon shared by HIV tat / rev, a nucleolus-localizing TAR decoy, and an anti-CCR5 specific hammerhead ribozyme can be used and / or adapted for the CRISPR-Cas system of the present invention. A minimum of 2.5 x 10 per kilogram of patient body weight can be used and / or adapted for the CRISPR-Cas system of the present invention. 6 2 x 10 CD34+ cells were harvested. 6 Cells can be pre-stimulated for 16-20 hours in X-VIVO15 medium (Lonza) containing 2 mM L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml), and thrombopoietin (10 ng / ml) (CellGenix) at a density of 100 cells / ml. Pre-stimulated cells are then cultured on fibronectin (25 mg / cm). 2 ) (RetroNectin, Takara Bio Inc.) coated 75cm 2Lentivirus can be transduced in tissue culture flasks at a multiplicity of infection of 5 for 16-24 hours.

[0116] Lentiviral vectors have been disclosed for the treatment of Parkinson's disease, for example, see U.S. Patent Application Publication No. 20120295960 and U.S. Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, for example, see U.S. Patent Application Publication Nos. 20060281180, 20090007284, 20110117189; 20090017543; 20070054961, 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.

[0117] RNA delivery RNA delivery: CRISPR enzymes, such as Cas9, and / or any of the RNAs, such as guide RNA, 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 from a β-globin-polyA tail (a series of 120 or more adenines) using a PCR cassette containing the following elements: T7 promoter-Kozak sequence (GCCACC)-Cas9-3'UTR. This cassette can be used for transcription with T7 polymerase. Guide RNA can also be transcribed using in vitro transcription from a cassette containing a T7 promoter-GG-guide RNA sequence.

[0118] To enhance expression and reduce toxicity, the CRISPR enzyme and / or guide RNA can be modified with pseudo-U or 5-methyl-C.

[0119] mRNA delivery methods currently show particular promise for liver delivery, and in particular AAV8 is particularly preferred for delivery to the liver.

[0120] nanoparticles The CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.

[0121] For example, Su X, Fricke J, Kavanagh DG, and 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 in which a poly(β-amino ester) (PBAE) core is surrounded by a phospholipid bilayer shell. These nanoparticles were developed for in vivo mRNA delivery. The pH-responsive PBAE component was selected to promote endosomal disruption, while the lipid surface layer was selected to minimize the toxicity of the polycation core. Therefore, this is preferred for delivering the RNA of the present invention.

[0122] In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are contemplated, which may be applied to oral, intravenous, and nasal delivery of peptides, all 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 protected and delivered to disease sites (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. MolPharm, 2012. 9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N.L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., 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). Depending on the target tissue, a dose of about 5 mg / kg is contemplated, in single or multiple doses.

[0123] In one embodiment, nanoparticles developed by 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. Specifically, the Anderson lab has developed a fully automated combinatorial system for the synthesis, purification, characterization, and formulation of novel biomaterials and nanoformulations. For example, Alabiet al.,Proc Natl Acad SciU S A.2013 Aug 6;110(32):12881-6;Zhanget al.,Adv Mater.2013 Sep 6;25(33):4641-5;Jianget al.,Nano Lett.2013 Mar 13;13(3):1059-64;Karagianniset See Whitehead et al., ACS Nano.2012 Aug 28;6(8):6922-9 and Leet al., Nat Nanotechnol.2012 Jun 3;7(6):389-93.

[0124] U.S. Patent Application Publication No. 20110293703 relates to lipidoid compounds particularly useful for administering polynucleotides, which can be applied to the delivery of the CRISPRCas system of the present invention. In one embodiment, an aminoalcohol lipidoid compound is combined with a drug to be delivered to a cell or subject to form a microparticle, nanoparticle, liposome, or micelle. The drug delivered by the particle, liposome, or micelle may be in gas, liquid, or solid form, and the drug may be a polynucleotide, protein, peptide, or small molecule. The aminoalcohol lipidoid compound may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles may then optionally be combined with pharmaceutical excipients to form pharmaceutical compositions.

[0125] US Patent Publication No. 0110293703 also provides a method for preparing aminoalcohol lipidoid compounds. The aminoalcohol lipidoid compounds of the present invention are formed by reacting one or more equivalents of an amine with one or more equivalents of an epoxide-terminated compound under suitable conditions. In certain embodiments, all amino groups on the amine react completely with the epoxide-terminated compound to form a tertiary amine. In other embodiments, not all amino groups on the amine react completely with the epoxide-terminated compound to form a tertiary amine, thus resulting in primary or secondary amines in the aminoalcohol lipidoid compound. These primary or secondary amines can remain or can be reacted with another electrophile, such as a different epoxide-terminated compound. As will be understood by those skilled in the art, reacting an amine with less than an excess of an epoxide-terminated compound can result in multiple different aminoalcohol lipidoid compounds with various 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 on various amino moieties of the molecule, resulting in 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 compound are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of amino alcohol lipidoid compounds may be carried out with or without solvent, and the synthesis may be carried out at elevated temperatures ranging from 30 to 100°C, preferably about 50 to 90°C. The prepared amino alcohol lipidoid compounds may optionally be purified. For example, a mixture of amino alcohol lipidoid compounds may be purified to produce an amino alcohol lipidoid compound with a specific number of epoxide-derived compound tails, or a mixture may be purified to produce a specific stereoisomer or positional isomer. The amino alcohol lipidoid compounds may also be alkylated and / or acylated using alkyl halides (e.g., methyl iodide) or other alkylating agents.

[0126] U.S. Patent Application Publication No. 0110293703 also provides libraries of amino alcohol lipidoid compounds prepared by the methods of the invention. These amino alcohol lipidoid compounds can be prepared and / or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, amino alcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into cells.

[0127] U.S. Patent Application Publication No. 20130302401 describes a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization. The PBAAs of the present invention can be used in biotechnological and biomedical applications as coatings (such as thin or multilayer 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 induced varying levels of inflammation both in vitro and in vivo depending on their chemical structure. The large chemical diversity of this material class made it possible to identify polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduced inflammatory cell recruitment and fibrosis after subcutaneous implantation of carboxylated polystyrene microparticles. These polymers can be used to form polyelectrolyte complex capsules for cell encapsulation. This invention may also have many other biological applications, such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Application Publication No. 20130302401 may be applied to the CRISPRCas system of the present invention.

[0128] In another embodiment, lipid nanoparticles (LNPs) are contemplated. Specifically, anti-transthyretin small interfering RNA encapsulated in lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29) may be applied to the CRISPR-Cas system of the present invention. Doses of about 0.01 to about 1 mg / kg body weight administered intravenously are contemplated. Medication to reduce the risk of injection-related reactions is contemplated, including dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine. Multiple doses of about 0.3 mg per kilogram every four weeks for five doses are also contemplated.

[0129] 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, pp. 363-470), and therefore, delivery of CRISPRCas to the liver is contemplated. A dosage of approximately four doses of 6 mg / kg LNP every two weeks may be contemplated. Tabernero et al. demonstrated that tumor regression was observed after the first two cycles of LNP administered at 0.7 mg / kg, and by the end of six cycles, the patient achieved a partial response, with complete regression of lymph node metastases and substantial shrinkage of liver tumors. This patient achieved a complete response after 40 doses, and the patient remained in remission after receiving doses for 26 months, completing treatment. The patient with RCC and two patients with extrahepatic disease sites, including kidney, lung, and lymph node disease, who had progressed after previous treatment with a VEGF pathway inhibitor, remained stable at all sites for approximately 8 to 12 months, and the patient with PNET and liver metastases continued on the extension study for 18 months (36 doses) with stable disease.

[0130] However, the charge of LNPs must be considered. Cationic lipids, when combined with negatively charged lipids, induce nonbilayer structures, thereby facilitating intracellular delivery. Because charged LNPs are rapidly removed from the circulation after intravenous injection, ionizable cationic lipids with pKa values ​​below 7 have been developed (see, e.g., Rosine et al., Molecular Therapy, vol. 19, no. 12, pp. 1286-2200, December 2011). Negatively charged polymers, such as siRNA oligonucleotides, can be loaded onto LNPs at low pH values ​​(e.g., pH 4), where the ionizable lipids exhibit a positive charge. However, at physiological pH values, LNPs exhibit a low surface charge, which is compatible with longer circulation times. Four ionizable cationic lipids have received attention: 1,2-dilineoyl-3-dimethylammonium propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). These lipid-containing LNP siRNA systems exhibit significantly different gene silencing properties in hepatocytes in vivo, and have been shown to differ in potency in the order DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP using a factor VII gene silencing model (see, for example, Rosine et al., Molecular Therapy, vol.19, no.12, pp.1286-2200, December 2011).Particularly for formulations containing DLinKC2-DMA, dosages of 1 μg / ml level can be contemplated.

[0131] Preparation of LNPs and CRISPRCas encapsulation using cationic lipids such as 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), and (3-o-[2'-(methoxypolyethylene glycol 2000) succinoyl]-1,2 R-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be provided by Tekmira Pharmaceuticals (Vancouver, Canada) or may be synthesized. Cholesterol can be purchased from Sigma (St. Louis, MO). Specific CRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (40:10:40:10 molar ratio of cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-c-DOMG). If necessary, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may be included to assess cellular uptake, intracellular delivery, and biodistribution. Encapsulation may be performed by dissolving a lipid mixture composed 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 ethanolic lipid solution may be added dropwise to 50 mmol / L citrate, pH 4.0, to yield a final concentration of 30% ethanol vol / vol to form multilamellar vesicles.Large unilamellar vesicles can be formed after extrusion of multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters 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 until the final RNA / lipid weight ratio was 0.06 / 1 wt / wt. Ethanol removal and neutralization of the formulation buffer were performed by dialysis against phosphate-buffered saline (PBS), pH 7.4, using a Spectra / Por2 regenerated cellulose dialysis membrane for 16 h. Nanoparticle size distribution can be determined by dynamic light scattering using a NICOMP370 particle sizer, vesicle / intensity mode, and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, CA). The particle size of all three LNP systems can be approximately 70 nm in diameter. The siRNA encapsulation efficiency can be determined by removing free siRNA from samples collected before and after dialysis using a VivaPure D Mini H column (Sartorius Stedim Biotech). The encapsulated RNA can be extracted from the eluted nanoparticles and quantified at 260 nm. The siRNA to lipid ratio was determined by measuring the cholesterol content in the vesicles using the Cholesterol E enzyme assay from WakoChemicals USA (Richmond, VA).

[0132] The preparation of large LNPs by Rosin et al., Molecular Therapy, vol. 19, no. 12, pp. 1286-2200, December 2011, can be used and / or adapted. A lipid premix solution (20.4 mg / ml total lipid concentration) can be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol in a 50:10:38.5 molar ratio. Sodium acetate can be added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The mixture can then be combined with 1.85 volumes of citrate buffer (10 mmol / L, pH 3.0) under vigorous stirring to hydrate the lipids, which can result in spontaneous liposome formation in an 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 and monitored for changes in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired size is reached, an aqueous PEG-lipid solution (stock = 10 mg / ml PEG-DMG in 35% (vol / vol) ethanol) can be added to the liposome mixture to achieve a final PEG molar concentration of 3.5% of the total lipid. Upon addition of the PEG-lipid, liposomes should be at that size, effectively preventing further growth. Loaded LNPs can then be formed by adding RNA to the empty liposomes at an siRNA-to-total lipid ratio of approximately 1:10 (wt:wt), followed by incubation at 37°C for 30 minutes. The mixture can then be dialyzed overnight in PBS and filtered through a 0.45 μm syringe filter.

[0133] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means of delivering CRISPR / Cas systems to intended targets. A large body of data demonstrates that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs, based on gold nanoparticles functionalized with nucleic acids, are superior to alternative platforms due to several keys to success, including: High in vivo stability: Due to its high density loading, the majority of the cargo (DNA or siRNA) remains bound to the construct inside the cell, conferring nucleic acid stability and resistance to enzymatic degradation. Deliverability: For all cell types studied (e.g., neurons, tumor cell lines, etc.), this construct demonstrates 99% transfection efficiency without the need for carriers or transfection agents. Therapeutic Targeting: The unique target binding affinity and specificity of the constructs allows for exquisite specificity to the matching target sequence (i.e., limited off-target effects). Superior efficacy: This construct significantly outperforms previous conventional transfection reagents (Lipofectamine 2000 and Cytofectin). Low toxicity: The construct is capable of entering a variety of cultured cells, primary cells, and tissues without apparent toxicity. No significant immune response. The construct induces minimal changes in global gene expression as measured by whole genome microarray studies and cytokine-specific protein assays. Chemical Tunability: Any single agent or combination of agents (eg, proteins, peptides, small molecules) can be used to tailor the surface of the construct.

[0134] This platform for nucleic acid-based therapeutics may be applicable to numerous disease states, including inflammation and infection, cancer, skin disorders and cardiovascular disease.

[0135] The chemical reactions of the chemicals are also available:Cutler et al.,.J.Am.Chem.Soc.2011 133:9254-9257;Hao et al.,Small.2011 7:3158-3162;Zhanget 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;Zhenget al.,Proc.Natl.Acad.Sci.USA.2012109:11975-80;Mirkin,Nanomedicine 2012 7:635-638;Zhang et al.,J.Am.Chem.Soc.2012 134:16488-1691;Weintraub,Nature 495:S14-S16;Choi et al.,Proc.Natl.Acad.Sci.USA.2013 110(19):7625-7630.Jensenet al.,Sci.Transl.Med.5,209ra152(2013) and Mirkin,etal.,Small,doi.org / 10.1002 / smll.201302143.

[0136] Self-assembling nanoparticles containing siRNA, constructed with polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG) as a means of targeting tumor angiogenesis-expressing integrins, can be used to deliver siRNA that inhibits vascular endothelial growth factor receptor 2 (VEGFR2) expression and thus tumor angiogenesis (see, e.g., Schiffel 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 ionizable 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, hence referred to herein as nanoplexes. A dosage of approximately 100-200 mg of CRISPRCas is envisioned for delivery in Schiffelerset al self-assembling nanoparticles.

[0137] 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 volumes of aqueous solutions of cationic polymer and nucleic acid to provide a net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) ranging from 2 to 6. The electrostatic interaction between the cationic polymer and nucleic acid results in the formation of polyplexes with an average particle size distribution of approximately 100 nm, which are thus 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-NHS ester) was ordered from Macrocyclics (Dallas, TX). Amine-modified RNA sense strands were added to a microcentrifuge tube along with a 100-fold molar excess of DOTA-NHS ester in carbonate buffer (pH 9). The contents were allowed to react by stirring at room temperature for 4 hours. The DOTA-RNA sense conjugates were ethanol precipitated, resuspended in water, and annealed with the unmodified antisense strand to yield DOTA-siRNA. All liquids were pretreated with Chelex-100 (Bio-Rad, Hercules, CA) to remove trace metal contamination. Tf-targeted and non-targeted siRNA nanoparticles can be formed using cyclodextrin-containing polycations. Typically, nanoparticles were formed in water at a charge ratio of 3 (±) and an siRNA concentration of 0.5 g / liter. One percent 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.

[0138] Davis et al. (Nature, Vol. 464, April 15, 2010) conducted an siRNA clinical trial using a targeted nanoparticle delivery system (clinical trial registration number NCT00689065). Patients with solid tumors refractory to standard treatment received doses of the targeted nanoparticles via 30-minute intravenous infusion on days 1, 3, 8, and 10 of a 21-day cycle. The nanoparticles consisted of a synthetic delivery system containing: (1) a linear cyclodextrin-based polymer (CDP); (2) a human transferrin protein (TF) targeting ligand displayed on the exterior of the nanoparticle to engage with the TF receptor (TFR) on the surface of cancer cells; (3) a hydrophilic polymer (polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids); and (4) an siRNA designed to reduce the expression of RRM2 (the sequence used in clinical trials was formerly known as siR2B+5). TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anticancer target. These nanoparticles (the clinical version is called CALAA-01) have been shown to be well tolerated in repeated-dose studies in non-human primates. While one patient with chronic myeloid leukemia has received siRNA via liposomal delivery, Davis et al.'s clinical trial is the first human trial to treat patients with solid tumors by systemically delivering siRNA with a targeted delivery system. To determine whether this targeted delivery system can provide effective delivery of functional siRNA to human tumors, Davis et al. examined biopsies from three patients in three different dosing cohorts: Patients A, B, and C (all patients had metastatic melanoma and received 18, 24, and 30 mg / mL siRNA, respectively). -2 (These patients received a CALAA-01 dose of siRNA. Similar doses may be contemplated for the CRISPR-Cas systems of the present invention. Delivery of the present invention may be achieved by nanoparticles containing linear cyclodextrin-based polymers (CDPs), human transferrin protein (TF) targeting ligands displayed on the exterior of the nanoparticles to engage with TF receptors (TFRs) on the surface of cancer cells, and / or hydrophilic polymers (e.g., polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids).

[0139] Exosomes Exosomes are endogenous nanovesicles that transport RNA and proteins and can deliver small interfering (si)RNA to the brain in mice. To reduce immunogenicity, Alvarez-Erviti et al. (2011, NatBiotechnol 29:341) used autologous dendritic cells for exosome production. Targeting was achieved by engineering dendritic cells to express Lamp2b (an exosomal membrane protein) fused to the neuron-specific RVG peptide 3. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes specifically delivered GAPDH siRNA to neurons, microglia, and oligodendrocytes in the brain, resulting in specific gene knockdown. Preexposure to RVG exosomes did not attenuate knockdown, and nonspecific uptake in other tissues was not 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.

[0140] 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 lacking T cell activating factors 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 following day, exosomes were purified from the culture supernatant using a well-established ultracentrifugation protocol. The exosomes produced were physically homogenous, with a size distribution peak of 80 nm in diameter as determined by nanoparticle tracking analysis (NTA) and electron microscopy. Alvarez-Erviti et al. 6 6-12 μg of exosomes (measured based on protein concentration) were obtained per cell.

[0141] Next, Alvarez-Erviti et al. investigated the possibility of loading modified exosomes with exogenous cargo using an electroporation protocol adapted for nanoscale applications. Because electroporation of membrane particles at the nanometer scale has not been well characterized, nonspecific Cy5-labeled siRNA was used for experimental optimization of the electroporation protocol. After ultracentrifugation and lysis of the exosomes, the amount of encapsulated siRNA was assayed. Electroporation at 400 V and 125 μF yielded the greatest siRNA retention and was used for all subsequent experiments.

[0142] Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNA encapsulated in 150 μg RVG exosomes to normal C57BL / 6 mice and compared knockdown efficiency with 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 with RVG-9R, an RVG peptide conjugated with nine D-arginines that electrostatically bind to siRNA. Cortical tissue samples were analyzed 3 days after administration and found significant protein knockdown (45%, P<0.05 vs. 62%, P<0.01) in both siRNA-RVG-9R-treated and siRNA-RVG exosome-treated mice, accompanied by a significant reduction in BACE1 mRNA levels (66% ± 15%, P<0.001 and 61% ± 13%, P<0.01, respectively). Furthermore, Applicants demonstrated a significant reduction (55%, P<0.05) in total β-amyloid 1-42 levels, the major component of amyloid plaques in Alzheimer's pathology, in RVG-exosome-treated animals. The observed reduction was greater than the reduction in β-amyloid 1-40 demonstrated after intracerebroventricular injection of a BACE1 inhibitor in normal mice. Alvarez-Erviti et al. performed rapid amplification of 5'-cDNA ends (RACE) on BACE1 cleavage products, which provided evidence of RNAi-mediated knockdown by siRNA.

[0143] Finally, Alvarez-Erviti et al. investigated whether siRNA-RVG exosomes induced immune responses in vivo by assessing serum concentrations of IL-6, IP-10, TNFα, and IFN-α. In contrast to siRNA-RVG-9R, which potently stimulated IL-6 secretion, siRNA-RVG exosome treatment, as with siRNA-transfection reagent treatment, showed no significant changes in all cytokines, confirming the immunologically inert profile of exosome therapy. 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 higher protein knockdown were achieved with 5-fold less siRNA without corresponding immune stimulation levels. This experiment demonstrates the therapeutic potential of RVG-exosome technology, potentially suitable for long-term silencing of genes associated with neurodegenerative diseases. The exosome delivery system of Alvarez-Erviti et al. can be applied to deliver the CRISPR-Cas system of the present invention to therapeutic targets, particularly neurodegenerative diseases. A dosage of about 100-1000 mg of CRISPR-Cas encapsulated in about 100-1000 mg of RVG exosomes can be contemplated for the present invention.

[0144] El-Andaloussi et al. (Nature Protocols 7, 2112-2126 (2012)) discloses how exosomes derived from cultured cells can be used for siRNA delivery in vitro and in vivo. The protocol first describes the creation of targeted exosomes by transfection of an expression vector containing an exosomal protein fused to a peptide ligand. Next, El-Andaloussi et al. explain how to purify and characterize exosomes from transfected cell supernatants. Next, El-Andaloussi et al. detail the crucial steps for loading siRNA into exosomes. Finally, El-Andaloussi et al. outline how exosomes can be used to efficiently deliver siRNA to the mouse brain in vitro and in vivo. In an example of expected results, exosome-mediated siRNA delivery is evaluated by functional assays, and imaging is also provided. The entire protocol takes approximately three weeks. Delivery or administration according to the present invention may also be carried out using exosomes produced from autologous dendritic cells.

[0145] In another embodiment, plasma exosomes from 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 the inward budding of late endosomes and then released into the extracellular environment upon fusion with the plasma membrane. Exosomes naturally transport RNA between cells, a property that may be useful in gene therapy.

[0146] Exosomes were prepared from plasma by centrifuging the buffy coat at 900g for 20 minutes to isolate the plasma, followed by collection of the cell supernatant, centrifugation at 300g for 10 minutes to remove cells, and centrifugation at 16,500g for 30 minutes, followed by filtration through a 0.22 mm filter. Exosomes were pelleted by ultracentrifugation at 120,000g for 70 minutes. Chemical transfection of exosomes with siRNA was performed using the RNAi Human / Mouse Starter Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. siRNA was added to 100 ml of PBS at a final concentration of 2 mmol / ml. After adding HiPerFect transfection reagent, the mixture was incubated at room temperature for 10 minutes. To remove excess micelles, exosomes were re-isolated using aldehyde / sulfate latex beads. Chemical transfection of CRISPRCas into exosomes can also be performed in the same manner as with siRNA. Exosomes can be co-cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Thus, it is contemplated that exosomes containing CRISPRCas can be introduced into human monocytes and lymphocytes and then reintroduced into humans. Thus, delivery or administration according to the present invention can be carried out using plasma exosomes.

[0147] Liposomes The delivery or administration of the present invention can be carried out using liposomes. Liposomes are spherical vesicular structures composed of a monolayer or multilayer lipid bilayer surrounding an inner 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 and the blood-brain barrier (BBB) ​​(see, for example, Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review).

[0148] Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to create liposomes as drug carriers. Liposome formation occurs spontaneously when a lipid film is mixed with an aqueous solution, but can also be promoted by applying force in the form of shaking using a homogenizer, sonicator, or extrusion device (see, for example, Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review).

[0149] Liposomes may also contain some other additives to improve their structure and properties.For example, to help stabilize the structure of liposomes and prevent the leakage of cargo inside liposomes, either cholesterol or sphingomyelin may be added to the liposome mixture.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 and 100 nm (for example, see Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review).

[0150] Conventional liposome formulations are primarily composed of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialogangliosides. Because these formulations are made solely of phospholipids, liposome formulations face many challenges, one of which is instability in plasma. Several attempts have been made to overcome these challenges, particularly in the area of ​​lipid membrane engineering. One of these attempts focused on the manipulation of cholesterol. The addition of cholesterol to conventional formulations reduces the rapid release of encapsulated bioactive compounds into the plasma or increases the stability of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review).

[0151] In particularly advantageous embodiments, Trojan horse liposomes (also known as molecular Trojan horses) are desirable; protocols can be found at http: / / cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long. These particles allow transgenes to be delivered to the entire brain after intravascular injection. Without being bound by any limitations, it is believed that neutral lipid particles conjugated to specific antibodies on their surface can cross the blood-brain barrier by endocytosis. The applicant hypothesizes that using Trojan horse liposomes to deliver CRISPR nuclease family molecules to the brain via intravascular injection may enable whole-brain transgenic animals without the need for embryonic manipulation. Approximately 1 to 5 g of DNA may be contemplated for in vivo administration in liposomes.

[0152] In another embodiment, the CRISPR Cas system may be administered in a liposome, such as a stable nucleic acid lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injection of about 1, 3, or 5 mg / kg / day of a specific CRISPR Cas targeted in SNALP is contemplated. Daily treatment can be for about 3 days, then weekly for about 5 weeks. In another embodiment, specific CRISPR Cas encapsulated in SNALP administered by intravenous injection at a dose of about 1 or 2.5 mg / kg is also contemplated (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). SNALP formulations can contain the lipids 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-!,2-dimyristyloxypropylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in a 2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

[0153] In another embodiment, stable nucleic acid lipid particles (SNALP) have been shown to be effective in delivering molecules to highly vascularized HepG2-derived liver tumors but not to poorly vascularized HCT-116-derived liver tumors (see, e.g., Li, GeneTherapy (2012) 19, 775-780). SNALP liposomes can be prepared by combining D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), cholesterol, and siRNA using a 25:1 lipid / siRNA ratio and a 48 / 40 / 10 / 2 molar ratio of cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA. The resulting SNALP liposomes are approximately 80-100 nm in size.

[0154] In yet another embodiment, the SNALP can include synthetic cholesterol (Sigma-Aldrich, St. Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010;375:1896-905). A total CRISPRCas dosage of about 2 mg / kg per administered dose can be contemplated, for example, as a bolus intravenous infusion.

[0155] In yet another embodiment, the SNALP can include synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; AvantiPolar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for in vivo testing can include a final lipid / RNA mass ratio of about 9:1.

[0156] The safety profile of RNAi nanomedicines has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, for example, Advanced Drug Delivery Reviews 64 (2012) 1730-1737). Stable nucleic acid lipid particles (SNALPs) are composed of four different lipids: a low-pH cationic ionizable lipid (DLinDMA), a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. The particles are approximately 80 nm in diameter and neutrally charged at physiological pH. During formulation, the ionizable lipid serves to condense the lipid with anionic siRNA during particle formation. Positively charged under increasingly acidic endosomal conditions, the ionizable lipid also mediates fusion of the SNALP with the endosomal membrane, allowing the siRNA to be released into the cytoplasm. The PEG-lipid stabilizes the particles, reducing aggregation during formation and providing a neutral hydrophilic exterior that subsequently improves pharmacokinetic properties.

[0157] To date, two clinical programs have been initiated using SNALP siRNA formulations. Tekmira Pharmaceuticals recently completed a Phase 1 single-dose study of SNALP-ApoB in adult volunteers with high LDL cholesterol. ApoB is primarily expressed in the liver and jejunum and is essential for the assembly and secretion of VLDL and LDL. Seventeen subjects received a single dose of SNALP-ApoB (escalated over seven dose levels). There was no evidence of liver toxicity (anticipated as a potential dose-limiting toxicity based on preclinical studies). One (of two) subjects developed flu-like symptoms consistent with immune system stimulation at the highest dose, leading to the decision to terminate the study.

[0158] Alnylam Pharmaceuticals is also advancing ALN-TTR01, which uses the SNALP technology described above to target hepatocyte production of both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR syndromes have been described: familial amyloidotic neuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC), both caused by autosomal dominant mutations in TTR; and senile systemic amyloidosis (SSA), caused by wild-type TTR. A placebo-controlled, single-ascending-dose phase I study of ALN-TTR01 in patients with ATTR was recently completed. ALN-TTR01 was administered as a 15-minute intravenous infusion to 31 patients (23 receiving the investigational drug and 8 receiving placebo) at doses ranging from 0.01 to 1.0 mg / kg (based on siRNA). Treatment was well tolerated, with no significant increases in liver function tests. Three of 23 patients experienced infusion-related reactions at ≥0.4 mg / kg; all patients responded to a reduced infusion rate and all continued on study. Minimal and transient elevations in serum cytokines IL-6, IP-10, and IL-1ra were observed in two patients at the highest dose of 1 mg / kg (as expected from preclinical studies and NHP studies). A reduction in serum TTR, the expected pharmacodynamic effect of ALN-TTR01, was observed at 1 mg / kg.

[0159] In yet another embodiment, SNALP may be produced by solubilizing cationic lipids, DSPC, cholesterol, and PEG-lipids in ethanol at a molar ratio of 40:10:40:10, respectively (see Semple et al., Nature Biotechnology, Volume 28, Number 2, February 2010, pp. 172-177). This lipid mixture was added with mixing to aqueous buffer (50 mM citrate, pH 4) to final ethanol and lipid concentrations of 30% (vol / vol) and 6.1 mg / ml, respectively, and equilibrated at 22°C for 2 minutes before extrusion. The hydrated lipids were extruded through two stacked 80 nm pore size filters (Nuclepore) using a Lipex extruder (Northern Lipids) at 22°C until vesicle diameters of 70-90 nm were obtained, as determined by dynamic light scattering analysis. This typically required one to three passes. siRNA (solubilized in 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to pre-equilibrated (35°C) vesicles at a rate of approximately 5 ml / min with mixing. After reaching a final target siRNA / lipid ratio of 0.06 (wt / wt), the mixture was incubated at 35°C for an additional 30 min to allow vesicle reconstitution and encapsulation of siRNA. The ethanol was then removed, and the external buffer was exchanged for PBS (155 mM NaCl, 3 mM NaHPO, 1 mM KHPO, pH 7.5) by dialysis or tangential flow diafiltration. siRNA was encapsulated into SNALP using a controlled serial dilution process. The lipid components of KC2-SNALP were DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; AvantiPolar Lipids), synthetic cholesterol (Sigma), and PEG-C-DMA used in a molar ratio of 57.1:7.1:34.3:1.4. Once the loaded particles were formed, the SNALP was dialyzed against PBS and filter-sterilized through a 0.2 μm filter before use.The average particle size was 75-85 nm, and 90-95% of the siRNA was encapsulated within the lipid particles. The final siRNA / lipid ratio of the formulation used for in vivo testing was approximately 0.15 (wt / wt). The LNP-siRNA system containing Factor VII siRNA was diluted to the appropriate concentration in sterile PBS immediately before use, and the formulation was administered intravenously via the lateral tail vein in a total volume of 10 ml / kg. This method can be extended to the CRISPR Cas system of the present invention.

[0160] Other lipids Other cationic lipids, such as the aminolipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), can be used to encapsulate CRISPR Cas similarly to siRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533). Preformed vesicles with the following lipid composition can be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate (PEG-lipid) in a molar ratio of 40 / 10 / 40 / 10, respectively, and a FVII siRNA / total lipid ratio of approximately 0.05 (w / w). To ensure a narrow particle size distribution in the 70-90 nm range and a low polydispersity index of 0.11-0.04 (n = 56), particles can be extruded up to three times through an 80 nm membrane before adding CRISPR-Cas RNA. Particles containing highly potent amino lipids 16 may also be used, where the molar ratio of the four lipid components 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5), can be further optimized to enhance in vivo activity.

[0161] Michael S.D. Kormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA in mice": Nature Biotechnology, Volume: 29, Pages: 154-157 (2011), published online January 9, 2011) describe the delivery of RNA using a lipid envelope. The use of a lipid envelope is also preferred in the present invention.

[0162] In another embodiment, lipids may be combined with the CRISPR Cas system of the present invention to form lipid nanoparticles (LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and the colipid distearoylphosphatidylcholine, cholesterol, and PEG-DMG, and may be combined with CRISPR Cas instead of siRNA using a spontaneous vesicle formation approach (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi:10.1038 / mtna.2011.3). The molar ratio of the components may be approximately 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / disteroylphosphatidylcholine / cholesterol / PEG-DMG). The final lipid: siRNA weight ratio can be about 12:1 and 9:1 for DLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively. The formulations can have a mean particle diameter of about 80 nm and an entrapment efficiency of greater than 90%. A 3 mg / kg dose can be contemplated.

[0163] Tekmira has a portfolio of approximately 95 patent families in the United States and abroad that relate to various aspects of LNPs and LNP formulations (e.g., U.S. Patent Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; See US Pat. Nos. 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and EP Patent Nos. 1766035; 1519714; 1781593 and 1664316), all of which may be used and / or adapted to the present invention.

[0164] The CRISPR Cas system may be delivered encapsulated in PLGA microspheres, such as those further described in U.S. Patent Application Publication Nos. 20130252281, 20130245107, and 20130244279 (assigned to Moderna Therapeutics), which relate to formulations of compositions containing modified nucleic acid molecules capable of encoding proteins, protein precursors, or partially or fully processed forms of proteins or protein precursors. The formulations may have a molar ratio of 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipid may be selected from, but not limited to, PEG-c-DOMG and PEG-DMG. The fusogenic lipid may be DSPC. See also Schrum et al., "Delivery and Formulation of Engineered Nucleic Acids," U.S. Patent Application Publication No. 20120251618.

[0165] Nanomerics' technology addresses the bioavailability challenges of a wide range of therapeutics, including small hydrophobic drugs, peptides, and nucleic acid-based therapeutics (plasmids, siRNA, miRNA). Specific routes of administration where this technology has demonstrated clear advantages include oral administration, transport across the blood-brain barrier, solid tumors, and ocular delivery. See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb 26;7(2):1016-26; Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10; and Lalatsa et al., 2012, J Control Release. 2012 Jul 20;161(2):523-36.

[0166] U.S. Patent Application Publication No. 20050019923 describes cationic dendrimers for delivering bioactive molecules, such as polynucleotide molecules, peptides and polypeptides, and / or pharmaceuticals, to the mammalian body. The dendrimers are suitable for targeting the delivery of bioactive molecules to, for example, the liver, spleen, lungs, kidneys, or heart. Dendrimers are synthetic three-dimensional macromolecules prepared in a stepwise manner from simple branched monomer units, allowing their properties and functionality to be easily controlled and varied. Dendrimers are synthesized by repeatedly adding building blocks to (divergent synthesis approach) or toward (convergent synthesis approach) a multifunctional core, with each addition of a three-dimensional shell of building blocks resulting in the formation of a higher-generation dendrimer. Polypropyleneimine dendrimers begin with a diaminobutane core, to which double the number of amino groups are added by a double Michael addition of acrylonitrile to a primary amine followed by hydrogenation of the nitrile, resulting in doubling of the amino groups. Polypropyleneimine dendrimers contain 100% protonatable nitrogen and up to 64 terminal amino groups (5th generation, DAB64). The protonatable groups are typically amine groups capable of accepting protons at neutral pH. The use of dendrimers as gene delivery agents has largely focused on the use of polyamidoamines and phosphorous-containing compounds with amine / amide mixtures or NP(O2)S as conjugation units, respectively; no studies have been reported on the use of lower-generation polypropyleneimine dendrimers for gene delivery. Polypropyleneimine dendrimers have also been investigated as pH-sensitive controlled-release systems for drug delivery and their encapsulation of guest molecules when chemically modified with peripheral amino acid groups. The cytotoxicity and interaction of polypropyleneimine dendrimers with DNA, as well as the transfection efficacy of DAB64, have also been investigated.

[0167] U.S. Patent Application Publication No. 20050019923 is based on the observation that, contrary to previous reports, cationic dendrimers, such as polypropyleneimine dendrimers, exhibit properties such as specific targeting and low toxicity that make them suitable for use in the targeted delivery of bioactive molecules, such as genetic material. In addition, derivatives of cationic dendrimers also exhibit properties that make them suitable for the targeted delivery of bioactive molecules. See also "Bioactive Polymers," U.S. Patent Application Publication No. 20080267903, which discloses that "various polymers, including cationic polyamine polymers and dendrimer polymers, have been shown to have antiproliferative activity and may therefore be useful in the treatment of disorders characterized by unwanted cell proliferation, such as neoplasms and tumors, inflammatory disorders (including autoimmune disorders), psoriasis, and atherosclerosis. Such polymers may be used alone as active agents or as delivery vehicles for other therapeutic agents, e.g., drug molecules or nucleic acids for gene therapy. In such cases, the intrinsic anti-tumor activity of the polymer itself may complement the activity of the delivered agent."

[0168] supercharged protein Supercharged proteins are a class of engineered or naturally occurring proteins that typically have a high positive or negative net theoretical charge. Both extremely negatively and extremely positively charged proteins exhibit a remarkable ability to resist thermally or chemically induced aggregation. Extremely positively charged proteins are also capable of penetrating mammalian cells. By associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, it may be possible to functionally deliver these macromolecules to mammalian cells both in vitro and in vivo. David Liu's laboratory reported the creation and characterization of supercharged proteins in 2007 (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110-10112).

[0169] Nonviral delivery of siRNA and plasmid DNA to mammalian cells is valuable for both research and therapeutic applications (Akince et al., 2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or other highly positively charged protein) is mixed with siRNA in an appropriate serum-free medium and allowed to form complexes before adding cells. The inclusion of serum at this stage inhibits the formation of supercharged protein-siRNA complexes and reduces the efficacy of the treatment. The following protocol has been found to be effective for various cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116). However, pilot experiments varying the protein and siRNA doses should be performed to optimize the procedure for a particular cell line. (1) On the day before treatment, 1 × 10 cells were placed in a 48-well plate. 5 Plate the cells. (2) On the day of treatment, dilute purified +36 GFP protein in serum-free medium to a final concentration of 200 nM. Add siRNA to a final concentration of 50 nM. Vortex to mix and incubate at room temperature for 10 minutes. (3) During incubation, aspirate the medium from the cells and wash once with PBS. (4) After incubation of +36GFP and siRNA, the protein-siRNA complex is added to the cells. (5) The cells are incubated with the complex at 37°C for 4 hours. (6) After incubation, aspirate the medium and wash three times with 20 U / mL heparin PBS. Incubate the cells with serum-containing medium for an additional 48 hours or longer depending on the knockdown assay. (7) Analyze the cells by immunoblot, qPCR, phenotypic assay, or other appropriate method.

[0170] David Liu's laboratory has further found that +36GFP is an effective plasmid delivery reagent in a variety of cells. Because plasmid DNA is a larger cargo than siRNA, proportionally more +36GFP protein is required to effectively complex the plasmid. For effective plasmid delivery, applicants have developed a variant of +36GFP with a C-terminal HA2 peptide tag, a known endosome-disrupting peptide derived from the influenza virus hemagglutinin protein. While the following protocol has been effective in a variety of cells, it is recommended that the dosage of plasmid DNA and supercharged protein be optimized for specific cell lines and delivery applications, as described above. (1) On the day before treatment, 1 × 10 cells were placed in a 48-well plate. 5 Plate the cells. (2) On the day of treatment, dilute purified p36 GFP protein in serum-free medium to a final concentration of 2 mM. Add 1 mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10 minutes. (3) During incubation, aspirate the medium from the cells and wash once with PBS. (4) After incubation of p36 GFP and plasmid DNA, the protein-DNA complex is gently added to the cells. (5) The cells are incubated with the complex at 37°C for 4 hours. (6) After incubation, aspirate the medium and wash with PBS. Incubate the cells in serum-containing medium for an additional 24-48 hours. (7) Analyze plasmid delivery (e.g., by plasmid drive gene expression) as needed.

[0171] See, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752 (2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011); Thompsons et al., Methods in Enzymology 503, 293-319 (2012); Thompson, DB, et al., Chemistry & Biology 19(7), 831-843 (2012). This method of supercharged proteins can be used and / or adapted for delivery of the CRISPR Cas system of the present invention.

[0172] Implantable Devices In another embodiment, implantable devices are also contemplated for delivering the CRISPR-Cas system. For example, US Patent Application Publication No. 20110195123 discloses that implantable medical devices for localized, long-term drug elution are provided, including several types of such devices, treatment embodiments, and implantation methods. The device is composed of a polymer substrate, such as a matrix, used as the device body, a drug, and in some cases, an additional scaffolding material, such as a metal or another polymer, as well as a material for enhancing visibility and imaging. The drug is selected based on the advantage of localized, long-term drug release, where the drug is released directly to the extracellular matrix (ECM) of affected areas such as tumors, inflammation, and degeneration, or for symptomatic purposes, or to damaged smooth muscle cells, or for prophylactic purposes. Some drugs are gene silencing drugs based on RNA interference (RNAi), including, but not limited to, siRNA, shRNA, or antisense RNA / DNA, ribozymes, and nucleoside analogs. Therefore, this system can be used and / or adapted to the CRISPR-Cas system of the present invention. In some embodiments, the implantation method is an existing implantation procedure currently being developed and used for other treatments, including brachytherapy and needle biopsy. In such cases, the dimensions of the new implant described in this invention are similar to the original implant. Typically, several devices are implanted during the same treatment procedure.

[0173] As described in US Patent Application Publication No. 20110195123, a drug delivery implantable or insertable system is provided, including a system applicable to a body cavity such as the peritoneal cavity, and / or any other type of administration involving a biostable and / or degradable and / or bioabsorbable polymer substrate (e.g., which may optionally be a matrix) where the drug delivery system is not tethered or attached. It should be noted that the term "insertion" also includes implantation. The drug delivery system is preferably implanted as a "loader" as described in US Patent Application Publication No. 20110195123.

[0174] The polymer or polymers are biocompatible, incorporate one or more drugs, and allow for the release of the drug at a controlled rate, where the total volume of the polymer substrate, e.g., matrix, is optionally and preferably in some embodiments no greater than the maximum volume that allows for reaching therapeutic levels of the drug. As a non-limiting example, such volume may be less than or equal to 0.1 m, as required by the volume for drug loading. 3 ~1000mm 3 The loader may in some cases be larger, for example, when incorporated into devices where size is dictated by functionality, such as, and without limitation, knee joints, intrauterine or cervical rings.

[0175] In some embodiments, drug delivery systems (for delivering compositions) are preferably designed to use degradable polymers, where the primary release mechanism is bulk erosion; or in some embodiments, non-degradable or slowly degrading polymers are used, where the primary release mechanism is diffusion rather than bulk erosion, with the outer portion acting as a membrane and the inner portion acting as a drug reservoir that is substantially unaffected by the environment for an extended period of time (e.g., from about one week to about several months). Combinations of different polymers with different release mechanisms may also be used. The concentration gradient at the surface preferably remains substantially constant for a significant portion of the total drug release period, and thus the diffusion rate is substantially constant (referred to as "zero-mode" diffusion). The term "constant" means that the diffusion rate preferably remains above the lower threshold for therapeutic efficacy, but may still optionally be characterized by an initial burst and / or may fluctuate, e.g., somewhat more or less. The diffusion rate is preferably maintained so over an extended period, which is considered to be somewhat constant to optimize the therapeutically effective period, e.g., the effective silencing period.

[0176] The drug delivery system is optionally and preferably designed to protect the nucleotide-based therapeutic agent from degradation, whether chemical in nature or due to attack from enzymes and other factors in the subject's body.

[0177] Drug delivery systems as described in US Patent Application Publication No. 20110195123 optionally involve sensing and / or actuation instruments that operate during and / or after implantation of the device, for example, optionally by non-invasive and / or minimally invasive actuation and / or acceleration / deceleration methods including, but not limited to, thermal heating and cooling, laser beams, and ultrasound and / or RF (radio frequency) methods or devices, including focused ultrasound.

[0178] According to some embodiments of U.S. Patent Application Publication No. 20110195123, local delivery sites can optionally include target sites characterized by high levels of cellular abnormal proliferation and inhibited apoptosis, such as tumors, active and / or chronic inflammation and infection, e.g., autoimmune conditions, degenerated tissues, e.g., muscle and nerve tissue, chronic pain, degenerative sites, and sites of fractures and other wounds where tissue regeneration is enhanced, as well as damaged cardiac, smooth, and striated muscle. Local delivery sites also optionally include sites that allow for prophylactic activities, including prevention of pregnancy, infection, and aging.

[0179] The implantation site, i.e., the target site, of the composition is preferably characterized by a radius, area, and / or volume that is sufficiently small for targeted local delivery. For example, the target site optionally has a diameter ranging from about 0.1 mm to about 5 cm.

[0180] The location of the target site is preferably selected to maximize therapeutic effectiveness, for example, the drug delivery system composition (optionally with an implantable device as described above) is optionally and preferably implanted within or adjacent to the tumor environment or its associated blood supply.

[0181] For example, the composition (optionally with a device) is optionally implanted in or adjacent to the pancreas, prostate, breast, liver, within the vascular system using a nipple, or the like.

[0182] The target location is optionally selected from the group consisting of (by way of non-limiting example only, as any site in the body may optionally be suitable for implantation of the Loder): 1. the brain, in areas of degeneration such as in Parkinson's or Alzheimer's disease, in the basal ganglia, white matter, and gray matter; 2. the spine, such as in amyotrophic lateral sclerosis (ALS); 3. the cervix to prevent HPV infection; 4. active and chronically inflamed joints; 5. the dermis, such as in psoriasis; 6. the sympathetic and sensory (s) nerves for analgesic effects. ensoric nerve sites; 7. intraosseous implants; 8. sites of acute and chronic infection; 9. intravaginal; 10. inner ear - auditory system, labyrinth of the inner ear, vestibular system; 11. intratracheal; 12. intracardiac; coronary arteries, epicardium; 13. bladder; 14. biliary system; 15. parenchymal tissues, for example, but not limited to, kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18. gums; 19. intra-articular (inside joints); 20. intraocular; 21. brain tissue; 22. ventricles; 23. body cavities, for example, the peritoneal cavity (for example, but not limited to, ovarian cancer); 24. intraesophageal and 25. intrarectal.

[0183] In some cases, insertion of the system (e.g., a device containing the composition) involves injecting material into the ECM at and near the target site, affecting the local pH and / or temperature at and near the target site and / or other biological factors that affect the diffusion and / or pharmacokinetics of the drug in the ECM.

[0184] Optionally, according to some embodiments, the release of the agent may involve sensing and / or actuating instruments that operate before and / or during and / or after insertion by non-invasive and / or minimally invasive and / or other actuating and / or acceleration / deceleration methods, including laser beams, radiation, thermal heating and cooling, and ultrasound, e.g., focused ultrasound and / or RF (radio frequency) methods or devices, and chemical activators.

[0185] According to other embodiments of US Patent Application Publication No. 20110195123, the drug preferably comprises a gene-silencing biological RNAi drug, as described below, for example, for cases of localized cancer in the breast, pancreas, brain, kidney, bladder, lung, and prostate. Furthermore, many drugs other than siRNA are amenable to encapsulation in a Loder and can be used in connection with this invention, provided such drugs can be encapsulated in a Loder substrate, such as a matrix. Such drugs include approved drugs currently delivered by methods other than those of this invention, including amphotericin B for fungal infections in Loders implanted near the spine for back pain; antibiotics for osteomyelitis; analgesics such as anesthetics; and anti-degenerative drugs for Alzheimer's disease or Parkinson's disease. Such systems can be used and / or adapted for delivery of the CRISPRCas system of the present invention.

[0186] For example, for certain applications, such as preventing the growth or regrowth of smooth muscle cells (which are damaged during stent placement procedures and tend to proliferate as a result), the drug may be an siRNA that silences smooth muscle cells, including H19 silencing, or a drug selected from the group consisting of taxol, rapamycin, and rapamycin analogs. In this case, the loader is preferably a drug-eluting stent (DES) that releases drugs at a constant rate over time, or a dedicated device that is implanted separately and associated with the stent. All of these can be used and / or adapted for use with the CRISPRCas system of the present invention.

[0187] Another example of specific application is the development of neurodegenerative diseases of the muscles and nerves due to abnormal gene expression.Local delivery of silencing RNA can have therapeutic properties for interfering with such abnormal gene expression.Local delivery of anti-apoptotic, anti-inflammatory and anti-degenerative drugs, including small molecule drugs and macromolecules, can also be effective in some cases.In this case, Loader is applied for long-term release at a constant rate and / or is applied via a dedicated device that is implanted separately.All of these can be used and / or adapted to the CRISPRCas system of the present invention.

[0188] As another example of specific application, psychiatric and cognitive disorders are treated with gene modifiers.Genetic knockdown by silencing RNA is a treatment option.Loder, which delivers nucleotide-based drugs locally to central nervous system sites, is a treatment option for psychiatric and cognitive disorders, including but not limited to psychosis, bipolar disorder, neurotic disorder and behavioral illness.Loder can also be implanted in specific brain sites and locally deliver drugs, including small molecule drugs and macromolecules.All of these can be used and / or adapted to the CRISPRCas system of the present invention.

[0189] As another example of a specific application, silencing innate and / or adaptive immune mediators at the local site can prevent transplant rejection. Local delivery of silencing RNA and immunomodulatory reagents by Loders implanted in the transplanted organ and / or transplant site activates local immunosuppression by repelling immune cells such as CD8. All of these can be used and / or adapted to the CRISPRCas system of the present invention.

[0190] As another example of a specific application, vascular growth factors, including VEGF and angiogenin, are essential for angiogenesis. Local delivery of factors, peptides, and peptidomimetics, or inhibition of their repressors, are important therapeutic modalities; local delivery of angiogenesis-stimulating factors, peptides, macromolecular and small molecule drugs and silencing of their repressors have therapeutic effects on peripheral, systemic, and cardiovascular diseases.

[0191] The method of insertion, e.g., implantation, may be one that is already used for other types of tissue transplantation and / or insertion and / or tissue harvesting, optionally without modification of such method or optionally with only minor modifications, optionally including, but not limited to, brachytherapy, biopsy, endoscopy with and / or without ultrasound, e.g., ERCP, stereotactic methods into brain tissue, laparoscopy, e.g., laparoscopic implantation into joints, abdominal organs, bladder wall, and body cavities.

[0192] CRISPR enzyme mRNA and guide RNA The CRISPR enzyme mRNA and guide RNA may also be delivered separately. To allow time for the CRISPR enzyme to be expressed, the CRISPR enzyme mRNA may be delivered before the guide RNA. The CRISPR enzyme mRNA may be administered 1 to 12 hours (preferably about 2 to 6 hours) before the guide RNA.

[0193] Alternatively, the CRISPR enzyme mRNA and 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 initial administration of the CRISPR enzyme mRNA + guide RNA.

[0194] To achieve the most efficient level of genome modification, additional administration of CRISPR enzyme mRNA and / or guide RNA may be useful.

[0195] To minimize toxicity and off-target effects, it is important to control the concentration of the delivered CRISPR enzyme mRNA and guide RNA. The optimal concentration of CRISPR enzyme mRNA and guide RNA can be determined by testing various concentrations in cells or animal models and using deep sequencing to analyze the degree of modification at potential off-target genomic loci. For example, for a guide sequence targeting 5'-GAGTCCGAGCAGAAGAAGAA-3' of the EMX1 gene in the human genome, deep sequencing can be used to evaluate the modification levels at the following two off-target loci: 1:5'-GAGTCCTAGCAGGAGAAGAA-3' and 2:5'-GAGTCTAAGCAGAAGAAGAA-3'. The concentration that achieves the highest on-target modification level while minimizing off-target modification level should be selected for in vivo delivery.

[0196] Alternatively, to minimize toxicity levels and off-target effects, the CRISPR enzyme nickase mRNA (e.g., S. pyogenes Cas9 with a D10A mutation) can be delivered along with a pair of guide RNAs targeting the site of interest. The two guide RNAs must be separated as follows: guide sequences in red (single underlined) and blue (double underlined), respectively (these examples are based on the PAM requirements of Streptococcus pyogenes Cas9).

[0197] [Table 2]

[0198] [Table 3]

[0199] [Table 4]

[0200] [Table 5]

[0201] [Table 6]

[0202] [Table 7]

[0203] Further investigation of this system has provided Applicants with evidence of a 5' overhang (see, e.g., Ranet et al., Cell. 2013 Sep 12;154(6):1380-9 and U.S. Provisional Patent Application No. 61 / 871,301, filed August 28, 2013). Applicants have further identified parameters associated with efficient cleavage by Cas9 nickase mutants in combination with two guide RNAs, including, but not limited to, the length of the 5' overhang. In embodiments 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 embodiments 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 or 1-34 base pairs. In another preferred method of the invention, a first guide sequence directs cleavage of one strand of a DNA duplex near a first target sequence, and a second guide sequence directs cleavage of the other strand near a second target sequence, resulting in a blunt end or a 3' overhang. In embodiments of the invention, the 3' overhang is at most 150, 100, or 25 base pairs, or at least 15, 10, or 1 base pair. In preferred embodiments, the 3' overhang is 1 to 100 base pairs.

[0204] Aspects of the invention relate to reducing the expression of a gene product, or introducing a template polynucleotide into a DNA molecule encoding the gene product, or precisely excising an intervening sequence by reannealing and ligating two 5' overhangs, or altering the activity or function of a gene product, or increasing the expression of a gene product. In one embodiment of the invention, the gene product is a protein.

[0205] Only sgRNA pairs that create 5' overhangs with less than 8 bp overlap (greater than -8 bp offset) between the guide sequences were able to mediate detectable indel formation. Importantly, each guide used in these assays was able to efficiently induce indels when paired with wild-type Cas9, indicating that the relative position of the guide pair is the most important parameter in predicting double-nicking activity.

[0206] Because Cas9n and Cas9H840A nick opposite strands of DNA, replacement of Cas9n with Cas9H840A with a given sgRNA pair should result in an inversion of the overhanging sequence. For example, an sgRNA pair that can generate a 5' overhang with Cas9n should, in principle, generate a corresponding 3' overhang instead. Thus, an sgRNA pair that results in the generation of a 3' overhang with Cas9n could be used to generate a 5' overhang with Cas9H840A. Unexpectedly, we tested Cas9H840A with a set of sgRNA pairs designed to generate both 5' and 3' overhangs (offset range -278 to +58 bp) but did not observe indel formation. Further research may be required to identify the design criteria necessary to combine sgRNA pairs to enable double nicking by Cas9H840A.

[0207] Liver, proprotein convertase subtilisin kexin 9 (PCSK9) Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. PCSK9 is primarily expressed by the liver and is critically important in downregulating hepatocyte LDL receptor expression. Plasma LDL-C levels are significantly elevated in individuals with PCSK9 gain-of-function mutations, classifying them as having severe hypercholesterolemia. Therefore, PCSK9 is an attractive target for CRISPR. CRISPR targeting PCS9K can be formulated into lipid particles and administered intravenously at, for example, approximately 15, 45, 90, 150, 250, and 400 μg / kg (see, e.g., http: / / www.alnylam.com / capella / wp-content / uploads / 2013 / 08 / ALN-PCS02-001-Protocol-Lancet.pdf).

[0208] Bailey et al. (J Mol Med (Berl). 1999 Jan;77(1):244-9) discloses insulin delivery by ex vivo somatic cell gene therapy, which involves removing non-B cell somatic cells (e.g., fibroblasts) from a diabetic patient and genetically altering them in vitro to produce and secrete insulin. The cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way can be evaluated before transplantation, and backup stocks can be cryopreserved. Because the patient's own cells are used, this procedure should obviate the need for immunosuppression and eliminate tissue supply issues while avoiding repeated cell destruction. Ex vivo somatic cell gene therapy requires an accessible and robust cell type that is suitable for multiple transfections and amenable to controlled proliferation. Particular problems associated with the use of non-B cell somatic cells include the processing of proinsulin to insulin and the conferring of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release. Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells, and ovarian (CHO) cells suggest that these challenges may be addressed and that ex vivo somatic cell gene therapy may provide a feasible approach to insulin replacement therapy. The Baileye et al. system can be used and / or adapted for delivery of the CRISPRCas system of the present invention to the liver.

[0209] The method of Sato et al. (Nature Biotechnology, Volume 26, Number 4, April 2008, Pages 431-442) can be applied to deliver the CRISPR-Cas system of the present invention to the liver. Sato et al. found that treatment with vitamin A-conjugated liposomes carrying siRNA almost completely resolved liver fibrosis and extended survival in rats with liver cirrhosis induced by otherwise lethal dimethylnitrosamine in a dose- and duration-dependent manner. Cationic liposomes (Lipotrust) containing O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14) as the cationic lipid, cholesterol, and dioleoylphosphatidylethanolamine in a 4:3:3 molar ratio (which has shown high transfection efficiency under serum-supplemented conditions for in vitro and in vivo gene delivery) were purchased from Hokkaido System Science. These liposomes were prepared using the freeze-dried empty liposome method and adjusted to a concentration of 1 mM (DC-16-4) by adding double-distilled water (DDW) to the freeze-dried lipid mixture under vortexing before use. To prepare VA-conjugated liposomes, 200 nmol of vitamin A (retinol, Sigma) dissolved in DMSO was mixed with the liposome suspension (100 nmol as DC-16-4) in a 1.5 ml tube by vortexing at 251°C. To prepare VA-conjugated liposomes carrying siRNAgp46 (VA-lip-siRNAgp46), a solution of siRNAgp46 (580 pmol / ml in DDW) was added to the retinol-conjugated liposome solution with stirring at 25°C. The siRNA to DC-16-4 ratio was 1:11.5 (mol / mol), and the siRNA to liposome ratio (wt / wt) was 1:1. Any free vitamin A or siRNA not incorporated into the liposomes was separated from the liposome preparation using a microdispensing system (VIVASPIN2 concentrator 30,000 MWCO PES, VIVASCIENCE). The liposome suspension was added to a filter and centrifuged three times at 1,500 g for 5 min at 25°C.Fractions were collected, and the material trapped on the filter was reconstituted with PBS to reach the desired dose for in vitro or in vivo use. Rats were administered three injections of 0.75 mg / kg siRNA every other day. The Sato et al. system can be used and / or adapted for hepatic delivery of the CRISPRCas system of the present invention by delivering approximately 0.5-1 mg / kg of CRISPRCas RNA in liposomes as described by Sato et al. in humans.

[0210] The method of Rozema et al. (PNAS, August 7, 2007, vol. 104, no. 32) for a vehicle for delivering siRNA to hepatocytes both in vitro and in vivo (which Rozema et al. termed siRNA dynamic polyconjugates) can also be applied to the present invention. Key features of this dynamic polyconjugate technology include membrane-active polymers, the ability to reversibly mask the activity of the polymer until it reaches the acidic environment of the endosome, and the ability to specifically target the modified polymer and its siRNA cargo to hepatocytes in vivo after simple low-pressure intravenous infusion. 5'-amine-modified siRNA is synthesized by reacting 1 weight equivalent (wteq) of N-succinimidyl-S-acetylthioacetate (SATA) reagent (Pierce) with 0.36 wteq of NaHCO3 in water at 4°C for 16 hours. The modified siRNA is then precipitated by adding 9 volumes of ethanol, which is then incubated at 80°C for 2 hours. The precipitate was resuspended in 1x siRNA buffer (Dharmacon) and quantified by measuring absorbance at 260 nm. PBAVE (30 mg / ml in 5 mM TAPS, pH 9) was modified by adding 1.5 wt% SMPT (Pierce). After 1 hour of incubation, 0.8 mg of SMPT-PBAVE was added to 400 μl of isotonic glucose solution containing 5 mM TAPS (pH 9). 50 μg of SATA-modified siRNA was added to this solution. For dose-response experiments with constant [PBAVE], various amounts of siRNA were added. The mixture was then incubated for 16 hours. 5.6 mg of Hepes free base was then added to this solution, followed by a mixture of 3.7 mg of CDM-NAG and 1.9 mg of CDM-PEG. The solution was then incubated at room temperature for at least 1 hour before injection. CDM-PEG and CDM-NAG are synthesized from acid chlorides generated by using oxalyl chloride.Addition of 1.1 molar equivalents of polyethylene glycol monomethyl ether (average molecular weight 450) to this acid chloride yields CDM-PEG, or addition of (aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-β-D-glucopyranoside yields CDM-NAG. The final product is purified using reverse-phase HPLC with a 0.1% TFA water / acetonitrile gradient. Approximately 25-50 μg of siRNA was delivered to mice. The Rozema et al. system can be adapted for liver delivery of the CRISPR Cas system of the present invention, for example, by assuming a dosage of approximately 50-200 mg of CRISPR Cas for human delivery.

[0211] bone Oakes and Lieberman (Clin OrthopRelat Res. 2000 Oct; (379 Suppl): S101-12) discuss gene delivery to bone. By transferring genes to cells at specific anatomical sites, the osteoinductive properties of growth factors can be used at physiological doses and for sustained periods to promote a more significant healing response. The specific anatomical site, bone quality, and soft tissue envelope influence the selection of target cells for local gene therapy. Gene therapy vectors delivered to the treatment site in osteoinductive carriers have produced promising results. Several researchers have shown exciting results using ex vivo and in vivo local gene therapy in animal models. Such systems can be used and / or adapted for delivery of the CRISPRCas system to bone.

[0212] brain The brain delivery option includes CRISPR enzyme and guide RNA in either DNA or RNA form, encapsulated in liposome, and conjugated with molecular Trojan horse, and then delivered through the blood-brain barrier (BBB).Molecular Trojan horse has been shown to be effective in delivering B-gal expression vector to the brain of non-human primates.The same method can be used to deliver the vector that contains CRISPR enzyme and guide RNA. For example, Xia CF and Boado RJ, Pardridge WM ("Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology," Mol Pharm. 2009 May-Jun;6(3):747-51. doi:10.1021 / mp800194) describe how the combination of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology enables delivery of small interfering RNA (siRNA) to cells in culture and in vivo. The authors also report that the bond between the targeting mAb and siRNA is stable with avidin-biotin technology, and that RNAi effects can be observed in distant sites, such as the brain, in vivo after intravenous administration of the targeted siRNA.

[0213] Zhang et al. (Mol Ther. 2003 Jan;7(1):11-8) described how an expression plasmid encoding a reporter such as luciferase was encapsulated inside an "artificial virus" composed of 85 nm PEGylated immunoliposomes that were targeted in vivo to the rhesus monkey brain by a monoclonal antibody (MAb) against the human insulin receptor (HIR). After intravenous injection, the HIRMAb enabled liposomes carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across neuronal membranes. The level of luciferase gene expression in the brain was 50-fold higher in rhesus monkeys compared with rats. Widespread neuronal expression of the β-galactosidase gene in the primate brain was demonstrated by both histochemistry and confocal microscopy. The authors demonstrated that this technique enabled reversible adult transgenic development within 24 hours. Therefore, the use of immunoliposomes is preferred. They are used in conjunction with antibodies to target specific tissues or cell surface proteins.

[0214] Other delivery means or RNA are also preferred, such as via nanoparticles (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 exosomes (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).

[0215] Indeed, exosomes have been shown to be particularly useful in the delivery of siRNA, a system somewhat similar 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) describe how exosomes are a promising tool for drug delivery across various biological barriers and can be used to deliver siRNA in vitro and in vivo. The authors' approach involves generating targeted exosomes by transfection with an expression vector containing an exosomal protein fused to a peptide ligand. The exosomes are then purified and characterized from the supernatant of transfected cells, and then loaded with siRNA. Delivery or administration according to the present invention can be carried out with exosomes, particularly to, but not limited to, the brain.

[0216] Vitamin E (α-tocopherol) can be conjugated to CRISPR Cas and delivered to the brain with high-density lipoprotein (HDL), similar to the method used by Uno et al. (HUMAN GENETHERAPY 22:711-719 (June 2011)) to deliver small interfering RNA (siRNA) to the brain. Mice were infused using 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). A brain infusion cannula was placed approximately 0.5 mm posterior to bregma on the midline to infuse into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce similar target reduction as the same ICV infusion method. Similar dosages of CRISPRCas conjugated to α-tocopherol and co-administered with brain-targeted HDL may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 μmol of CRISPRCas targeted to the brain.

[0217] Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) described a lentivirus-mediated delivery method of short hairpin RNA targeting PKCγ for in vivo gene silencing in the rat spinal cord. They administered 1 × 10 RNA by intrathecal catheter. 9 Approximately 10 μl of recombinant lentivirus was administered, with a titer of transducing units (TU) / ml. Similar dosages of CRISPRCas expressed in lentiviral vectors targeted to the brain can be contemplated for humans in the present invention, e.g., 1×10 9 Approximately 10-50 ml of CRISPRCas targeted to the brain in lentivirus with a titer of transducing units (TU) / ml can be contemplated.

[0218] Targeted deletion, therapeutic application Targeted deletion of genes is preferred. Examples are shown in Example 18. Therefore, among other disorders, genes involved in cholesterol biosynthesis, fatty acid biosynthesis, and other metabolic diseases, genes encoding misfolded proteins involved in amyloid diseases and other diseases, oncogenes that cause cell transformation, latent viral genes, and genes that cause dominant-negative disorders are preferred. As exemplified herein, the applicants prefer to use either viral or nanoparticle delivery systems to deliver genes to the liver, brain, eye, epithelium, hematopoietic, or other tissues of subjects or patients in need who suffer from metabolic diseases, amyloidosis and protein aggregation-related diseases, cell transformation caused by gene mutations and translocations, dominant-negative effects of gene mutations, latent viral infections, and other related conditions.

[0219] Therapeutic applications of the CRISPR-Cas system include glaucoma, amyloidosis, and Huntington's disease, which are exemplified in Example 20, and the features described therein, either alone or in combination, are preferred.

[0220] Another example of a polyglutamine expansion disease that can be treated by the present invention is spinocerebellar ataxia type 1 (SCA1). Intracerebellar injection of a recombinant adeno-associated virus (AAV) vector expressing a short hairpin RNA significantly improves motor coordination, restores cerebellar morphology, and resolves the characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice (see, for example, Xia et al., Nature Medicine, Vol. 10, No. 8, August 2004). Specifically, AAV1 and AAV5 vectors are preferred, with approximately 1 x 10 12 An AAV titer of vector genomes / ml is desirable.

[0221] As an example, chronic infection with HIV-1 can be treated or prevented. To achieve this, CRISPR-Cas guide RNAs can be engineered to target the majority of the HIV-1 genome, taking into account HIV-1 strain variants to maximize coverage and efficacy. Delivery of the CRISPR-Cas system can be traditionally achieved through adenovirus- or lentivirus-mediated infection of the host's immune system. Depending on the approach, host immune cells can be a) isolated, transduced with CRISPR-Cas, selected, and reintroduced into the host, or b) transduced in vivo by systemic delivery of the CRISPR-Cas system. The first approach allows for the creation of a resistant immune population, while the second approach is more likely to target latent viral reservoirs within the host. This is discussed in more detail in the Examples section.

[0222] In another example, U.S. Patent Application Publication No. 20130171732, assigned to Sangamo BioSciences, Inc., relates to the insertion of an anti-HIV transgene into the genome, and this method may be applied to the CRISPRCas system of the present invention. In another embodiment, the CXCR4 gene may be targeted, and the TALE system of U.S. Patent Application Publication No. 20100291048, assigned to Sangamo BioSciences, Inc., may be modified for the CRISPRCas system of the present invention. The methods of U.S. Patent Application Publication Nos. 20130137104 and 20130122591, assigned to Sangamo BioSciences, Inc. and U.S. Patent Application Publication No. 20100146651, assigned to Cellectis, relate to the modification of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to increase gene modification frequency and may therefore be more generally applicable to transgene expression.

[0223] It is also envisioned that the present invention creates a gene knockout cell library, where each cell can have a knockout of a single gene. This is exemplified in Example 23.

[0224] A library of ES cells may be generated, in which each cell has a knockout of a single gene, and the entire library of ES cells has every gene knocked out. This library is useful for screening gene function in cellular processes and diseases. To generate this cell library, Cas9 driven by an inducible promoter (e.g., a doxycycline-inducible promoter) may be integrated into ES cells. Additionally, a single guide RNA targeting a specific gene may be integrated into ES cells. To generate an ES cell library, ES cells may simply be mixed with a library of genes encoding guide RNAs targeting each gene in the human genome. First, a single BxB1attB site may be introduced into the AAVS1 locus of human ES cells. BxB1 integrase may then be used to promote integration of individual guide RNA genes into the BxB1attB site in the AAVS1 locus. To promote integration, each guide RNA gene may be contained on a plasmid carrying a single attP site. In this way, BxB1 may recombine the genomic attB site with the attP site on the guide RNA-containing plasmid. To generate a cell library, one can take a library of cells that have a single integrated guide RNA and induce Cas9 expression, after which Cas9 mediates a double-strand break at the site specified by the guide RNA.

[0225] Chronic administration of protein therapeutics can induce unacceptable immune responses against specific proteins. The immunogenicity of protein drugs can be attributed to several immunodominant helper T lymphocyte (HTL) epitopes. Reducing the MHC binding affinity of these HTL epitopes contained within these proteins can create drugs with lower immunogenicity (Tangri S, et al. ("Rationally engineered therapeutic proteins with reduced immunogenicity") J Immunol. 2005 Mar 15;174(6):3187-96). In the present invention, the immunogenicity of CRISPR enzymes can be reduced, specifically, following the approach first demonstrated in Tangri et al. with respect to erythropoietin and subsequently expanded upon. Thus, directed evolution or rational design can be used to reduce the immunogenicity of CRISPR enzymes (e.g., Ca9) in host species (humans or other species).

[0226] In Example 28, the applicants used three target guide RNAs and were able to visualize efficient DNA cleavage in vivo, which occurred only in a small proportion of cells. Essentially, what the applicants demonstrate here is targeted in vivo cleavage. Specifically, this provides proof of concept that specific targeting in higher organisms, such as mammals, can also be achieved. This also highlights the multiplexing aspect in that multiple guide sequences (i.e., distinct targets) can be used simultaneously (in the sense of co-delivery). In other words, the applicants used a multiplexing approach, in which several different sequences are targeted simultaneously but independently.

[0227] A suitable example of a production protocol for AAV, a preferred vector of the present invention, is provided in Example 34.

[0228] Trinucleotide repeat disorders are preferred conditions to treat and are also exemplified herein.

[0229] For example, U.S. Patent Application Publication No. 20110016540 describes the genetic modification of cells, animals, and proteins associated with trinucleotide repeat expansion disorders using zinc finger nucleases. Trinucleotide repeat expansion disorders are complex, progressive disorders that involve developmental neurobiology and often affect cognitive and sensorimotor function.

[0230] Trinucleotide repeat expansion proteins are a diverse group of proteins associated with susceptibility to the development of trinucleotide repeat expansion disorders, the presence of trinucleotide repeat expansion disorders, the severity of trinucleotide repeat expansion disorders, or any combination thereof. Trinucleotide repeat expansion disorders are divided into two types determined by the type of repeat. The most common repeat is the triplet CAG, which, when present in the coding region of a gene, encodes the amino acid glutamine (Q). Therefore, these disorders are referred to as polyglutamine (polyQ) disorders, and include the following diseases: Huntington's disease (HD); spinal-bulbar muscular atrophy (SBMA); spinocerebellar ataxias (SCA types 1, 2, 3, 6, 7, and 17); and dentatorubral-pallidoluysian atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders do not involve CAG triplets or the CAG triplet is not in the coding region of the gene and are therefore referred to as non-polyglutamine disorders, including Fragile X syndrome (FRAXA), Fragile XE mental retardation (FRAXE), Friedreich ataxia (FRDA), myotonic dystrophy (DM), and spinocerebellar ataxia (SCA types 8 and 12).

[0231] Proteins associated with trinucleotide repeat expansion disorders are typically selected based on the experimental association between proteins associated with trinucleotide repeat expansion disorders and trinucleotide repeat expansion disorders.For example, in a population with trinucleotide repeat expansion disorders, the production rate or circulating concentration of proteins associated with trinucleotide repeat expansion disorders may be increased or decreased compared with a population without trinucleotide repeat expansion disorders.The difference in protein level can be evaluated using proteomics techniques, including but not limited to Western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry.Alternatively, proteins associated with trinucleotide repeat expansion disorders can be identified by obtaining gene expression profiles of the genes encoding these proteins using genomic techniques, including but not limited to DNA microarray analysis, sequence analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

[0232] Non-limiting examples of proteins associated with trinucleotide repeat expansion disorders include AR (androgen receptor), FMR1 (Fragile X mental retardation 1), HTT (huntingtin), DMPK (myotonic dystrophy protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat-containing 6A), PABPN1 (poly(A)-binding protein, nuclear 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1), and ATXN3 (ataxin 3). , TBP (TATA box binding protein), CACNA1A (calcium channel, voltage-gated, P / Q type, α1A subunit), ATXN80S (ATXN8 reverse chain (non-protein coding)), PPP2R2B (protein phosphatase 2, regulatory subunit B, β), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat-containing 6B), TNRC6C (trinucleotide repeat-containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer, non-polyposis type 1 (E. coli)coli), TMEM185A (Transmembrane protein 185A), SIX5 (SIX homeobox 5), CNPY3 (Canopy 3 homolog (zebrafish)), FRAXE (Fragile site, folate type, rare, fra(X)(q28)E), GNB2 (Guanine nucleotide-binding protein (G protein), beta polypeptide 2), RPL14 (Ribosomal protein L14), ATXN8 (Ataxin 8), INSR (Insulin receptor), TTR (Transthyretin), EP400 (E1A-binding protein p400), GIGYF2 (GRB 10 interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (metallopeptidase M20 family)), C10orf2 (chromosome 10 open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DKC1 (dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX (with transcription activation domain) interacting protein 1), CASK (calcium / calmodulin-dependent serine Protein kinase (MAGUK family)), MAPT (microtubule-associated protein τ), SP1 (Sp1 transcription factor), POLG (polymerase (DNA-directed), γ), AFF2 (AF4 / FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGGBP triplet repeat binding protein 1), ABT1 (activator of basal transcription 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN ( jun oncogene), KCNN3 (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folate-type, rare, fra(X)(q27.3)A (megalorchidism, mental retardation)), KBTBD10 (Kelch repeat and BTB(POZ) domain containing 10), MBNL1 (muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, Escherichia coli)) (S. cerevisiae)cerevisiae), NCOA3 (nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG / CTG1), TSC1 (tuberous sclerosis complex 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ​​ligase, catalytic subunit), RRAD (Ras-related associated with diabetes), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood type), CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (tripartite motif-containing 22), WT1 (Wilms' tumor 1), AHR (AHR) 1-(2-hydroxybenzoate)-1, 2 ... Drosophila (like), FABP2 (fatty acid binding protein 2, intestine), EN2 (engrailed homeobox 2), CRYGC (crystallin, γC), SRP14 (signal recognition particle 14 kDa (homologous AluRNA-binding protein)), CRYGB (crystallin, γB), PDCD1 (programmed cell death 1), HOXA1 (homeobox A1), ATXN2L (ataxin 2-like), PMS2 (PMS2 post-meiotic segregation increased 2) in S. cerevisiae (S.cerevisiae), GLA (galactosidase, alpha), CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), FTH1 (ferritin, heavy polypeptide 1), IL12RB2 (interleukin-12 receptor, beta2), OTX2 (orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNA-directed), gamma2, accessory subunit), DLX2 (distal-less homeobox 2), SIRPA (signal regulatory protein alpha), OTX1 (orthodenticle homeobox 1), AHRR (aryl hydrocarbon receptor repressor), MANF (mesencephalic astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene / pseudogene)), and ENSG00000078687.

[0233] Preferred proteins associated with trinucleotide repeat expansion disorders include HTT (huntingtin), AR (androgen receptor), FXN (frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7 (ataxin), Atxn10 (ataxin), DMPK (myotonic dystrophy protein kinase), Atn1 (atrophin 1), CBP (CREB binding protein), VLDLR (very low density lipoprotein receptor), and any combination thereof.

[0234] According to another aspect, a gene therapy method for treating a subject with a mutation in the CFTR gene is provided, which comprises administering a therapeutically effective amount of CRISPR-Cas gene therapy particles to the subject's cells, optionally via a biocompatible pharmaceutical carrier. Preferably, the target DNA contains the mutation ΔF508. Generally, it is preferred that the mutation be repaired to wild type. In this case, the mutation is a deletion of three nucleotides including the phenylalanine (F) codon at position 508. Therefore, repair in this case requires reintroducing the missing codon into the mutant.

[0235] To achieve this gene repair strategy, an adenovirus / AAV vector system is preferably introduced into a host cell, cell, or patient. Preferably, this system comprises Cas9 (or Cas9 nickase) and a guide RNA, along with an adenovirus / AAV vector system containing a homology repair template containing the F508 residue. This can be introduced into a subject by one of the delivery methods discussed above. The CRISPR-Cas system can be guided by a CFTRΔ508 chimeric guide RNA, which targets a specific site in the CFTR genomic locus to be nicked or cleaved. After cleavage, the repair template is inserted into the cleavage site by homologous recombination, repairing the deletion that results in cystic fibrosis or cystic fibrosis-related symptoms. This strategy, which directs the delivery of the CRISPR system with the appropriate guide RNA and results in its systemic introduction, can be used to target genetic mutations and edit or otherwise manipulate genes that cause metabolic, liver, kidney, and protein diseases and disorders, such as those listed in Table B.

[0236] Genome editing The CRISPR / Cas9 system of the present invention can be used to correct gene mutations that have previously been attempted using TALENs and ZFNs with limited success. For example, Duke University's published application, International Publication No. 2013163628 A2, entitled "Genetic Correction of Mutated Genes," describes attempts to repair frameshift mutations that result in premature stop codons and truncated gene products, such as those involved in Duchenne muscular dystrophy (DMD), a recessive, fatal, X-linked disorder that causes muscle degeneration due to mutations in the dystrophin gene. The majority of dystrophin mutations that cause DMD are exon deletions, which disrupt the reading frame and cause premature translation termination of the dystrophin gene. Dystrophin is a cytoplasmic protein that provides structural stability to the dystroglycan complex in the cell membrane, which is involved in regulating the integrity and function of muscle cells. The dystrophin gene, or "DMD gene," as used interchangeably herein, is 2.2 megabases long and located at locus Xp21. The primary transcript is approximately 2,400 kb long, and the mature mRNA is approximately 14 kb long. 79 exons encode the protein, which is more than 3,500 amino acids long. Exon 51 is often adjacent to frame-breaking deletions in DMD patients, and this has been targeted in clinical trials involving oligonucleotide-based exon skipping. A clinical trial involving the exon 51 skipping compound eteplirsen recently reported significant functional benefit over 48 weeks, with an average of 47% dystrophin-positive fibers compared to baseline. Exon 51 mutations are ideally suited for permanent repair by NHEJ-based genome editing.

[0237] The method of U.S. Patent Application Publication No. 20130145487, assigned to Cellectis, which relates to meganuclease variants that cleave target sequences from the human dystrophin gene (DMD), can also be modified for the CRISPRCas system of the present invention.

[0238] blood The present invention also contemplates delivering the CRISPR-Cas system to the blood.

[0239] Wahlgren et al.'s plasma exosomes (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) have previously been described and can be used to deliver CRISPR Cas systems to the blood.

[0240] The CRISPR Cas systems of the present invention are also contemplated for treating hemoglobinopathies, such as thalassemia and sickle cell disease. See, e.g., WO 2013 / 126794 for potential targets that can be targeted by the CRISPR Cas systems of the present invention.

[0241] U.S. Patent Application Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881, and 20090222937, assigned to Cellectis, relate to CREI mutants, wherein at least one of the two I-CreI monomers has at least two substitutions, one in each of two functional subdomains of the LAGLIDADG core domain located at positions 26-40 and 44-77 of I-CreI, respectively, and wherein the mutant is capable of cleaving a DNA target sequence from the human interleukin-2 receptor gamma chain (IL2RG) gene, also known as the common cytokine receptor gamma chain gene or gamma gene. The target sequences identified in U.S. Patent Application Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881, and 20090222937 can be utilized in the CRISPRCas system of the present invention.

[0242] Severe combined immunodeficiency (SCID) results from a defect in T lymphocyte maturation, always associated with a functional defect in B lymphocytes (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). The overall incidence is estimated at 1 in 75,000 live births. Untreated SCID patients are prone to multiple opportunistic microbial infections and generally do not survive more than one year. SCID can be cured by allogeneic hematopoietic stem cell transplantation from a family donor. Donor histocompatibility can vary widely. In one form of SCID, adenosine deaminase (ADA) deficiency, patients can be treated by infusion of recombinant adenosine deaminase enzyme.

[0243] Since the ADA gene was shown to be mutated in patients with SCID (Giblett et al., Lancet, 1972, 2, 1067-1069), several other genes involved in SCID have been identified (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). There are four major causes of SCID: (i) the most common form of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutations in the IL2RG gene, resulting in the absence of mature T lymphocytes and NK cells. IL2RG encodes the γC protein, a component common to at least five interleukin receptor complexes (Noguchi et al., Cell, 1993, 73, 147-157). These receptors activate several targets via the JAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), and inactivation of this JAK3 kinase results in the same syndrome as γC inactivation; (ii) mutations in the ADA gene result in a defect in purine metabolism that is lethal to lymphoid precursor cells, resulting in a pseudo-deficiency of B, T, and NK cells; (iii) V(D)J recombination is an essential step in the maturation of immunoglobulin and T lymphocyte receptors (TCRs). Mutations in three genes involved in this process, recombination activating genes 1 and 2 (RAG1 and RAG2) and Artemis, result in a lack of mature T and B lymphocytes; and (iv) mutations in other genes involved in T cell-specific signaling, such as CD45, have also been reported, but represent a minority of cases (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109).

[0244] Since its genetic basis was identified, various forms of SCID have become paradigms for gene therapy approaches for two main reasons (Fischer et al., Immunol. Rev., 2005, 203, 98-109). First, as with any hematological disorder, ex vivo therapy is conceivable. Hematopoietic stem cells (HSCs) can be harvested from the bone marrow and retain their pluripotent properties over several cell divisions. Thus, HSCs can be processed in vitro and then reinfused into the patient, where they repopulate the bone marrow. Second, because lymphocyte maturation is impaired in SCID patients, repaired cells have a selective advantage. Thus, a small number of repaired cells can restore a functional immune system.This hypothesis is supported by (i) partial restoration of immune function following reversion of the mutation in SCID patients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan et al., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl., Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103, 4565-4572), and (ii) correction of SCID-X1 deficiency in hematopoietic cells in vitro (Candottiet al., Blood, 1996, 87, 3097-3102; Cavazzana-Calvo et al., Blood, 1996, 88, 3901-3909; Taylor et al., Blood, 1996, 87, 3103-3107; Hacein-Beyet et al., Blood, 1998, 92, 4090-4097), (iii) in vivo in animal models, SCID-X1 (Soudaiset et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3 (Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Blood, 1998, 5, 59-60), and (iv) in vivo in animal models, SCID-X1 (Soudaiset et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), and JAK-3 (Bunting et al., Nat. Med., 1998, 4, 58-64). al., Hum. Gene Ther., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100, 3942-3949) defects, and (iv) by the results of gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000, 288, 669-672; Aiutiet al., Nat. Med., 2002; 8, 423-425; Gaspar et al., Lancet, 2004, 364, 2181-2187).

[0245] U.S. Patent Application Publication No. 20110182867, assigned to Children's Medical Center Corporation and President and Fellows of Harvard College, relates to methods and uses for regulating fetal hemoglobin (HbF) expression in hematopoietic progenitor cells using inhibitors of BCL11A expression or activity, such as RNAi and antibodies. Targets disclosed in U.S. Patent Application Publication No. 20110182867, such as BCL11A, can be targeted by the CRISPR Cas system of the present invention to regulate fetal hemoglobin expression. For additional BCL11A targets, see also Bauer et al. (Science 11 October 2013: Vol. 342 no. 6155 pp. 253-257) and Xuet et al. (Science 18 November 2011: Vol. 334 no. 6058 pp. 993-996).

[0246] ear The present invention also contemplates delivery of the CRISPR-Cas system to one or both ears.

[0247] Researchers are investigating whether gene therapy can be used to supplement the current treatment for hearing loss: cochlear implants. Hearing loss is often caused by missing or damaged hair cells that are unable to relay signals to auditory neurons. In such cases, cochlear implants can be used to respond to sound and transmit electrical signals to nerve cells. However, these neurons often degenerate and recede from the cochlea due to reduced release of growth factors by the damaged hairs.

[0248] US Patent Application Publication No. 20120328580 describes the injection of pharmaceutical compositions into the ear (e.g., pinna administration), for example, into the cochlear cavity (e.g., scala media, scala vestibuli, and scala tympani), for example, using a syringe, e.g., a single-dose syringe. For example, one or more of the compounds described herein can be administered by intratympanic injection (e.g., into the middle ear) and / or by injection into the outer ear, middle ear, and / or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics to the human ear. Injection can be, for example, through the round window of the ear or through the cochlear capsule. Other methods of inner ear administration are known in the art (see, e.g., Salt and Plontke, Drug Discovery Today, 10:1299-1306, 2005).

[0249] In another administration method, the pharmaceutical composition can be administered in situ via a catheter or pump. The catheter or pump can, for example, direct the pharmaceutical composition to the cochlear cavity or the round window of the ear and / or the lumen of the colon. Exemplary drug delivery devices and methods suitable for administering one or more of the compounds described herein to the ear, for example, the human ear, are described by McKenna et al. (U.S. Patent Application Publication No. 2006 / 0030837) and Jacobsen et al. (U.S. Patent No. 7,206,639). In some embodiments, the catheter or pump can be placed in, for example, a patient's ear (e.g., the outer ear, middle ear, and / or inner ear) during a surgical procedure. In some embodiments, the catheter or pump can be placed in, for example, a patient's ear (e.g., the outer ear, middle ear, and / or inner ear) without the need for a surgical procedure.

[0250] Alternatively or additionally, one or more of the compounds described herein can be administered in combination with a mechanical device attached to the outer ear, such as a cochlear implant or hearing aid. An exemplary cochlear implant suitable for use in the present invention is described by Edge et al. (U.S. Patent Application Publication No. 2007 / 0093878).

[0251] In some embodiments, the administration methods described above may be combined in any order, and may be simultaneous or interspersed.

[0252] Alternatively or additionally, the present invention may be administered in accordance with any of the methods approved by the Food and Drug Administration, e.g., as described in the CDER Data Standards Manual, 4th Edition (available at fda.give / cder / dsm / DRG / drg00301.htm).

[0253] Generally, the cell therapy methods described in U.S. Patent Application Publication No. 20120328580 can be used to promote the complete or partial differentiation of cells in vitro into or toward mature cell types of the inner ear (e.g., hair cells). Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment. Cell culture methods necessary to carry out such methods are described below, including methods for identifying and selecting suitable cell types, promoting the complete or partial differentiation of selected cells, identifying fully or partially differentiated cell types, and implanting fully or partially differentiated cells.

[0254] Cells suitable for use in the present invention include, but are not limited to, cells capable of fully or partially differentiating into mature cells of the inner ear, such as hair cells (e.g., inner hair cells and / or outer hair cells), when contacted, for example, in vitro, with one or more of the compounds described herein. Exemplary cells capable of differentiating into hair cells include, but are not limited to, stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and adipose-derived stem cells), progenitor cells (e.g., inner ear progenitor cells), supporting cells (e.g., Deiters cells, pillar cells, inner phalangeal cells, tectal cells, and Hensen cells), and / or germ cells. The use of stem cells to replenish inner ear sensory cells is described in Li et al. (U.S. Patent Application Publication No. 2005 / 0287127) and Li et al. (U.S. Patent Application No. 11 / 953,797). The use of bone marrow-derived stem cells to replenish inner ear sensory cells is described in Edge et al., PCT / US Patent Application Publication No. 2007 / 084654. iPS cells are described, for example, in Cell, Volume 131, Issue 5, Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science 318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106 (2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007).

[0255] Such suitable cells can be identified by analyzing (e.g., qualitatively or quantitatively) the presence of one or more tissue-specific genes. For example, gene expression can be detected by detecting the protein product of one or more tissue-specific genes. Protein detection techniques include staining proteins (e.g., using cell extracts or whole cells) using antibodies against an appropriate antigen. In this case, the appropriate antigen is the protein product of tissue-specific gene expression. While in principle the primary antibody (i.e., the antibody that binds to the antigen) can be labeled, it is more common (and visualization is improved) to use a secondary antibody (e.g., anti-IgG) that targets the primary antibody. This secondary antibody is conjugated with a fluorescent dye or with an appropriate enzyme for colorimetric reaction, or with gold beads (for electron microscopy), or with a biotin-avidin system, which allows for recognition of the location of the primary antibody and thus the antigen.

[0256] The CRISPR Cas molecules of the present invention can be delivered to the ear by applying a pharmaceutical composition directly to the outer ear, such as by compositions modified from US Patent Publication No. 20110142917. In some embodiments, the pharmaceutical composition is applied to the ear canal. Delivery to the ear can also be referred to as aural delivery or otic delivery.

[0257] In some embodiments, the RNA molecules of the invention are delivered in liposomal or lipofectin formulations, etc., which 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.

[0258] Delivery systems specifically targeted at enhancing and improving 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 Simeon et al., NAR 2003, 31, 11:2717-2724) and may be applied to the present invention. siRNA has recently been successfully used to inhibit gene expression in primates (see, e.g., Tolentino et al., Retina 24(4):660), which can also be applied to the present invention.

[0259] Qi et al. have disclosed a method for efficient siRNA transfection into the inner ear through the intact round window using a novel proteidic delivery technique, which can be applied to the CRISPRCas system of the present invention (see, e.g., Qi et al., Gene Therapy (2013), 1-9). Specifically, the TAT double-stranded RNA binding domain (TAT-DRBD), which can transfect Cy3-labeled siRNA into cells of the inner ear, such as inner and outer hair cells, the crista ampullaris, the utricular macula, and the sacculus, via penetration through the intact round window, has been successfully used in vivo to deliver double-stranded siRNA to treat various inner ear ailments and maintain hearing function. Approximately 40 μl of 10 mM RNA can be contemplated as a dosage for administration to the ear.

[0260] According to Rejali et al. (Hear Res. 2007 Jun;228(1-2):180-7), cochlear implant function can be improved by better preserving spiral ganglion neurons, the target of electrical stimulation by the implant, and it has previously been shown that brain-derived neurotrophic factor (BDNF) enhances spiral ganglion survival in experimentally deafened ears. Rejali et al. tested an improved cochlear implant electrode design that included a fibroblast coating transduced with a viral vector carrying a BDNF gene insert. To achieve this type of ex vivo gene transfer, Rejali et al. transduced guinea pig fibroblasts with an adenovirus carrying a BDNF gene cassette insert, determined that these cells secreted BDNF, and then attached the BDNF-secreting cells to a cochlear implant electrode in an agarose gel and implanted the electrode into the scala tympani. Rejalie et al. determined that this BDNF-expressing electrode was able to maintain significantly more spiral ganglion neurons in the basal turn of the cochlea 48 days after implantation compared with control electrodes, demonstrating the feasibility of combining cochlear implant therapy with ex vivo gene transfer to enhance spiral ganglion neuron survival. Such a system can be applied to deliver the CRISPRCas system of the present invention to the ear.

[0261] Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5, 2010) reported that knockdown of NOX3 using small interfering (si)RNA reversed cisplatin-induced hearing loss, as evidenced by protection of OHCs from damage and a reduced threshold shift in auditory brainstem responses (ABRs). Various doses of siNOX3 (0.3, 0.6, and 0.9 μg) were administered to rats, and NOX3 expression was assessed by real-time RT-PCR. The lowest dose used (0.3 μg) of NOX3 siRNA did not show any inhibition of NOX3 mRNA compared with transtympanic administration of scrambled siRNA or untreated cochleae. However, administration of higher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expression compared with the control scrambled siRNA. Such a system may be applied to the CRISPR Cas system of the present invention for transtympanic administration at a dosage of about 2 mg to about 4 mg of CRISPR Cas for administration to humans.

[0262] Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 Apr. 2013) demonstrated that Hes5 levels in the utricle were reduced after siRNA application, and that the number of hair cells in those utricles was significantly higher than after control treatment. This data suggests that siRNA technology may be useful for inducing repair and regeneration in the inner ear, and that the Notch signaling pathway is a potentially useful target for inhibiting specific gene expression. Jung et al. injected 8 μg of Hes5 siRNA, prepared by adding sterile normal saline to lyophilized siRNA, into the vestibular epithelium of the ear in a volume of 2 μl. This system can be applied to the CRISPR-Cas system of the present invention for administration to the vestibular epithelium of the ear at a dosage of approximately 1 to approximately 30 mg of CRISPR-Cas for human administration.

[0263] eye The present invention also contemplates delivery of the CRISPR-Cas system to one or both eyes.

[0264] In yet another embodiment of the present invention, the CRISPR-Cas system can be used to correct eye defects caused by several genetic mutations, as further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

[0265] For ocular administration, lentiviral vectors, particularly equine infectious anemia virus (EIAV), are particularly preferred.

[0266] 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, published online November 21, 2005, Wiley InterScience (www.interscience.wiley.com). DOI:10.1002 / jgm.845). The vectors are contemplated to have a cytomegalovirus (CMV) promoter driving expression of the target gene. Intracameral, subretinal, intraocular, and intravitreal injections are all contemplated (see, e.g., Balagaan, J Gene Med 2006;8:275-285, published online November 21, 2005, Wiley InterScience (www.interscience.wiley.com). DOI:10.1002 / jgm.845). Intraocular injections are performed with the aid of an operating microscope. For subretinal and intravitreal injections, the eye may be explanted using gentle digital pressure, and the fundus may be visualized using a contact lens system consisting of a drop of propagation medium solution on the cornea covered by a glass microscope slide coverslip. For subretinal injections, the tip of a 10 mm 34-gauge needle attached to a 5 μl Hamilton syringe may be advanced tangentially from the superior equatorial sclera toward the posterior pole under direct visualization until the needle hole is visible in the subretinal space. Next, 2 μl of vector suspension may be injected to create a superior bullous retinal detachment, thus confirming subretinal vector administration. This procedure creates a self-sealing sclerotomy, allowing the vector suspension to remain in the subretinal space until absorbed by the RPE, usually within 48 hours of the procedure. This procedure may be repeated in the inferior hemisphere to create an inferior retinal detachment. This technique exposes approximately 70% of the neurosensory retina and RPE to the vector suspension. For intravitreal injections, the needle tip may be advanced through the sclera 1 mm posterior to the corneoscleral limbus and 2 μl of vector suspension may be injected into the vitreous cavity. For intracameral injections, the needle tip may be advanced through the corneoscleral limbus and directed toward the central cornea, and 2 μl of vector suspension may be injected.For intracameral injection, the tip of the needle can be advanced through the corneal scleral limbus, directed toward the central cornea, and 2 μl of vector suspension can be injected. These vectors are 1.0–1.4 × 10 cells / mL. 10 or 1.0 to 1.4 × 10 9 The antibody may be injected at any titer of transducing units (TU) / ml.

[0267] In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector expressing the antiangiogenic proteins endostatin and angiostatin delivered by subretinal injection for the treatment of wet age-related macular degeneration, is also contemplated (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)). Such vectors can be modified for the CRISPR-Cas system of the present invention. 1.1 x 10 per eye 5 Each eye may be treated with a dose of transducing units / eye (TU / eye) of RetinoStat® in a total volume of 100 μl.

[0268] In another embodiment, E1-deleted, partially E3-deleted, and E4-deleted adenoviral vectors may be contemplated for ocular delivery. Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) received a single intravitreal injection of an E1-deleted, partially E3-deleted, and E4-deleted adenoviral vector (AdPEDF.11) expressing human pigment epithelium-derived factor (see, e.g., Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)). 6 ~10 9.5 A range of particle unit (PU) doses has been examined, and there have been no serious adverse events or dose-limiting toxicities associated with AdPEDF.ll (see, e.g., Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)). Adenoviral vector-mediated intraocular gene transfer appears to be a viable approach for the treatment of ocular disorders and may be applicable to the CRISPR Cas system.

[0269] In another embodiment, RXi Pharmaceuticals' sd-rxRNA® system can be used and / or adapted for delivery of CRISPR Cas to the eye. In this system, a single intravitreal administration of 3 μg of sd-rxRNA results in sequence-specific reduction of PPIB mRNA levels for 14 days. The sd-rxRNA® system can be applied to the CRISPR Cas system of the present invention, contemplating a dose of approximately 3-20 mg of CRISPR administered to humans.

[0270] Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 Apr. 2011) describe adeno-associated virus (AAV) vectors for delivering RNA interference (RNAi)-based rhodopsin inhibitors and codon-modified rhodopsin replacement genes that are resistant to suppression due to nucleotide changes at degenerate positions across the RNAi target site. 6.0 × 10 8 vp or 1.8 × 10 10 Injections of either vpAAV were subretinal injected into the eye by Millington-Ward et al. The AAV vector of Millington-Ward et al. administered to humans was approximately 2 × 10 11 ~Approx. 6×10 13 vp doses may be contemplated and applied to the CRISPRCas system of the present invention.

[0271] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also described in vivo directed evolution to generate AAV vectors that deliver wild-type versions of defective genes throughout the retina after non-invasive injection into the vitreous humor of the eye. Dalkara described AAV libraries constructed by DNA shuffling of a 7-mer peptide display library and the cap genes of AAV1, 2, 4, 5, 6, 8, and 9. These rcAAV libraries and rAAV vectors expressing GFP under the CAG or Rho promoter were packaged, and deoxyribonuclease-resistant genomic titers were obtained by quantitative PCR. The libraries were pooled and subjected to two rounds of evolution, each consisting of an initial library diversification step followed by three in vivo selection steps. In each step, P30rho-GFP mice were inoculated with 2 ml of iodixanol-purified, phosphate-buffered saline (PBS)-dialyzed library containing approximately 1 × 10 12 The AAV vectors of Dalkara et al. were injected intravitreally at a genome titer of approximately 1 × 10 15 ~Approx. 1×10 16 Doses of 1000 mg / ml are contemplated and may be applied to the CRISPRCas system of the present invention.

[0272] In another embodiment, the rhodopsin gene may be targeted to treat retinitis pigmentosa (RP), where the system of U.S. Patent Application Publication No. 20120204282, assigned to SangamoBioSciences, Inc., may be modified according to the CRISPR Cas system of the present invention.

[0273] In another embodiment, the method of U.S. Patent Application Publication No. 20130183282, assigned to Cellectis, for cleaving a target sequence from the human rhodopsin gene may also be modified for the CRISPRCas system of the present invention.

[0274] U.S. Patent Application Publication No. 20130202678, assigned to Academia Sinica, relates to a method for treating retinopathies and sight-threatening ophthalmological disorders involving delivery of the Puf-A gene, which is expressed in retinal ganglion cells and pigmented cells of ocular tissues and exhibits unique anti-apoptotic activity, to the subretinal or intravitreal space of the eye. Specifically, desirable targets are zgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1, and HtrA2, all of which can be targeted by the CRISPR Cas system of the present invention.

[0275] Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA that directed Cas9 to a single base pair mutation that causes cataracts in mice, which induced DNA cleavage. Then, using either the other wild-type allele or an oligo provided to the zygotic repair machinery, the sequence of the broken allele in the mutant mice was repaired, and the genetic defect that causes cataracts was corrected.

[0276] U.S. Patent Application Publication No. 20120159653 describes the genetic modification of cells, animals, and proteins associated with macular degeneration (MD) using zinc finger nucleases. Macular degeneration (MD) is a leading cause of visual impairment in the elderly but is also a prominent symptom of childhood diseases with onset as early as infancy, such as Stargardt's disease, Sorsby's fundus, and fatal pediatric neurodegenerative disorders. Macular degeneration results from retinal damage, leading to loss of vision in the central visual field (macula). As this disease is observed in humans, existing animal models do not recapitulate key features of the disease. Available animal models containing mutant genes encoding proteins associated with MD also produce highly variable phenotypes, complicating interpretation and therapeutic development for human disease.

[0277] One aspect of US Patent Application Publication No. 20120159653 relates to editing any chromosomal sequence encoding a protein associated with MD, which can be applied to the CRISPRCas system of the present invention. MD-associated proteins are typically selected based on experimental association between the MD-associated protein and MD disorder. For example, the production rate or circulating concentration of the MD-associated protein may be increased or decreased in a population with MD disorder compared to a population without MD disorder. Differences in protein levels may be assessed using proteomic techniques, including but not limited to Western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, MD-associated proteins may be identified by obtaining gene expression profiles of the genes encoding those proteins using genomic techniques, including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

[0278] As non-limiting examples, proteins associated with MD include, but are not limited to, the following proteins: (ABCA4) ATP-binding cassette, subfamily A (ABC1), member 4 ACHM1 achromatopsia (rod monochromacy) 1 ApoE apolipoprotein E (ApoE) C1QTNF5 (CTRP5) C1q and tumor necrosis factor-related protein 5 (C1QTNF5) C2 complement component 2 (C2) C3 complement component (C3) CCL2 chemokine (CC motif) ligand 2 (CCL2) CCR2 chemokine (CC motif) receptor 2 (CCR2) CD36 cluster of differentiation 36 CFB complement factor B CFH complement factor CFH H CFHR1 complement factor H-related 1 CFHR3 complement factor H-related 3 CNGB3 cyclic nucleotide-gated channel β3 CP ceruloplasmin (CP) CRP C-reactive protein (CRP) CST3 Cystatin C or cystatin 3 (CST3) CTSD Cathepsin D (CTSD) CX3CR1 Chemokine (C-X3-C motif) receptor 1 ELOVL4 Very long chain fatty acid elongation 4 ERCC6 Excision repair cross-complementing rodent repair deficiency, complementation group 6 FBLN5 Fibulin 5 FBLN5 Fibulin 5 FBLN6 Fibulin 6 FSCN2 Fascin (FSCN2) HMCN1 Hemicentrin 1 HMCN1 Hemicentin 1 HTRA1 HtrA serine peptidase 1 (HTRA1) HTRA1 HtrA serine peptidase 1 IL-6 Interleukin 6 IL-8 Interleukin 8 LOC387715 hypothetical protein PLEKHA1 Pleckstrin homology domain-containing family A member 1 (PLEKHA1) PROM1 Prominin 1 (PROM1 or CD133) PRPH2 peripherin-2 RPGR retinitis pigmentosa GTPase regulator SERPING1 serpin peptidase inhibitor, clade G, member 1 (C1-inhibitor) TCOF1 Treacle TIMP3 metalloproteinase inhibitor 3 (TIMP3) TLR3 Toll-like receptor 3.

[0279] The identity of the MD-associated protein whose chromosomal sequence is edited can and will be different. In a preferred embodiment, the MD-associated protein whose chromosomal sequence is edited can be an ATP-binding cassette encoded by the ABCR gene, a subfamily A (ABC1) member 4 protein (ABCA4), an apolipoprotein E protein (APOE) encoded by the APOE gene, a chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene, a chemokine (CC motif) receptor 2 protein (CCR2) encoded by the CCR2 gene, a ceruloplasmin protein (CP) encoded by the CP gene, a cathepsin D protein (CTSD) encoded by the CTSD gene, or a metalloproteinase inhibitor 3 protein (TIMP3) encoded by the TIMP3 gene. In an exemplary embodiment, the genetically modified animal is a rat, and the edited chromosomal sequence encoding the MD-associated protein can be: (ABCA4) ATP-binding cassette, NM_000350 subfamily A (ABC1), member 4 APOE apolipoprotein E NM_138828 (APOE) CCL2 chemokine (C-CNM_031530 motif) ligand 2 (CCL2) CCR2 chemokine (CC NM_021866 motif) receptor 2 (CCR2) CP ceruloplasmin (CP) NM_012532 CTSD cathepsin D (CTSD) NM_134334 TIMP3 inhibitor of metalloproteinase NM_012886 (TIMP3). The animal or cell can comprise 1, 2, 3, 4, 5, 6, 7 or more disrupted chromosomal sequences encoding MD-associated proteins and 0, 1, 2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding disrupted MD-associated proteins.

[0280] The edited or integrated chromosomal sequence may be modified to encode an altered MD-associated protein. Several mutations in MD-associated chromosomal sequences have been associated with MD. Non-limiting examples of mutations in chromosomal sequences associated with MD include E471K (i.e., glutamic acid at position 471 is changed to lysine), R1129L (i.e., arginine at position 1129 is changed to leucine), T1428M (i.e., threonine at position 1428 is changed to methionine), R1517S (i.e., arginine at position 1517 is changed to serine), I1562T (i.e., isoleucine at position 1562 is changed to threonine), and G1578R (i.e., glycine at position 1578 is changed to arginine) in the ABCR protein; V64I (i.e., valine at position 192 is changed to isoleucine) in the CCR2 protein; and V64I (i.e., valine at position 192 is changed to isoleucine) in the CP protein. In the TIMP3 protein, G969B (i.e., glycine at position 969 is changed to asparagine or aspartic acid); S156C (i.e., serine at position 156 is changed to cysteine), G166C (i.e., glycine at position 166 is changed to cysteine), G167C (i.e., glycine at position 167 is changed to cysteine), Y168C (i.e., tyrosine at position 168 is changed to cysteine), S170C (i.e., serine at position 170 is changed to cysteine), Y172C (i.e., tyrosine at position 172 is changed to cysteine), and S181C (i.e., serine at position 181 is changed to cysteine) cause MD. Other associations of genetic mutations in MD-related genes and diseases are known in the art.

[0281] heart The present invention also contemplates delivering the CRISPR-Cas system to the heart. For the heart, myocardial adeno-associated viruses (AAVMs), particularly AAVM41, which have shown preferential gene transfer in the heart, are preferred (see, for example, Lin-Yanga et al., PNAS, March 10, 2009, vol. 106, no. 10). Administration can be systemic or local. Systemic administration involves administering approximately 1 to 10 × 1014 Dosages of vector genomes are contemplated. See, e.g., Eulalio et al. (2012) Nature 492:376 and Somasuntharam et al. (2013) Biomaterials 34:7790.

[0282] For example, US Patent Application Publication No. 20110023139 describes the genetic modification of cells, animals, and proteins associated with cardiovascular disease using zinc finger nucleases. Cardiovascular disease generally includes high blood pressure, heart attack, heart failure, and stroke and TIA. The methods described in this disclosure can utilize any chromosomal sequence associated with cardiovascular disease or proteins encoded by any chromosomal sequence associated with cardiovascular disease. Cardiovascular-related proteins are typically selected based on experimental associations between cardiovascular-related proteins and the development of cardiovascular disease. For example, the production rate or circulating concentration of cardiovascular-related proteins may be increased or decreased in a population with cardiovascular disorders compared with a population without cardiovascular disorders. Differences in protein levels can be evaluated using proteomic techniques, including, but not limited to, Western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, cardiovascular-related proteins can be identified by obtaining gene expression profiles of the genes encoding those proteins using genomic techniques, including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

[0283] Exemplary chromosomal sequences include, but are not limited to, IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, subfamily G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), KCN J11 (potassium inward rectifier channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB (platelet-derived growth factor receptor, β polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor β polypeptide (simian sarcoma virus (v-sis) oncogene homolog)), KCNJ5 (potassium inward rectifier channel, subfamily J, member 5), KCNN3 (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B (adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain 5), PARP14 (poly(ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (C. elegans)), ACE angiotensin I-converting enzyme (peptidylcholinesterase) peptidase A)1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, beta2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, C1Q and collagen domain containing), APOB (apolipoprotein B (containing Ag(x) antigen)), APOE (apolipoprotein E), LEP (leptin),MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein AI), EDN1 (endothelin 1), NPPB (natriuretic peptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cells)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (plasminogen activator, tissue), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglandin G / H synthase and cyclooxygenase)), CETP (cholesteryl ester transfer protein Protein, plasma), AGTR1 (Angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (Insulin-like growth factor 1 (somatomedin C)), SELE (Selectin E), REN (Renin), PPARA (Peroxisome proliferator-activated receptor α), PON1 (Paraoxonase 1), KNG1 (Kininogen 1), CCL2 (Chemokine (CC motif) ligand 2), LPL (Lipoprotein lipase), VWF (von Willebrand factor), F2 (Coagulation factor II (Thrombin) ), ICAM1 (Intercellular adhesion molecule 1), TGFB1 (Transforming growth factor, beta 1), NPPA (Natriuretic peptide precursor A), IL10 (Interleukin 10), EPO (Erythropoietin), SOD1 (Superoxide dismutase 1, soluble), VCAM1 (Vascular cell adhesion molecule 1), IFNG (Interferon, gamma), LPA (Lipoprotein, Lp(a)), MPO (Myeloperoxidase), ESR1 (Estrogen receptor 1), MAPK1 (Mitogen-activated protein kinase 1), HP (Haptoglobin), F3 (Coagulation factor I II factor (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (component 2 of the oligomeric Golgi complex), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (decycling) 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8),PROK1 (prokineticin 1), CBS (cystathionine beta synthase), NOS2 (nitric oxide synthase 2, inducible), TLR4 (Toll-like receptor 4), SELP (selectin P (granule membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette, subfamily A (ABC1), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low-density lipoprotein receptor), GPT (glutamic pyruvate transaminase (alanine aminotransferase)) erase), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin-18 (interferon-γ-inducible factor)), NOS1 (nitric oxide synthase 1 (neuronal type)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen β chain), HGF (hepatocyte growth factor (hepapoetin A; scatter factor)), IL1A (interleukin-1, α), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogene) Child homolog 1), LIPC (lipase, liver), HSPD1 (heat shock 60 kDa protein 1 (chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, β3 (platelet glycoprotein 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (clotting factor X), CP (ceruloplasmin (ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily, member -11b), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)), PLG (plasminogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viral oncogene homolog (avian)),FN1 (fibronectin 1), CMA1 (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic, beta-2-, receptor, surface), APOA5 (apolipoprotein AV), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (proaccelerin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonic acid receptor) donate 5-lipoxygenase), HLA-DRB1 (major histocompatibility complex, class II, DRβ1), PARP1 (poly(ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (receptor for advanced glycation end products specific), IRS1 (insulin receptor substrate 1), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G / H synthase and cyclooxygenase)), ECE1 (endothelin-converting enzyme 1), F7 (coagulation factor VII (serum prothrombin time) Transformation-promoting factor), URN (Interleukin-1 receptor antagonist), EPHX2 (Epoxide hydrolase 2, cytoplasmic), IGFBP1 (Insulin-like growth factor binding protein 1), MAPK10 (Mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP-binding cassette, subfamily B (MDR / TAP), member 1), JUN (jun oncogene), IGFBP3 (Insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (Phosphodiesterase transmembrane kinase 5A, cGMP specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT (lecithin cholesterol acyltransferase), CCR5 (chemokine (CC motif) receptor 5), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM (adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription 3 (acute phase responder)),MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN (elastin), USF1 (upstream transcription factor 1), CFH (complement factor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenergic β-1-receptor) body), CYBA (cytochrome b-245, alpha polypeptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamyltransferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), CXCR4 (chemokine (CXC motif) receptor 4), PROC (protein C (inactivator of coagulation factors Va and VIIIa)), SCARB1 (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-related chain α), PLTP (phospholipid transfer protein), ADD1 (adducin 1(α)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid A1), KCNH2 (potassium voltage-gated channel, subfamily H (eag-related), member 2), DPP4 (dipeptidyl peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A / guanylate cyclase A (atrial natriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (F BJ murine osteosarcoma viral oncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isomerase G (cyclophilin G)), IL1R1 (interleukin-1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate homocysteine ​​methyltransferase),RBP4 (retinol-binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, α2, (CD49B, α2 subunit of VLA-2 receptor)), CABIN1 (calcineurin-binding protein 1), SHBG (sex hormone-binding globulin), HMGB1 (high mobility group box 1), HSP90B2P (heat shock protein 90kDa β (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gap junction protein, α1, 43kDa), CAV1 (caveolin 1, caveolar protein, 22kDa), ESR2 (estrogen receptor ERβ), LTA (lymphotoxin α (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (β polypeptide)), SP1 (Sp1 transcription factor), TGIF1 (TGFB-inducible factor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), EGF (epithelial growth factor factor (β-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline kinase α), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursor protein), CTNNB1 (catenin (cadherin-associated protein), β1, 88 kDa), IL2( Interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, β1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member A1), CX3CR1 (chemokine (C-X3-C motif) receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A),PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferase μ1), DMD (dystrophin), GATA4 (GATA-binding protein 4), F13A1 (coagulation factor XIII, A1 polypeptide), TTR (transthyretin), FABP4 (fatty acid-binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein CI), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine esterase inhibitor), Tryptamine (serotonin) receptor 2A), CSF3 (colony-stimulating factor 3 (granulocytes)), CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony-stimulating factor 2 (granulocyte-macrophage)), KDR (kinase insert domain receptor (type III receptor tyrosine kinase)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T cell specific, HMG box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor (erythroid-derived 2)-like 2), NOTCH1 (Notch homolog 1, translocation associated (Drosophila)), UGT1A1 (UD P-glucuronosyltransferase 1 family, polypeptide A1), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin (silent mating type signaling regulatory 2 homolog) 1 (S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, papalisin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC (natriuretic peptide precursor C),AHSP (alpha hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin-13), MTOR (mechanistic target of rapamycin (serine / threonine kinase)), ITGB2 (integrin, beta2 (complement component 3 receptor 3 and 4 subunit)), GSTT1 (glutathione S-transferase theta1), IL6ST (interleukin-6 signaling factor (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, furunculosis) Family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, α), SLC6A4 (solute carrier family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solute carrier family 8 (sodium / calcium exchanger), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldo-keto reductase Enzyme family 1, member A1 (aldehyde reductase), ALDH9A1 (aldehyde dehydrogenase 9 family, member A1), BGLAP (bone gamma-carboxyglutamic acid (gla)-containing protein), MTTP (microsomal triglyceride transfer protein), MTRR (5-methyltetrahydrofolate homocysteine ​​methyltransferase reductase), SULT1A3 (sulfotransferase family, cytosolic type, 1A, phenol-selective, member 3), RAGE (renal tumor antigen), C4B (complement component 4B (Chi do) blood type), P2RY12 (purinergic receptor P2Y, G protein-coupled, 12), RNLS (linalase, FAD-dependent amine oxidase), CREB1 (cAMP response element binding protein 1), POMC (proopiomelanocortin), RAC1 (ras-related C3 botulinum toxin substrate 1 (rho family, small GTP-binding protein Rac1)), LMNA (lamin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V, α subunit), CYP1B1 (cytochrome P450,Family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation inhibitor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 22 (lymphocytes)), MYH14 ( Myosin, heavy chain 14, non-muscle), MBL2 (Mannose-binding lectin (protein C) 2, soluble (opsonization-deficient)), SELPLG (Selectin P ligand), AOC3 (Amine oxidase, copper-containing 3 (vascular adhesion protein 1)), CTSL1 (Cathepsin L1), PCNA (Proliferating cell nuclear antigen), IGF2 (Insulin-like growth factor 2 (Somatomedin A)), ITGB1 (Integrin, β1 (fibronectin receptor, β polypeptide, antigen CD29 contains MDF2, MSK12)), CAST (Calpastatin), CXCL12 (Chemokine (CXC motif) ligand 12 (stromal cell-derived factor 1), IGHE (immunoglobulin heavy chain constant epsilon), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen, type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (N ADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solute carrier family 2 (facilitative glucose transporter), member 1), IL2RA (interleukin 2 receptor, α), CCL5 (chemokine (CC motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like regulator of apoptosis), CALCA (calcitonin-related polypeptide α), EIF4E (eukaryotic translation initiation factor 4E),GSTP1 (glutathione S-transferase π1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (chemokine (CC motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic, beta, receptor kinase 1), NR4A2 (nuclear receptor subfamily Milli 4, Group A, Member 2), MMP8 (Matrix metallopeptidase 8 (Neutrophil collagenase)), NPR2 (Natriuretic peptide receptor B / Guanylate cyclase B (Atrial natriuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (Glutamyl-prolyl-tRNA synthetase), PPARGC1A (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha), F12 (Coagulation factor XII (Hageman factor)), PECAM1 (Platelet / endothelial cell adhesion molecule), CCL4 (Chemoca in (CC motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gap junction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcription termination factor, RNA polymerase II), PROS1 (protein S(alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, beta (43 kDa dystrophin-associated glycoprotein)), YME1L1 ( YME1-like 1 (S. cerevisiae), CAMP (cathelicidin antimicrobial peptide), ZC3H12A (zinc finger CCCH type-containing 12A), AKR1B1 (aldo-keto reductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrix metallopeptidase 7 (matrilysin, uterus)), AHR (aryl hydrocarbon receptor), CSF1 (colony-stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (connective tissue growth factor),KCNMA1 (large conductance calcium-activated potassium channel, subfamily M, alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol-beta-methyltransferase), S100B (S100, Calcium-binding protein B), EGR1 (Early growth response 1), PRL (Prolactin), IL15 (Interleukin-15), DRD4 (Dopamine receptor D4), CAMK2G (Calcium / calmodulin-dependent protein kinase II gamma), SLC22A2 (Solute carrier family 22 (organic cation transporter), member 2), CCL11 (Chemokine (CC motif) ligand 11), PGF (B321 placental growth factor), THPO (Thrombopoietin), GP6 (Glycoprotein VI (platelet)), TACR1 (Tachykinin receptor receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, Shal-related subfamily, member 1), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelets)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ALO X12 (arachidonate 12-lipoxygenase), AHSG (α-2-HS-glycoprotein), BHMT (betaine-homocysteine ​​methyltransferase), GJA4 (gap junction protein, α4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial transport carrier; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonate 5-lipoxygenase-activating protein), NUMA1 (nuclear mitotic apparatus protein 1), CYP27B1 (cytokinin receptor activator), Chromium P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin / obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (Kelch repeat and BTB(POZ) domain containing 10), TNC (tenascin C),TYMS (thymidylate synthetase), SHCl (SHC (Src homology 2 domain-containing) transforming protein 1), LRP1 (low-density lipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokine signaling 3), ADH1B (alcohol dehydrogenase 1B (class I), beta polypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin B2) lupin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, αM (complement component 3 receptor subunit 3)), PITX2 (paired-like homeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fc fragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase) oxidase 1), GOT2 (glutamic oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclear receptor subfamily 1, group I, member 2), CRH (corticotropin-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-dependent anion channel 1), HPSE (heparanase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP-binding cascade PTK, subfamily B (MDR / TAP)), RNF123 (Ring finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase, receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5,Group A, member 1), GJB2 (gap junction protein, β2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium / hydrogen exchanger), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin / kexin type 9), FCGR2A (Fc fragment of IgG, low affinity IIa, receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (α-2 antiplasmin, pigment epithelium-derived factor), member 1), EDN3 (endothelin 3), DHFR (dihydroleaflet acid reductase), GAS6 (growth arrest specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidic lysosome), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)), TFAP2A (transcription factor AP-2α (activated enhancer binding protein 2α)), C4BPA (complement component 4 binding protein, α), SERPINF2 (serpin peptidase inhibitor, clade F (α-2 antiplasmin, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placenta ( Regan isozyme), CXCR2 (chemokine (CXC motif) receptor 2), SLC39A3 (solute carrier family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, subfamily G (WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11 (coagulation factor XI), ATP7A (ATPase, C u++ transport, alpha polypeptide), CR1 (complement component (3b / 4b) receptor 1 (Knops blood group)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated, coiled-coil-containing protein kinase 1), MECP2 (methyl-CpG-binding protein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCHE (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like),CXCR3 (chemokine (CXC motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloid polypeptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1), PTHLH (parathyroid hormone-like hormone), NRG1 (neuregulin 1), VEG FC (vascular endothelial growth factor C), ENPEP (glutamyl aminopeptidase (aminopeptidase A)), CEBPB (CCAAT / enhancer-binding protein (C / EBP), β), NAGLU (N-acetylglucosaminidase, α-), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradykinin receptor B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil globule expression), ENPP2 (ectonucleotide pyrophosphatase / phosphodiesterase 2), CISH (cytokine-inducible SH2-containing protein), GAST (gastrin), MYOC (myocilin, trabecular meshwork-induced glucocorticoid response), ATP1A2 (ATPase, Na+ / K+ transport, α2 polypeptide), NF1 (neurofibromin 1), GJB1 (gap junction protein, β1, 32 kDa), MEF2A (myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogenetic protein receptor, type II (serine / threonine)), onin kinase), TUBB (tubulin, β), CDC42 (cell division cycle 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock transcription factor 1), MYB (v-myb myeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase, AMP-activated, α2 catalytic subunit), ROCK2 (Rho-associated, coiled-coil-containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1 (protein kinase, cGMP-dependent,Type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), δ1), CTH (cystathionase (cystathionine γ-lyase)), CTSS (cathepsin S), VAV2 (vav2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase α1), PPIA (peptide amphotericin A (cyclophilin A), APOH (apolipoprotein H (beta-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin-11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1 (fibulin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide) , CHGB (chromogranin B (secretogranin 1)), PRKCB (protein kinase C, beta), SRD5A1 (steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5α-steroid Δ4-dehydrogenase α1)), HSD11B2 (hydroxysteroid (11-β) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) ferase 2 (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 4), PIK3C2A (phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DR beta 5), ​​BNIP3 (BCL2 / adenovirus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium-binding protein A12),PADI4 (peptidylarginine deiminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemokine (CXC motif) receptor 1), H19 (H19, imprinted maternally expressed transcript (non-protein coding)), KRTAP19-3 (keratin-associated protein 19-3), IDDM2 (insulin-dependent euglucanase, urinary disease 2), RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP-binding protein Rac2)), RYR1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine β-hydroxylase (dopamine β-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, α4), CACNA1C (calcium channel, voltage-gated, L-type, α1C subunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalytic subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (mortalin)), CYSLTR1 (cysteinyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I, α), OPRL1 (opiate receptor-like 1), IMPA1 (inositol (myo)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (macropain) subunit, α type 6), PS MB8 (proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (cartilage glycoprotein-39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member B1), PARP2 (poly(ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein), LBP (lipopolysaccharide-binding protein), ABCC6 (ATP-binding cassette, subfamily C (CFTR / MRP), member 6), RGS2 (regulator of G protein signaling 2,24kDa), EFNB2 (ephrin-B2), GJB6 (gap junction protein, β6, 30kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferlin, limb-girdle muscular dystrophy 2B (autosomal recessive)), FDFT1 (farnesyl diphosphate farnesyltransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (CC motif) receptor 6), GJB3 (gap junction protein, β3, 31kDa), IL1RL1 (interleukin-1 receptor receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin, EGFLAG seven-transmembrane G-type receptor 2 (flamingo homolog, Drosophila)), F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)) , ATF6 (activating transcription factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine / spermine N1-acetyltransferase 1), GGH (γ-glutamyl hydrolase (conjugase, folylpolyγglutamyl hydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute carrier family 4, sodium-bicarbonate cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B, cGMP-inhibited) sex), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and senescent cell antigen-like domain 1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phospholipase domain containing 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junction protein, gamma 1, 45 kDa),SLC17A5 (solute carrier family 17 (anion / sugar transporter), member 5), FTO (fat mass and obesity related), GJD2 (gap junction protein, δ2, 36 kDa), PSRC1 (proline / serine-rich coiled-coil 1), CASP12 (caspase 12 (gene / pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain-containing serine / threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles These proteins may include PI3K (Primer-like protein homolog 1) (Drosophila), PBX4 (Pre-B cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate lamina protein 2), TERC (telomerase RNA component), GGT2 (gamma-glutamyltransferase 2), MT-CO1 (mitochondrially encoded cytochrome c oxidase I), and UOX (urate oxidase, pseudogene).

[0284] In further embodiments, the chromosomal sequences may further be selected from Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), ApoB-100 (apolipoprotein B-100), ApoA (apolipoprotein(a)), ApoA1 (apolipoprotein A1), CBS (cystathione B-synthase), glycoprotein IIb / IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH), and combinations thereof. In one iteration, the chromosomal sequences and proteins encoded by the chromosomal sequences involved in cardiovascular disease may be selected from Cacna1C, Sod1, Pten, Ppar(α), ApoE, leptin, and combinations thereof.

[0285] kidney The present invention also contemplates delivering the CRISPR-Cas system to the kidney. Delivery strategies for inducing cellular uptake of therapeutic nucleic acids include physical force or vector systems, such as virus-based, lipid-based, or complex-based delivery, or nanocarriers. Since early applications of limited potential clinical relevance when nucleic acids were delivered systemically to kidney cells by hydrodynamic high-pressure injection, a wide range of gene therapy viral and non-viral carriers have already been applied to target post-transcriptional events in vivo in various animal kidney disease models (CsabaRevesz and Peter Hamar (2011), "Delivery Methods to Target RNAs in the Kidney", GeneTherapy Applications, Prof. Chunsheng Kang (Ed.), ISBN: 978-953-307-541-9, InTech, available from: http: / / www.intechopen.com / books / gene-therapy-applications / delivery-methods-to-target-rnas-in-the-kidney). Delivery methods to the kidney are summarized as follows:

[0286] [Table 8]

[0287] [Table 9]

[0288] [Table 10]

[0289] Yuan et al. (Am J Physiol Renal Physiol 295:F605-F617, 2008) investigated whether in vivo delivery of small interfering RNA (siRNA) targeting the 12 / 15-lipoxygenase (12 / 15-LO) pathway of arachidonic acid metabolism could ameliorate renal injury and diabetic nephropathy (DN) in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo delivery and siRNA expression in the kidney, Yuan et al. used a cholesterol-conjugated double-stranded 12 / 15-LO siRNA oligonucleotide. Approximately 400 μg of siRNA was subcutaneously injected into mice. The method of Yuan et al. can be applied to the CRISPR-Cas system of the present invention, contemplating subcutaneous injection of 1-2 g of cholesterol-conjugated CRISPR-Cas for kidney delivery in humans.

[0290] Molitoris et al. (J AmSoc Nephrol 20:1754-1764, 2009) used proximal tubule cells (PTCs) as a site of oligonucleotide reabsorption within the kidney to test the efficacy of siRNA targeted to p53, a central protein in the apoptotic pathway, in preventing renal injury. Naked synthetic siRNA against p53, injected intravenously 4 hours after ischemic injury, maximally protected both PTCs and renal function. Molitoris et al.'s data demonstrate rapid delivery of siRNA to proximal tubule cells after intravenous administration. For dose-response analysis, rats were injected with siP53 at doses of 0.33; 1, 3, or 5 mg / kg at the same four time points, resulting in cumulative doses of 1.32; 4, 12, and 20 mg / kg, respectively. All siRNA doses tested produced SCr-lowering effects on day 1, and higher doses remained effective for approximately 5 days compared with PBS-treated ischemic control rats. Cumulative doses of 12 and 20 mg / kg provided the best protective effect. The method of Molitoris et al. can be applied to the CRISPRCas system of the present invention, contemplating cumulative doses of 12 and 20 mg / kg for renal delivery to humans.

[0291] Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012) reported the toxicity and pharmacokinetic properties of the synthetic small interfering RNA I5NP after intravenous administration in rodents and non-human primates. I5NP is designed to temporarily inhibit the expression of the pro-apoptotic protein p53 by acting via the RNA interference (RNAi) pathway and is being developed to protect cells from acute ischemia / reperfusion injury, such as acute kidney injury that can occur during major cardiac surgery and delayed graft function that can occur after kidney transplantation. Doses of 800 mg / kg I5NP in rodents and 1,000 mg / kg I5NP in non-human primates were required to induce adverse effects, which were determined to lead to hematological effects in monkeys, including subclinical activation of complement and a slight increase in clotting time. No additional adverse effects were observed in rats with the rat analog of I5NP, indicating that these effects likely represent class effects of synthetic RNA duplexes rather than toxicity related to the intended pharmacological activity of I5NP. Collectively, these data support clinical trials of intravenous administration of I5NP for preserving renal function after acute ischemia / reperfusion injury. The no-observed-adverse-effect level (NOAEL) in monkeys was 500 mg / kg. No effects on cardiovascular, respiratory, or neurological parameters were observed after intravenous administration at dose levels up to 25 mg / kg in monkeys. Therefore, similar dosages may be contemplated for intravenous administration of CRISPRCas to the human kidney.

[0292] Shimizu et al. (J Am Soc Nephrol 21:622-633, 2010) developed a system for targeted delivery of siRNA to the glomerulus using a poly(ethylene glycol)-poly(L-lysine)-based vehicle. The siRNA / nanocarrier complexes were approximately 10-20 nm in diameter, a size that could allow the complexes to pass through fenestrated endothelium and reach the mesangium. After intraperitoneal injection of fluorescently labeled siRNA / nanocarrier complexes, Shimizu et al. detected siRNA in the blood circulation for a long period of time. Repeated intraperitoneal administration of mitogen-activated protein kinase 1 (MAPK1) siRNA / nanocarrier complexes suppressed glomerular MAPK1 mRNA and protein expression in a mouse model of glomerulonephritis. To examine siRNA accumulation, BALB-c mice were administered Cy5-labeled siRNA complexed with PIC nanocarriers (0.5 ml, 5 nmol siRNA content), naked Cy5-labeled siRNA (0.5 ml, 5 nmol), or Cy5-labeled siRNA encapsulated in HVJ-E (0.5 ml, 5 nmol siRNA content). The Shimizu et al. method can be applied to the CRISPR Cas system of the present invention, allowing for intraperitoneal administration and delivery to the kidney of approximately 10-20 μmol CRISPR Cas complexed with nanocarriers in approximately 1-2 liters of blood.

[0293] lung The present invention also contemplates delivering the CRISPR-Cas system to one or both lungs.

[0294] Although AAV-2-based vectors were initially proposed for CFTR delivery to CF airways, other serotypes, such as AAV-1, AAV-5, AAV-6, and AAV-9, have demonstrated improved gene transfer efficiency in various lung epithelial models (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 December 2009). AAV-1 transduces human airway epithelial cells approximately 100-fold more efficiently than AAV-2 and AAV-5 in vitro,5 however, AAV-1 transduces mouse tracheal epithelium with comparable efficiency to AAV-5 in vivo. Other studies have shown that AAV-5 delivers genes 50-fold more efficiently to human airway epithelium (HAE) in vitro and significantly more efficiently to mouse lung airway epithelium in vivo than AAV-2. AAV-6 has also been shown to be more efficient than AAV-2 in human airway epithelial cells in vitro and in mouse airways in vivo. The recent isolate AAV-9 has been shown to exhibit higher gene transfer efficiency than AAV-5 in mouse nasal and alveolar epithelia in vivo, with gene expression detected for up to 9 months, suggesting that AAV may be capable of achieving long-term gene expression in vivo, a desirable characteristic for a CFTR gene delivery vector. Furthermore, it has been demonstrated that AAV-9 can be readministered to mouse lungs without loss of CFTR expression and with minimal immunological consequences. 100 μl of AAV vector can be inoculated onto the apical surface of CF and non-CFHAE cultures for several hours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077, December 2009). MOIs range from 1 × 10 to 1 × 10 depending on the virus concentration and experimental objectives. 3 From 4×10 5 Even the vector genome / cell may vary. The above-cited vectors are contemplated for delivery and / or administration of the present invention.

[0295] Zamora et al. (Am J Respir Crit CareMed Vol 183, pp 531-538, 2011) reported the application of RNA interference therapeutics to the treatment of human infections and also a randomized trial of antiviral drugs in lung transplant recipients infected with respiratory syncytial virus (RSV). Zamora et al. conducted a randomized, double-blind, placebo-controlled trial in LTX recipients with RSV airway infection. Patients were allowed to receive standard treatment for RSV. Aerosolized ALN-RSV01 (0.6 mg / kg) or placebo was administered daily for three days. This study demonstrated that RNAi therapeutics targeting RSV can be safely administered to LTX recipients with RSV infection. Three daily doses of ALN-RSV01 did not result in exacerbation of respiratory tract symptoms or pulmonary dysfunction, and did not exhibit systemic pro-inflammatory effects such as induction of cytokines or CRP. Pharmacokinetics showed only low and transient systemic exposure after inhalation, consistent with preclinical animal data showing that ALN-RSV01 administered intravenously or by inhalation is rapidly cleared from the circulation by exonuclease-mediated digestion and renal excretion. The method of Zamora et al. can be applied to the CRISPR Cas system of the present invention, and aerosolized CRISPR Cas can be used in the present invention at a dosage of, for example, 0.6 mg / kg.

[0296] For an example of a CFTR Δ508 chimeric guide RNA, see Example 22, which demonstrates the use of adeno-associated virus (AAV) particles to transduce or deliver genes using a CRISPR-Cas system in the airways of a subject or patient in need who suffers from cystic fibrosis or a cystic fibrosis (CF)-related condition. Specifically, a strategy for repairing the cystic fibrosis ΔF508 mutation is exemplified. This type of strategy should be applicable across all organisms. With particular reference to CF, suitable patients may include: humans, non-human primates, dogs, cats, cattle, horses, and other livestock. In this example, applicants utilized a CRISPR-Cas system containing the Cas9 enzyme to target ΔF508 or other CFTR-induced mutations.

[0297] In this example, the subject receives intrabronchial delivery of a pharmaceutically effective amount of aerosolized AAV vector system per lung while breathing spontaneously. Thus, aerosolized delivery is generally preferred for AAV delivery. Adenovirus or AAV particles can be used for delivery. Suitable gene constructs, each operably linked to one or more regulatory sequences, can be cloned into the delivery vector. In this example, the following constructs are provided as examples: a Cbh or EF1a promoter for Cas9, a U6 or H1 promoter for a chimeric guide RNA: a preferred configuration uses a chimeric guide targeting CFTR Δ508, a repair template for the ΔF508 mutation, and a codon-optimized Cas9 enzyme (preferably a Cas9 with nuclease or nickase activity), optionally with one or more nuclear localization signals or sequences (NLS), for example, two NLSs. NLS-free constructs are also envisioned.

[0298] To identify Cas9 target sites, Applicants analyzed the human CFTR genomic locus and identified Cas9 target sites. Preferably, in general, and for this CF, the PAM may contain an NGG or NNAGAAW motif.

[0299] Thus, in the case of CF, the method involves: Delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding the composition for expression of the c...

Claims

1. 1. A method for altering expression of one or more gene products, comprising introducing into a non-human eukaryotic organism an engineered, non-naturally occurring CRISPR-Cas vector system; The vector system comprises: a) a first regulatory element operably linked to a nucleotide sequence encoding a Cas9 protein comprising one or more nuclear localization signaling (NLS); and b) a second regulatory element operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA, wherein the guide RNA comprises a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of a CRISPR-Cas complex to the target sequence, a tracr mate sequence capable of hybridizing to a tracr sequence, and a tracr sequence. and one or more vectors comprising components (a) and (b) are located on the same or different vectors of the system, and the target sequence is involved in the expression of the one or more gene products; method.

2. 2. The method of claim 1, wherein the tracr sequence comprises at least 40 nucleotides in length.

3. 2. The method of claim 1, wherein the tracr sequence comprises at least 50 nucleotides in length.

4. The method according to any one of claims 1 to 3, wherein the non-human eukaryote is a mammal.

5. 5. The method of any one of claims 1 to 4, wherein the Cas9 protein is mutated relative to a corresponding wild-type Cas9 protein, and the mutant protein is a nickase that lacks the ability to cleave one strand of a target polynucleotide.

6. 6. The method of claim 5, wherein the Cas9 protein comprises one or more mutations in the RuvCI, RuvCII, or RuvCIII catalytic domain.

7. 6. The method of claim 5, wherein the Cas9 protein comprises a mutation selected from the group consisting of D10A, H840A, N854A, and N863A with reference to the position numbering of the Streptococcus pyogenes Cas9 (SpCas9) protein.

8. 8. The method of any one of claims 1 to 7, wherein the Cas9 protein comprises two or more NLSs.

9. 9. The method of claim 8, wherein at least one NLS is present at or near the amino terminus of the Cas9 protein and / or at least one NLS is present at or near the carboxy terminus of the Cas9 protein.

10. 10. The method of claim 9, wherein at least one NLS is present at or near the amino terminus of the Cas9 protein and at least one NLS is present at or near the carboxy terminus of the Cas9 protein.

11. 11. The method of any one of claims 1 to 10, wherein the nucleotide sequence encoding Cas9 is codon-optimized for expression in a eukaryotic cell.

12. The method of any one of claims 1 to 11, wherein the one or more vectors are viral vectors.

13. 13. The method of claim 12, wherein the viral vector is a retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vector.

14. The method of any one of claims 1 to 13, wherein components (a) and (b) are located on the same vector.

15. The method of any one of claims 1 to 13, wherein components (a) and (b) are located on different vectors.

16. 16. The method of any one of claims 1 to 15, wherein the guide RNA is a chimeric RNA comprising a guide sequence, a tracr sequence, and a tracr mate sequence in a single transcript.

17. The method of any one of claims 1 to 16, wherein the expression of said one or more gene products is reduced.

18. 1. A method for altering expression of one or more gene products, comprising introducing an engineered, non-naturally occurring CRISPR-Cas system into a non-human eukaryote; The system is a) a Cas9 protein comprising one or more NLSs; and b) A CRISPR-Cas system guide RNA comprising a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of a CRISPR-Cas complex to the target sequence, a tracr mate sequence capable of hybridizing to a tracr sequence, and a tracr sequence at least 40 nucleotides in length. Including, the target sequence is involved in the expression of the one or more gene products; method.

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