Delivery, use and therapeutic applications of crispr-cas systems and compositions for genome editing

Abstract

To provide alternative and robust systems for therapeutic strategies that are able to modify nucleic acids within disease-affected cells and tissues.SOLUTION: A method of modeling a disease associated with a genomic locus in a eukaryotic organism or a non-human organism comprises manipulation of a target sequence within a coding, non-coding or regulatory element of the genomic locus comprising a step of delivering a non-naturally occurring or engineered composition comprising (A) a CRISPR-Cas system RNA polynucleotide sequence, and a polynucleotide sequence encoding Cas9, optionally comprising at least one or more nuclear localization sequences, or (B) polynucleotides comprising a polynucleotide sequence encoding Cas9 and a polynucleotide sequence comprising a tracr sequence.SELECTED DRAWING: None

Description

Technical field

[0001] Related Applications and Incorporation by Reference 61 / 915,192; 61 / 915,215; 61 / 915,107, each filed December 12, 2013. 61 / 915,145; 61 / 915,148; and 61 / 915,153.

[0002] the above applications, and all documents cited in these applications or cited during the prosecution of these applications ("application citations"), and all documents cited or referenced in this application citations; and All documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in the cited documents herein This specification, along with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned in any document incorporated by reference, are hereby incorporated by reference and used in the practice of this invention. can be used. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] The present invention generally relates to systems, methods for controlling gene expression involving sequence targeting involving clustered regularly spaced short palindromic repeats (CRISPR) and their constituents, such as genome perturbation or gene editing. , and delivery, engineering, optimization and therapeutic applications of the compositions. Specifically, the present invention provides in vitro, ex vivo and / or in vivo systems, methods and compositions for delivering CRISPR-Cas systems for therapeutic benefit from genome editing in animals, including mammals. about things.

[0004] STATEMENT ON FEDERALLY SPONSORED RESEARCH This invention is based on an NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health and grant 1DP1OD009552 also awarded by the National Institutes of Health. It was done with the support of the federal government. The federal 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 associated with a diverse range of biological functions and diseases. Precise genomic targeting techniques enable systematic reverse engineering of causative gene variants by allowing selective perturbation of individual genetic elements, as well as for synthetic biology, biotechnology and pharmaceutical applications. needed to advance Genome editing techniques such as designer zinc fingers (ZFNs), transcription activator-like effectors (TALEs), or homing meganucleases are available for the production of targeted genomic perturbations, but are inexpensive and easy to set up. There remains a need for new genome engineering techniques that are scalable and amenable to targeting multiple locations within the eukaryotic genome. [Outline of the invention] [Means for solving the problem]

[0006] Despite valid therapeutic hypotheses and strong efforts in drug development, there are a limited number of successful examples of using small molecules to treat diseases with strong genetic involvement. Therefore, there is an urgent need for alternative and robust systems towards therapeutic strategies capable of modifying nucleic acids within disease-affected cells and tissues. The addition of the CRISPR-Cas system to the repertoire of therapeutic genome engineering methods greatly simplifies methodologies and increases the ability to enumerate and map genetic factors associated with various biological functions and diseases, and to identify inherited diseases. The ability to develop animal models and to develop safe and effective therapeutic options will be facilitated. Understanding the manipulation, optimization and cell-type / tissue / organ-specific delivery of these genome engineering tools is critical for the effective use of CRISPR-Cas systems for genome editing without adverse effects. are aspects of the claimed invention. Aspects of the present invention address this need and provide related advantages.

[0007] An exemplary CRISPR complex can include a CRISPR enzyme (eg, Cas9) complexed with a guide sequence that hybridizes to a target sequence within a target polynucleotide. The guide sequence is linked to the tracr mate sequence, which in turn hybridizes to the tracr sequence. Applicants have optimized the components of the CRISPR-Cas genome engineering system, including using SaCas9 from Staphylococcus aureus. A variety of delivery means can be used to deliver the components of the CRISPR-Cas system to cells, tissues and organs ex vivo and / or in vivo. Applicants have effectively packaged CRISPR-Cas system components (including, for example, SaCas9) into viral delivery vectors, such as AAV, which are used to extract endogenous genomic sequences of mammalian cells in vivo. It proves that it can be changed. An important feature of Applicants' invention is that it effectively addresses the problem of low in vivo delivery efficiency (of therapeutic components) and low homology dependent repair (HDR) efficiency, in particular: A challenge associated with co-delivery is the small Cas9, yellow grape, which can be easily packaged into a single adeno-associated virus (AAV) vector to express both the Cas9 protein and its corresponding one or more sgRNAs. It is solved by SaCas9 from Staphylococcus aureus. Furthermore, importantly, applicants show that introduction of the small SaCas9 reduced the number of viral vectors required to perform HDR from three vectors to two. In embodiments of the invention, particles may be used to deliver one or more components of the CRISPR-Cas system. And the number of particles contacted can be one or two. In one aspect, the invention provides methods of using one or more elements of the CRISPR-Cas system. The CRISPR complexes of the invention provide an effective means of altering target polynucleotides at genomic loci, where the genomic loci contain mutations associated with abnormal protein expression or disease conditions or conditions. , associated with the mutation. The CRISPR complexes of the invention include modifying (e.g., deleting, inserting) a target polynucleotide within a genomic locus, e.g., within a coding, non-coding or regulatory element of such target locus. It has a wide range of utility, including activating, translocating, inactivating, activating). The CRISPR complexes of the invention therefore have a wide range of applications, for example, in gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis. Embodiments of the present invention are Cas9 enzymes with improved targeting specificity in CRISPR-Cas9 systems having guide RNAs of optimal activity that are shorter in length compared to the wild-type Cas9 enzyme and the nucleic acid molecule encoding it, and chimeric Cas9 enzymes A Cas9 enzyme and a method of improving the target specificity of the Cas9 enzyme or designing a CRISPR-Cas9 system, wherein the step of designing or preparing a guide RNA of optimal activity and / or size or size relative to wild-type Cas9. selecting or preparing a Cas9 enzyme that is smaller in length and therefore less encoded in the delivery vector compared to wild-type Cas9, making packaging of the nucleic acid encoding it in the delivery vector more favorable; and / or chimeras. A method comprising making a Cas9 enzyme. Also provided is the use of the sequences, vectors, enzymes or systems of the invention in medicine. Also provided is its use in gene or genome editing.

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

[0009] It will be understood that the terms Cas and CRISPR enzyme are generally used synonymously herein unless otherwise specified. 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 appreciated that the invention includes even more Cas9 from other microbial species such as SpCas9, SaCas9, St1Cas9. Further examples are provided herein. Those skilled in the art will be able to determine the appropriate corresponding residues in Cas9 enzymes other than SpCas9 by comparison of relevant amino acid sequences. Thus, unless it is clear from the context that when a particular amino acid substitution is referred to using SpCas9 numbering, it is intended not to refer to other Cas9 enzymes, this disclosure does not refer to other Cas9 enzymes. It is intended to cover corresponding modifications. Examples of human-optimized (ie, optimized for human expression) codon-optimized sequences in this case are provided herein (see SaCas9 human codon-optimized sequences). Although this is preferred, it is understood that other examples are possible and codon optimization for the host species is known. The invention encompasses methods wherein Cas9 is a chimeric Cas9 protein. These methods may involve one or more N-terminal fragments of one Cas9 homologue together with one or more C-terminal fragments of one or more other or distinct Cas9 homologues. In the present methods, it will be appreciated that where the organism is an animal, the modification may be performed ex vivo or in vitro, eg, in cell culture, optionally other than in vivo. In other embodiments, the modification can occur in vivo. The invention encompasses, in some embodiments, a composition of the invention or a CRISPR enzyme thereof (including or alternatively to an mRNA encoding the CRISPR enzyme), wherein the target sequence is specifically Cas9. If it is (or is derived from) S. pyogenes or S. aureus Cas9, it is flanked at its 3' end by a PAM (protospacer adjacent motif) sequence containing a 5'-motif. . For example, a suitable PAM is 5'-NRG or 5'-NNGRR (where N is any nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes). It will be appreciated that SpCas9 or SaCas9 is derived from or derived from S. pyogenes or S. aureus Cas9.

[0010] In one aspect, the invention provides: A) I. A CRISPR-Cas based chimeric RNA (chiRNA) polynucleotide sequence comprising: (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence a polynucleotide sequence comprising II. Polynucleotide Sequences Encoding CRISPR Enzymes Containing At Least One or More Nuclear Localization Sequences [(a), (b) and (c) are aligned in the 5′ to 3′ direction, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces 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 a polynucleotide sequence encoding the CRISPR enzyme. is DNA or RNA] providing a method of modifying an organism or non-human organism by manipulation of a target sequence at a genomic locus of interest associated with mutations associated with aberrant protein expression or a disease condition or condition, comprising; The method can optionally also include delivering the HDR template, eg, via a viral delivery vector or particle, wherein the HDR template directs expression of the normal or low-abnormal protein. result; "normal" may be with respect to wild type, and "abnormal" may be protein expression that causes a pathology or disease state; and Optionally, the method comprises isolating or obtaining cells expressing said abnormal protein from an organism or non-human organism, optionally expanding this cell population, one or more viral vectors or particles and said cells. to obtain a population of modified cells, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism.

[0011] In one aspect, the invention provides I. (a) a guide sequence hybridizable to a target sequence in HSCs, and (b) at least one or more tracr mate sequences, II. and III. a polynucleotide sequence comprising a tracr sequence [the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex comprises 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. to produce an organism or non-human organism by manipulating a target sequence at a genomic locus of interest associated with aberrant protein expression or mutations associated with a disease condition or condition, comprising contacting the cell with a viral vector or particle containing provide a way to modify; and The method can optionally also include delivering the HDR template, eg, via a viral delivery vector or particle, wherein the HDR template directs expression of the normal or low-abnormal protein. result; "normal" may be with respect to wild type, and "abnormal" may be protein expression that causes a pathology or disease state; and Optionally, the method comprises isolating or obtaining cells expressing said abnormal protein from an organism or non-human organism, optionally expanding this cell population, one or more viral vectors or particles and said cells. to obtain a population of modified cells, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism.

[0012] This delivery can be any one of the CRISPR complexes, e.g., via one or more particles containing a vector containing one or more polynucleotides operably linked to one or more regulatory elements. Delivery of one or more polynucleotides encoding any of the above or all, advantageously linked to one or more regulatory elements for expression in vivo. Part or all of the polynucleotide sequence encoding the CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence may be RNA. When reference is made to a polynucleotide that is RNA and is said to "comprising" such tracr mate sequence characteristics, it will be understood that RNA sequences possess those characteristics. Where the polynucleotide is DNA and is said to contain the characteristic of such a tracr mate sequence, the DNA sequence is or can be transcribed into RNA containing the characteristic of interest. If the feature is a protein, such as a CRISPR enzyme, the DNA or RNA sequence referred to is (in the case of DNA, first transcribed and then) translated or can be translated.

[0013] In certain embodiments, the present invention comprises delivering a non-naturally occurring or engineered composition to a cell or cell population, e.g., by contacting a genomic locus of interest, e.g. or a method of modifying an organism, e.g., a mammal, including a human, or a non-human mammal or organism by manipulation of a target sequence at a genomic locus of interest associated with a mutation associated with a disease condition or condition, wherein the composition The article comprises one or more delivery vectors or particles comprising one or more viral, plasmid or nucleic acid molecule vectors (e.g. RNA) functionally encoding the composition to express the composition, the composition (A) I. A first regulatory element operably linked to a CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence is (a) a target sequence in a eukaryotic cell; a guide sequence, (b) a tracr mate sequence, and (c) a tracr sequence, and II. at least one or more nuclear localization sequences (or in some embodiments A second regulatory element operably linked to an enzyme-encoding sequence encoding a CRISPR enzyme [(a) , (b) and (c) are aligned in the 5' to 3' direction, components I and II are located in the same or different vectors of the system, and when transcribed the tracr mate sequence hybridizes to the tracr sequence. and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) a tracr sequence that hybridizes to the tracr sequence. CRISPR enzyme complexed with a mate sequence], or (B) a non-naturally occurring or engineered composition wherein I. (a) a guide is hybridizable 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 operably linked to an enzyme coding sequence encoding a CRISPR enzyme. and III. A third regulatory element operably linked to the tracr sequence [Components I, II and III are located in the same or different vectors of the system and, when transcribed, the tracr mate sequence becomes the tracr sequence. and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex hybridizes to (1) the guide sequence that hybridizes to the target sequence and (2) the tracr sequence. comprising a CRISPR enzyme complexed with a soybean tracr mate sequence]; the method optionally includes contacting, e.g., with a cell or cell population It can also include delivering the HDR template via the delivery vector or particle or by contacting the cell, cell or cell population with another delivery vector or particle containing the HDR template, wherein the HDR template results in normal or mildly abnormal protein expression; "normal" refers to wild type and "abnormal" may be protein expression that causes a pathology or disease state; isolating or obtaining from an organism or non-human organism cells expressing said aberrant protein; optionally expanding said cell population; expressing said aberrant protein with one or more delivery vectors or particles; Contacting said cells to obtain a population of modified cells, optionally expanding the population of modified cells, and optionally administering the modified cells to the organism or non-human organism. In some embodiments, components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on a separate vector. In other embodiments, components I and III are located on the same vector, while component II is located on a separate vector. In other embodiments, components II and III are located on the same vector, while component I is located on a separate vector. In other embodiments, each of components I, II and III are located on different vectors. The invention also provides viral or plasmid vector systems as described herein.

[0014] By target sequence manipulation, Applicants also mean epigenetic manipulation of the target sequence. This may be through modification of the methylation status of the target sequence (i.e. methylation or methylation patterns or addition or removal of CpG islands), histone modifications, increased or decreased accessibility to the target sequence, or by tertiary folding. It may also be manipulation of the chromatin state of the target sequence, such as by promoting Where reference is made to a method of modifying an organism or mammal, including humans, or a non-human mammal or organism by manipulation of a target sequence at a genomic locus of interest, this may apply to the organism (or mammal) as a whole. , or (if the organism is a multicellular organism) it may apply only to a single cell or cell population of the organism. For example, in the case of humans, Applicants specifically envisage 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, of course in vivo embodiments are also envisioned. And the invention is particularly advantageous with respect to eye cells, retinal cells, vascular cells, epithelial cells, endothelial cells, and cochlear cells.

[0015] The present invention provides, in some embodiments, for example: I. A first CRISPR-Cas-based chimeric RNA (chiRNA) polynucleotide sequence comprising: (a) a first guide sequence hybridizable to the first target sequence; (b) a first tracr mate sequence, and (c) the first tracr sequence a first polynucleotide sequence comprising II. A second CRISPR-Cas-based chiRNA polynucleotide sequence comprising: (a) a second guide sequence hybridizable to a second target sequence; (b) a second tracr mate sequence, and (c) a second tracr sequence a second polynucleotide sequence comprising, and III. A polynucleotide sequence encoding a CRISPR enzyme comprising at least one nuclear localization sequence and comprising one or more mutations [(a), (b) and (c) 5' to 3' line up in the direction of]; or IV. One or more expression products of one or more of I. to III., such as first and second tracr mate sequences, CRISPR enzyme; [When transcribed, the first and second tracr mate sequences hybridize to the first and second tracr sequences, respectively, and the first and second guide sequences form the first and second CRISPR complexes, respectively. and the first and second target sequences, wherein the first CRISPR complex comprises (1) a first guide sequence that hybridizes to the first target sequence, and (2) a second a CRISPR enzyme complexed with a first tracr mate sequence that hybridizes to one tracr sequence, the second CRISPR complex comprising: (1) a second guide sequence that hybridizes to a second target sequence; and (2) a CRISPR enzyme complexed with a second tracr mate sequence that hybridizes to a second tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA; The guide sequence induces cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence induces cleavage of the other strand near the second target sequence to form two by contacting a cell or population of cells with a delivery vector, such as a viral vector or particle, comprising a non-naturally occurring or engineered composition comprising opposite strands of a DNA duplex at a genomic locus of interest in a cell or population of cells, for example associated with aberrant protein expression or a mutation associated with a disease condition or condition. includes a method of modifying an organism or non-human organism by manipulation of the above first and second target sequences; It can also include delivering the HDR template via a delivery vector that contacts the population or by contacting the cell or cell population with another delivery vector that contains the HDR template, wherein the HDR template is normally result in the expression of a type or mildly aberrant form of the protein; "normal" may be with respect to wild-type, and "abnormal" may be protein expression that causes a pathology or disease state; Isolating or obtaining a cell or cell population from an organism or non-human organism, optionally expanding this cell population, contacting one or more delivery vectors or particles with the cell or cell population to modify it. obtaining a modified cell population, optionally expanding the population of modified cells. A method of modeling a disease associated with a genomic locus in a eukaryotic or non-human organism comprising a viral vector system comprising one or more viral vectors functionally encoding a composition for expression thereof delivering a non-naturally occurring or engineered composition comprising (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence comprising (a) a guide sequence hybridizable to the target sequence; (b) a tracr mate sequence, and (c) tracr sequence a first regulatory element comprising, and II. A second regulatory element operably linked to the enzyme coding sequence encoding SaCas9, optionally comprising at least one or more nuclear localization sequences [(a), (b) and (c) are aligned in the 5′ to 3′ direction, Components I and II are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) SaCas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence. a composition comprising a system; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element comprising (a) a guide sequence hybridizable to the target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to the enzyme coding sequence encoding SaCas9, and III. A Third Regulatory Element Functionally Linked to the tracr Sequence [Components I, II and III are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) SaCas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence. manipulating a target sequence within a coding, non-coding or regulatory element of said genomic locus, comprising comprising a composition comprising the system, and optionally administering the modified cell to an organism or non-human organism. method including. In some methods of the invention, part or all of a polynucleotide sequence encoding a CRISPR enzyme, first and second guide sequences, first and second tracr mate sequences, or first and second tracr sequences is RNA. In a further embodiment of the invention, the polynucleotide encoding the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or the first and second tracr sequences is RNA. Yes, delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, delivery is advantageously by viral vectors or particles. In certain embodiments of the 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, a polynucleotide can be contained within a vector system that includes one or more vectors. In a preferred embodiment of the invention the CRISPR enzyme is a Cas9 enzyme, eg SpCas9 or SaCas9. In embodiments of the invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, wherein the one or more mutations are from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A with reference to SpCas9. Selected, for example the D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme and the second CRISPR enzyme is such that the enzyme is a non-complementary strand nicking enzyme. have one or more mutations such as Alternatively, the first enzyme may be a non-complementary strand nicking enzyme and the second enzyme may be a complementary strand nicking enzyme. In a preferred method of the invention, a first guide sequence induces cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence near a second target sequence. A 5' overhang is generated by inducing cleavage of the other strand. 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.

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

[0017] The present invention provides, in some embodiments, for example: I. A first regulatory element comprising (a) a first guide sequence hybridizable to the first target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element comprising (a) a second guide sequence hybridizable to a second target sequence, and (b) at least one tracr mate sequence a second regulatory element operably linked to III. A third regulatory element operably linked to an enzyme-coding sequence that encodes a CRISPR enzyme, and IV. a fourth regulatory element operably linked to the tracr sequence; one or more expression products of one or more of V.I.-IV., such as first and second tracr mate sequences, CRISPR enzyme; [When components I, II, III and IV are located in the same or different vectors of the system and are transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the first and second guide and a first guide sequence that induces sequence-specific binding of the first and second CRISPR complexes to a second target sequence, the first CRISPR complex hybridizing to (1) the first target sequence and (2) a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to the tracr sequence, the second CRISPR complex comprising: (1) a second guide sequence that hybridizes to a second target sequence; and (2) a CRISPR enzyme complexed with a tracr mate sequence that hybridizes to the tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and the first guide sequence is the first Inducing cleavage of one strand of a DNA duplex near the target sequence and a second guide sequence inducing cleavage of the other strand near the second target sequence to produce a double-strand break , thereby modifying the organism or non-human organism] by contacting the cell or cell population with one or more delivery vectors or particles comprising the non-naturally occurring or engineered composition a first and a first on opposite strands of a DNA duplex at a genomic locus of interest in a cell or population of cells that is associated with aberrant protein expression or a mutation associated with a disease condition or condition; and optionally via a delivery vector or particle that contacts a cell or cell population containing the HDR template, e.g. , or by contacting the cell or cell population with another particle containing the HDR template, wherein the HDR template expresses a normal or low-abnormal form of the protein. "Normal" may refer to wild-type, and "abnormal" may be protein expression that causes a pathology or disease state; optionally expanding the cell; contacting the cell or cell population with one or more delivery vectors or particles to obtain a modified cell population; and optionally administering the modified HSCs to the organism or non-human organism.

[0018] The invention also provides vector systems as described herein. The system may contain 1, 2, 3 or 4 different vectors. Components I, II, III and IV may therefore be located in 1, 2, 3 or 4 different vectors, and all possible combinations of component positions are envisaged herein, For example: components I, II, III and IV may be located on the same vector; components I, II, III and IV may each be located on different vectors in all possible combinations of positions. ; components I, II, III and IV may be located in a total of 2 or 3 different vectors, and so on. In some methods of the invention, part or all of a polynucleotide sequence encoding a CRISPR enzyme, first and second guide sequences, first and second tracr mate sequences, or first and second tracr sequences is RNA. In further embodiments of the 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 invention the CRISPR enzyme is a Cas9 enzyme, eg SpCas9. In embodiments of the invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, wherein the one or more mutations are from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A with reference to SpCas9. Selected; for example the D10A mutation. In a preferred embodiment, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme and the second CRISPR enzyme is such that the enzyme is a non-complementary strand nicking enzyme. have one or more mutations such as Alternatively, the first enzyme may be a non-complementary strand nicking enzyme and the second enzyme may be a complementary strand nicking enzyme. In further embodiments of the invention, one or more of the viral vectors are delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; however, viral or particle delivery is advantageous.

[0019] In a preferred method of the invention, a first guide sequence induces cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence near a second target sequence. A 5' overhang is generated by inducing cleavage of the other strand. 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.

[0020] The invention provides, in some embodiments, for example, a Cas protein with one or more mutations and two guides that target the first and second strands, respectively, of a DNA molecule in a cell or cell population. One or more delivery vectors or particles comprising RNA are introduced into the cell or cell population by contacting the cell or cell population, whereby the guide RNA targets the DNA molecule, and the Cas protein targets the DNA molecule. By nicking each of the first strand and the second strand, thereby altering the target in the cell or cell population (and the Cas protein and the two guide RNAs do not naturally exist together), the cell or A method of altering a genomic locus of interest in a cell population, for example associated with aberrant protein expression or a mutation associated with a disease condition or condition, and the method ( mthod) optionally, e.g., via a delivery vector or particle in contact with a cell or cell population containing the HDR template, or into another delivery vector or particle containing the HDR template. , wherein the HDR template results in expression of a normal or low-aberrant protein; "normal" refers to wild-type; and An "abnormality" may be a protein expression that causes a pathology or disease state; and optionally the method comprises isolating or obtaining cells from an organism or non-human organism, optionally expanding this cell population. contacting cells with one or more delivery vectors or particles to obtain a population of modified cells; optionally expanding the population of modified cells; A step of administering to the organism can be included. In preferred methods of the invention, the Cas protein nicks each of the first and second strands of the DNA molecule to create a 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. Embodiments of the invention also include a guide sequence and a guide RNA comprising a tracr sequence fused to a tracr mate sequence. In embodiments of the invention, the Cas proteins are 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, eg a Cas9 protein. In a highly preferred embodiment the Cas protein is a Cas9 protein, eg SpCas9 or SaCas9. In embodiments of the invention, the Cas protein is based on SpCas9 and has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A; for example the D10A mutation. Aspects of the invention allow for reduced expression of the gene product, or further introduction of a template polynucleotide into the DNA molecule encoding the gene product, or reannealing and ligation of the two 5' overhangs. This relates to the precise excision of the intervening sequence, or to altering the activity or function of the gene product, or to increasing the expression of the gene product. In some embodiments of the invention the gene product is a protein.

[0021] The present invention provides, in some embodiments, for example: a) a first operably linked to each of two CRISPR-Cas system guide RNAs that target the first and second strands, respectively, of a double-stranded DNA molecule of cells within a cell or population of cells; the regulatory element of, and b) a second regulatory element operably linked to the Cas protein, or c) one or more expression products of a) or b), introducing into a cell or cell population by contacting the cell or cell population one or more delivery vectors or particles comprising [components (a) and (b) are located on the same or different vectors of the system]; By thereby targeting the guide RNA to a DNA molecule of a cell or cell within a population of cells and the Cas protein nicking each of the first and second strands of the DNA molecule of that cell or cells within that cell (and the Cas protein and the two guide RNAs do not naturally exist together), the genomic locus of interest in a cell or population of cells, e.g. and optionally via a delivery vector or particle in contact with, for example, a cell or cell population containing the HDR template, or another particle containing the HDR template. A step of delivering the HDR template by contacting the cell or cell population with a and the "abnormality" may be a protein expression that causes a pathology or disease state; and optionally the method comprises isolating or obtaining a cell from an organism or non-human organism; expanding the population, contacting the cells with one or more delivery vectors or particles to obtain a modified cell population, optionally expanding the population of modified cells, and optionally modifying A step of administering the cell to an organism or non-human organism can be included. In embodiments of the invention, the guide RNA may comprise a guide sequence and a tracr sequence fused to a tracr mate sequence. In certain embodiments of the invention, the Cas protein is a type II CRISPR-Cas protein. In embodiments of the invention, the Cas proteins are 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, eg a Cas9 protein. In a highly preferred embodiment the Cas protein is a Cas9 protein, eg SpCas9 or SaCas9. In embodiments of the invention, the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A relative to SpCas9; for example the D10A mutation. Aspects of the invention allow for reduced expression of the gene product, or further introduction of a template polynucleotide into the DNA molecule encoding the gene product, or reannealing and ligation of the two 5' overhangs. This relates to the precise excision of the intervening sequence, or to altering the activity or function of the gene product, or to increasing the expression of the gene product. In some embodiments of the invention the gene product is a protein. In a preferred embodiment of the invention, the vector of the system is a viral vector. In further embodiments, the vectors of the system are delivered by liposomes, nanoparticles, exosomes, microvesicles, or gene guns; and particles are preferred. In one aspect, the invention provides a method of modifying a target polynucleotide in a cell or cell population. In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide, causing cleavage of said target polynucleotide, thereby modifying the target polynucleotide, wherein The CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within said target polynucleotide, said guide sequence linked to a tracr mate sequence which in turn is linked to a tracr sequence. hybridize to In some embodiments, said cleaving comprises cleaving one or two strands at the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, said repair comprising one or more nucleotides of said target polynucleotide resulting in mutations, including insertions, deletions, or substitutions of In some embodiments, said mutation results in one or more amino acid changes in the protein expressed from the gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors or one or more expression products thereof to said cell or cell population, e.g., via one or more delivery vectors or particles, Here, the one or more vectors drive expression of one or more of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, the vector is delivered to a cell or cell population of interest. In some embodiments, the modifying step is performed on the cell or cell population in cell culture. In some embodiments, the method further comprises isolating said cell or cell population from a subject prior to said modifying step. In some embodiments, the method further comprises returning said cell or cell population and / or cells derived therefrom to said subject.

[0022] In one aspect, the invention provides a method of making a cell or cell population comprising a mutated disease gene. In some embodiments, a disease gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises the steps of: (a) introducing one or more vectors or one or more expression products thereof into a cell or cell population, e.g., via one or more delivery vectors or particles; the one or more vectors driving expression of one or more of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence; and (b) allowing the CRISPR complex to bind to the target polynucleotide. A step wherein cleavage of a target polynucleotide within the disease gene occurs [the CRISPR complex hybridizes to (1) a guide sequence that hybridizes to a target sequence within the target polynucleotide, and (2) a tracr sequence; a CRISPR enzyme complexed with a tracr mate sequence that provides a mutated disease gene], thereby producing a cell or population of cells containing the mutated disease gene. In some embodiments, said cleaving comprises cleaving one or two strands at the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, said repair comprising one or more nucleotides of said target polynucleotide resulting in mutations, including insertions, deletions, or substitutions of In some embodiments, said mutation results in one or more amino acid changes in protein expression from the gene comprising the target sequence. In some embodiments, the modified cell or cell population is administered to an animal, thereby creating an animal model.

[0023] In one aspect, the invention provides a method of modifying a target polynucleotide in a cell or cell population. In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide, causing cleavage of said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex The body comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within said target polynucleotide, said guide sequence linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. soy. In another embodiment, the invention provides a method of altering expression of a polynucleotide in a eukaryotic cell resulting from a cell or cell population that expresses an aberrant protein. The method comprises increasing or decreasing expression of a target polynucleotide using a CRISPR complex that binds to a polynucleotide in a cell or cell population; Delivered via a vector or particle.

[0024] In some methods, alteration of expression in a cell or cell population can occur by inactivating the target polynucleotide. For example, when the CRISPR complex binds to a target sequence in a cell, the target polynucleotide is inactivated so that the sequence is no longer transcribed, no encoded protein is produced, or the sequence does not function like the wild-type sequence. no longer.

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

[0026] The invention further encompasses a composition of the invention or a CRISPR complex or enzyme thereof or RNA thereof (including or alternatively mRNA encoding a CRISPR enzyme) for use in medicine or therapy. In some embodiments, the present invention encompasses compositions of the present invention or components thereof used in methods of the present invention. In some embodiments, the present invention may optionally be subsequently introduced into the original organism or non-human organism from which the cell or cell population was obtained, or into another organism or non-human organism of the same species. Use of the compositions of the invention or CRISPR complexes or enzymes thereof or RNAs thereof (including or alternatively mRNAs encoding CRISPR enzymes) in ex vivo gene or genome editing in a cell or population of cells is provided. In certain embodiments, the present invention provides compositions of the invention or CRISPR complexes or enzymes thereof or RNAs thereof (including Alternatively or alternatively, the use of mRNA encoding the CRISPR enzyme is included. In certain embodiments, the present invention provides for disease states caused by defects in target sequences of genomic loci of interest in subjects (e.g., mammals or humans) or non-human subjects (e.g., mammals) in need thereof. A method of treating or inhibiting is provided, comprising modifying a cell or cell population of a subject or non-human subject by manipulation of a target sequence in the cell or cell population, and administering the modified cell to the subject or non-human subject. Advantageously, the step of modifying the cell comprises contacting the cell with a delivery vector (e.g., a viral delivery vector) or particle containing the CRISPR complex or its constituents, advantageously through a specific In embodiments the delivery vector (viral delivery vector) or particle also provides the HDR template, or another particle or vector provides the HDR template, and the condition is amenable to treatment or inhibition by manipulation of the target sequence. .

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

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

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

[0030] In one aspect, the invention provides a method of modeling a disease associated with a genomic locus in a eukaryotic or non-human organism, which functionally encodes the composition to express the composition. manipulating target sequences within coding, non-coding or regulatory elements of said genomic locus comprising delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors that the composition comprising (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence comprising (a) a guide sequence hybridizable to the target sequence; (b) a tracr mate sequence, and (c) tracr sequence a first regulatory element comprising, and II. A second regulatory element operably linked to the enzyme coding sequence encoding Cas9 (preferably SaCas9), optionally comprising at least one or more nuclear localization sequences [(a), (b) and (c) are aligned in the 5′ to 3′ direction, Components I and II are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence. a composition comprising a system; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element comprising (a) a guide sequence hybridizable to the target sequence, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to the enzyme coding sequence encoding Cas9, and III. a third regulatory element operably linked to the tracr sequence; [Components I, II and III are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence. Includes compositions that include the system.

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

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

[0033] In one aspect, the invention provides any of the methods of the invention (e.g., a method of modeling a disease associated with a locus in a eukaryotic or non-human organism), wherein a coding element of said locus, a non- Compositions for use in methods involving manipulation of target sequences within coding or regulatory elements are provided. In certain embodiments, the invention provides uses of the compositions in ex vivo or in vivo gene or genome editing, including therapeutic applications.

[0034] In one aspect, the invention is used in methods for modifying organisms or non-human organisms by in vitro, ex vivo or in vivo gene or genome editing agents or manipulation of targeted sequences of genomic loci associated with disease. or compositions for use in the manufacture of medicaments for use in methods of treating or inhibiting conditions or diseases caused by mutations in one or more genomic loci in eukaryotes or non-human organisms provide.

[0035] In one aspect, the invention provides: (A)-I. A CRISPR-Cas system RNA polynucleotide sequence comprising: (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence a polynucleotide sequence comprising II. Polynucleotide sequences encoding Cas9, preferably Sa Cas9, optionally including at least one or more nuclear localization sequences [(a), (b) and (c) are aligned in the 5′ to 3′ direction, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises Cas9 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 Cas9 is DNA or is RNA], or (B) I. A polynucleotide, (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell, and (b) at least one tracr mate sequence a polynucleotide comprising II. A polynucleotide sequence encoding Cas9, preferably SaCas9, and III. A polynucleotide sequence comprising a tracr sequence; [When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence induces sequence-specific binding between the CRISPR complex and the target sequence, and The CRISPR complex comprises SaCas9 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 Cas9 is DNA or is RNA]; used in medicine or therapy; or in methods of modifying organisms or non-human organisms by manipulation of targeted sequences of genomic loci associated with diseases or disorders; or diseases in eukaryotic or non-human organisms. for use in methods of treating or inhibiting disease states caused by one or more mutations in genetic loci associated with ; or for use in in vitro, ex vivo or in vivo gene or genome editing. .

[0036] In one aspect, the invention provides therapeutic genome editing methods for treating or inhibiting conditions or diseases caused by mutations in one or more genomic loci in eukaryotes or non-human organisms, the methods comprising: within the coding, non-coding or regulatory elements of said genomic locus in a target sequence in a subject or non-human subject in need thereof, comprising modifying the subject or non-human subject by manipulation of the target sequence. A condition or disease involving manipulation of the target sequence, and delivering a non-naturally occurring or engineered composition comprising an AAV or lentiviral vector system comprising one or more AAV or lentiviral vectors functionally encoding the composition to express the composition amenable to treatment or inhibition by manipulation of the target sequence, the target sequence being manipulated by the composition upon expression, the composition comprising: (A) a non-naturally occurring or engineered composition, I. A first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, the polynucleotide sequence comprising: (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell; (b) a tracr mate sequence, and (c) tracr sequence a first regulatory element comprising, and II. A second regulatory element operably linked to the enzyme coding sequence encoding Cas9, preferably SaCas9, comprising at least one or more nuclear localization sequences [(a), (b) and (c) are aligned in the 5′ to 3′ direction, Components I and II are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to the tracr sequence. a composition comprising a vector system comprising a vector; or (B) a non-naturally occurring or engineered composition, I. A first regulatory element comprising (a) a guide sequence hybridizable to a target sequence in a eukaryotic cell, and (b) at least one tracr mate sequence a first regulatory element operably linked to II. A second regulatory element operably linked to the enzyme coding sequence encoding SaCas9, and III. A Third Regulatory Element Functionally Linked to the tracr Sequence [Components I, II and III are located on the same or different vectors of the system, the tracr mate sequence hybridizes to the tracr sequence when transcribed, and the guide sequence induces sequence-specific binding of the CRISPR complex to the target sequence; and The CRISPR complex comprises (1) a guide sequence that hybridizes to the target sequence, and (2) Cas9 complexed with a tracr mate sequence that hybridizes to the tracr vector system comprising one or more vectors. A composition comprising

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

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

[0039] In the discussion herein regarding targets associated with mutations or disease states, such mutations or disease states include, for example, neurological diseases; eye diseases (e.g., retinal diseases such as retinitis pigmentosa; color blindness ( achromtaopsia); age-related macular degeneration; vision impairment), hearing disorders (eg, cochlear cell-related disorders, hearing impairment, deafness), and the like.

[0040] Accordingly, it is an object of the present invention not to include within the scope of the present invention any product, process for making the product, or method of use for the product previously described, and Applicants agree that any known product, Any rights in the process or method are reserved but hereby expressly disclaimed. This invention covers any product, process, or manufacture of such product that does not meet the description and enablement requirements of the USPTO (Section 112, first paragraph) or the EPO (Section 83 of the EPC), or Methods of using such products are not intended to be within the scope of this invention, and Applicants reserve the right to any products, processes for making products, or methods of using products previously described. , which expressly disclaims it here.

[0041] In this disclosure, and particularly in the claims and / or paragraphs, words such as "comprises," "comprised," and "comprising" have their meanings according to United States patent law. for example, these words mean "includes," "included," and "including," etc.; and "consisting of." Terms such as "essentially of" and "consists essentially of" have the meaning under U.S. patent law, e.g. It should be noted that elements which are required or which affect basic or novel features of the invention are excluded. In the practice of this invention, it may be advantageous to comply with Article 53(c) EPC and Regulations 28(b) and (c) EPC. There is no covenant herein.

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

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

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

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

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

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

[0048] Additionally, a method of preparing sgRNA-Cas9 protein-containing particles, comprising adding a mixture comprising sgRNA and Cas9 protein (and optionally an HDR template) to surfactants, phospholipids, biodegradable polymers, lipoproteins and alcohols. mixing with a mixture comprising, consisting essentially of, or consisting of; and particles from such a method; DISEASES USING PARTICLE DELIVERY COMPONENTS", Attorney Docket No. 47627.99.2060 and BI-2013 / 107 (U.S. Provisional Patent Application No. 62 / 010,441 filed June 10, 2014; and 61 / 915,118 and 61 / 915,215, respectively, filed December 12, 2013. claiming priority from one or more or all of the specification and US Pat. No. 61 / 915,148) (“particle delivery PCT”) (incorporated herein by reference). For example, Cas9 protein and sgRNA, advantageously in a sterile nuclease-free buffer, such as 1×PBS, at a suitable molar ratio, such as 3:1 to 1:3 or 2:1 to 1:2 or 1:1. ratios and mixed together at a suitable temperature, such as 15-30° C., such as 20-25° C., such as room temperature, for a suitable time, such as 15-45, such as 30 minutes. separately surfactants such as cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); phospholipids such as dimyristoylphosphatidylcholine (DMPC); biodegradable polymers such as ethylene glycol polymers. Alternatively, the particle constituents such as or comprising PEG and lipoproteins, such as low density lipoproteins such as cholesterol, are alcohols, advantageously C 1~6 Dissolved in an alkyl alcohol such as methanol, ethanol, isopropanol such as 100% ethanol. These two solutions were mixed together to form particles containing Cas9-sgRNA complexes. Thus, the sgRNA may be pre-complexed with the Cas9 protein prior to forming the entire complex as a particle. Various molar ratios of various components (e.g., 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn- Formulations may be made with glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol), for example with a molar ratio of DOTAP:DMPC:PEG:cholesterol of DOTAP100, DMPC0, PEG0, cholesterol 0; DOTAP90, DMPC0, PEG10, 0 cholesterol; or DOTAP90, DMPC0, PEG5, cholesterol 5, DOTAP100, DMPC0, PEG0, 0 cholesterol. The application thus encompasses the step of mixing sgRNA, Cas9 protein and the components that form the particles; as well as particles from such mixing steps. An aspect of the invention is the formation of particles; For example, particles using methods similar to particle delivery PCT, and particles from such mixing steps (or of course other particles containing sgRNA and / or Cas9 as in the present invention) may be included.

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

[0050] The advantage of this method is that this CRISPR system avoids off-target binding and consequent side effects. This is accomplished using systems designed to have a high degree of sequence specificity for target DNA.

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

[0052] Of the approximately 25,000 annotated genes in the human genome, mutations in over 3,000 genes have already been associated with disease phenotypes (www.omim.org / statistics / geneMap), with more disease relevance. high genetic variation is becoming apparent with alarming rapidity. Now that the cost of sequencing has plummeted, the Human Genome Project has been completed, and the genome sequencing data from patients is growing exponentially, the role of genetics in human health is becoming increasingly important for targeted therapeutics. has become a major focus area for research, clinical medicine, and development in the field [Lander, E.S. Nature 470, 187-197 (2011)]. This advance in Applicants' understanding of the genetic basis of disease has improved Applicants' understanding of disease mechanisms and directed attention to potential therapeutic strategies. However, despite valid therapeutic hypotheses and strong efforts in drug development, there are only a limited number of successful examples of using small molecules to treat diseases with strong genetic involvement [Thoene, J.G. therapy for genetic disease, (Cambridge University Press, Cambridge, UK; New York, 2010)]. Therefore, alternative approaches are needed. Emerging therapeutic strategies capable of altering nucleic acids within diseased cells and tissues have enormous therapeutic potential. Due to the well-defined genetics and the lack of safe and effective therapeutic options in many cases, the high penetrance of monogenic diseases such as severe combined immunodeficiency SCID), hemophilia, and certain enzyme deficiencies are the focus of such treatment.

[0053] Two of the most potent gene therapy strategies developed to date mediate viral gene therapy, which can complement defective gene function through transgene expression, and targeted suppression of defective genes by knockdown of targeted mRNAs. RNA interference (RNAi) (reviewed in Kay, M.A. Nature reviews. Genetics 12, 316-328 (2011) and Vaishnaw, A.K., et al. Silence 1, 14 (2010)). Viral gene therapy leads to the treatment of single-gene recessive disorders that affect the hematopoietic system, such as SCID and Wiskott-Aldrich syndrome, by semi-randomly integrating a functional copy of the affected gene into the genome of hematopoietic stem / progenitor cells. [Gaspar, H. B., et al. Science translational medicine 3, 97ra79 (2011), Howe, S. J., et al. The Journal of clinical investigation 118, 3143-3150 (2008), Aiuti, A. Science 341, 1233151 (2013)]. RNAi has been used to silence the function of genes involved in cancer, age-related macular degeneration, and TTR amyloidosis, among others, and has shown therapeutic benefit in clinical trials (www.clinicaltrials.gov, trial number: NCT00689065). , NCT01961921 and NCT00259753.Despite their promise and recent success, viral gene therapy and RNAi have limitations that hinder their usefulness for many diseases. The Journal of clinical investigation 118, 3143-3150 (2008)], or RNAi can only suppress the expression of the target gene, thus its use. are limited to targets where knockdown is beneficial, and RNAi often fails to completely suppress gene expression and is therefore beneficial to diseases that require complete loss of gene function for treatment. If there is a drastic alternative that could overcome these limitations, it would be the precise modification of the genome of the target cell to remove or correct deleterious mutations or insert protective mutations. Cartier.

[0054] Watts, "Hematopoietic Stem Cell Expansion and Gene Therapy." Cytotherapy 13(10):1164-1171.doi:10.3109 / 14653249.2011.620748 (2011) (with its citation, all given incorporated herein by reference) are hematologic conditions, immunodeficiencies including HIV / AIDS, and other genetic disorders such as lysosomal storage diseases, e.g., SCID-X1, ADA-SCID, Hematopoiesis as a highly attractive therapeutic option for many disorders, including β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD). Stem cell (HSC) gene therapy, such as virus-mediated hematopoetic stem cell (HSC) thereapy, is discussed.

[0055] Williams, "Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies", Cell Stem Cell 13:263-264 (2013) (with its citations, all incorporated by reference). incorporated herein) are HSC / P from patients with metachromatic leukodystrophic disease (MLD) of lysosomal storage disease, an inherited disease caused by a deficiency of arylsulfatase A (ARSA) and resulting in neuronal demyelination. lentiviral-mediated gene transfer into cells; and patients with Wiskott-Aldrich syndrome (WAS), who have defects in the WAS protein, which is an effector of the small GTPase CDC42 that regulates cytoskeletal function in blood cell lineages, thus recurrent Lentiviral-mediated genes into HSCs in patients with infection-associated immunodeficiency, autoimmune conditions, and thrombocytopenia with abnormally small, dysfunctional platelets leading to excessive bleeding and increased risk of leukemia and lymphoma. reporting the introduction. In contrast to the use of lentiviruses, one of ordinary skill in the art, based on the knowledge in the art and the teachings of this disclosure, would know for MLD (deficiency in arylsulfatase A (ARSA)) a mutation (deficiency in arylsulfatase A (ARSA)). ) (including, for example, a suitable HDR template that delivers the coding sequence for ARSA) can be used to modify HSCs. In contrast to the use of lentiviruses, one of ordinary skill in the art, based on the knowledge in the art and the teachings of this disclosure, will have a CRISPR-Cas9 system (e.g., A suitable HDR template that delivers the coding sequence for the WAS protein) can be used to modify HSCs; and HDR can provide proper WAS protein expression coding.

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

[0057] Zinc finger nuclease (Urnov, F.D., et al. Nature reviews. Genetics 11, 636-646 (2010)), transcription activator-like effector nuclease (Bogdanove, A.J. & Voytas, D.F. Science 333, 1843-1846 (2011) ), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 (Hsu, P.D., et al. Cell 157, 1262-1278 (2014)). reviewed) are opening up the possibility of achieving therapeutic genome editing in diseased cells and tissues. Applicants provide a recent review herein.

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

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

[0060] Once a DSB is generated, its damage can be repaired by either NHEJ or HDR depending on the cellular state and the presence of repair templates. NHEJ can repair lesions by directly rejoining the two DSB ends in a process that does not require a repair template. Although NHEJ-mediated DSB repair can be precise, repeated repair of the same DSB by the NHEJ machinery due to nuclease activity ultimately results in the formation of small insertion or deletion mutations spanning the break site [Bibikova, M., et al. Genetics 161, 1169-1175 (2002)]. Such insertions or deletions (indels) that are introduced into the coding sequence of the gene cause frameshift mutations that lead to mRNA degradation via a nonsense mutation-dependent degradation machinery, depleting the functional gene or rendering it non-functional. of truncated proteins can be produced [Hentze, M.W. & Kulozik, A.E. Cell 96, 307-310 (1999)]. Thus, NHEJ can be used to silence gene function in a manner similar to RNAi, however, gene expression continues to be silenced in target cells by introducing permanent covalent modifications to the genome by NHEJ.

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

[0062] Considerations for therapeutic application A primary consideration in genome editing therapy is the selection of sequence-specific nucleases. Each nuclease platform has its own set of strengths and weaknesses, many of which must be balanced for maximum therapeutic benefit in the therapeutic context (Figure 12).

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

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

[0065] Cell fitness and results With respect to fitness, editing can result in three outcomes for a treated cell relative to its unedited counterpart: increased fitness, intermediate fitness, or decreased fitness. When fitness is increased (eg, in treatment of SCID-X1), the modified hematopoietic progenitor cells are selectively expanded relative to their unedited counterparts. SCID-X1 is a disease caused by mutations in the IL2RG gene, whose function is required for normal development of hematopoietic and lymphocyte lineages [Leonard, W.J., et al. Immunological reviews 138, 61-86 (1994 ); Kaushansky, K. & Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trials with patients undergoing viral gene therapy for SCID-X1, and in rare cases of spontaneous correction of SCID-X1 mutations, corrected hematopoietic progenitor cells reversed this developmental arrest, showing a By enlarging it was able to mediate therapy [Bousso, P., et al. Proceedings of the National Academy of Sciences of the United States of America 97, 274-278 (2000); The New England journal of medicine 346, 1185-1193 (2002); Gaspar, H.B., et al. Lancet 364, 2181-2187 (2004)]. In this case, the edited cells have a selective advantage and even a small number of edited cells can be amplified through expansion, resulting in therapeutic benefit to the patient. In contrast, editing of other hematopoietic diseases, such as chronic granulomatous disease (CGD), does not result in changes in the fitness of edited hematopoietic progenitor cells and therapeutic modification thresholds may be increased. CGD is caused by mutations in the gene encoding the phagocyte oxidase protein, which is used by neutrophils to generate reactive oxygen species that normally kill pathogens [Mukherjee, S. & Thrasher, A. J. Gene 525, 174-181. 2013)]. Dysfunction of these genes does not affect the fitness or development of hematopoietic progenitor cells, but only the ability of mature hematopoietic cell types to fight infection, thus preferential expansion of edited cells in this disease. It seems not. Indeed, no selective advantage for gene-modified CGD cells has been observed in gene therapy trials, making long-term cell engraftment difficult [Malech, H.L., et al. Sciences of the United States of America 94, 12133-12138 (1997); Kang, H.J., et al. Molecular therapy: the journal of the American Society of Gene Therapy 19, 2092-2101 (2011)]. Thus, treatment of diseases such as CGD, in which editing results in an intermediate fitness advantage compared to diseases in which editing results in increased fitness in target cells, may require significantly higher levels of editing. When editing imposes a fitness disadvantage, as is the case for restorative function on tumor suppressor genes in cancer cells, the modified cell is outmatched by its diseased counterpart, resulting in low therapeutic benefit relative to editing rate. obtain. This latter class of diseases can be particularly difficult to treat with genome editing therapies. X-linked chronic granulomatosis (CGD) is an inherited disorder of host defense due to lack or reduced activity of phagocytic NADPH oxidase. From this disclosure and the knowledge in the art, one skilled in the art will be able to target and correct mutations (absence or reduction in activity of phagocytic NADPH oxidase) in a CRISPR-Cas9 system (e.g., delivering a coding sequence for phagocytic NADPH oxidase). Specifically, sgRNAs can target mutations that cause CGD (deficiency in phagocytic NADPH oxidase), and HDRs can be used , can result in coding for proper phagocytic NADPH oxidase expression.

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

[0067] Disorders where subtle changes in gene product levels can affect clinical outcome, and diseases in which edited cells have a fitness advantage, require therapeutic modification to allow high response rates given current technology. The threshold is sufficiently low that it is an ideal target for genome editing therapy.

[0068] Targeting these diseases has now resulted in successful editing therapies in preclinical and Phase I clinical trials (see table below). Extending these promising results to diseases with intermediate fitness dominance for edited cells, or those in which high abundance of the gene product is required for treatment, requires manipulation of DSB repair pathways and improved nuclease delivery. Become. The table below shows examples of the application of genome editing to therapeutic models.

[0069]

table 1

[0070] In certain embodiments, hematopoetic stem cells carrying hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or inherited tyrosinemia mutations are treated with hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or genomic loci of interest for hereditary tyrosinemia (e.g. those in Li, Genovese or Yin) and contact with Cas9 protein; contacting with a suitable HDR template to modify the .

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

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

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

[0074] However, the ex vivo technique has two major drawbacks that limit its application to a small number of diseases. First, the target cells must have the ability to survive manipulation in vitro. For many tissues, such as the brain, culturing cells in vitro is a major challenge because the cells are either not viable or lose properties necessary for their function in vivo. Therefore, ex vivo therapy is generally limited to tissues with adult stem cell populations suitable for ex vivo culture and manipulation, such as the hematopoietic system. Second, cultured cells often have poor engraftment upon reintroduction into the patient, reducing therapeutic efficacy. However, engraftment can be enhanced by an ablation conditioning regimen that depletes host cells prior to transplantation, which is clinically feasible but poses significant risks to the patient [Bunn, H.F. & Aster, J. Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)]

[0075] In vivo genome editing involves directing delivery of the editing system to cell types in their native tissue (Figure 14, bottom panel). In vivo editing allows treatment of diseases in which the affected cell population is not amenable to ex vivo manipulation. Furthermore, since the nuclease is delivered to cells in situ, treatment of multiple tissues and cell types is possible. Perhaps these properties make in vivo therapy more applicable to a wider range of diseases than ex vivo therapy.

[0076] To date, in vivo editing has largely been achieved through the use of viral vectors with defined tissue-specific tropisms. Such vectors are currently limited in terms of cargo-carrying capacity and tropism, and this therapy is limited to organ systems such as liver, muscle, and eyes where transduction with clinically useful vectors is efficient. [Kotterman, M.A. & Schaffer, D.V. Nature reviews. Genetics 15, 445-451 (2014); Nguyen, T. H. & Ferry, N. Gene therapy 11 Suppl 1, S76-84 (2004); al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 509-519 (2013)].

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

[0078] Successful Genome Editing Therapy Strategy ex vivo editing therapy Long-standing clinical observations on hematopoietic cell purification, culture and transplantation have made diseases affecting the blood system such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome and sickle cell anemia the focus of ex vivo editing therapy. It's here. Another reason for the focus on hematopoietic cells is that relatively highly efficient delivery systems already exist, thanks to previous efforts attempting to design gene therapies for blood disorders. Despite these advantages, the low engraftment efficiency of cells upon engraftment in many cases necessitates this treatment because edited cells have a fitness advantage and therefore fewer engrafted edited cells. has been applied to diseases that can be expanded to treat diseases.

[0079] Fanconi anemia: at least 15 genes (FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ / BACH1 / BRIP1, FANCL / PHF9 / POG, FANCM, FANCN / PALB2, FANCO / Rad51C and FANCP / SLX4 / BTBD12) mutations can cause Fanconi anemia. Proteins produced from these genes participate in cellular processes known as the FA pathway. The FA pathway is turned on (activated) when the process of making new copies of DNA, called DNA replication, is interrupted due to DNA damage. The FA pathway delivers specific proteins to the damaged area, triggering them to initiate DNA repair so that DNA replication can continue. The FA pathway is particularly responsive to a particular type of DNA damage known as interstrand crosslinks (ICLs). ICL occurs when two DNA building blocks (nucleotides) on opposite strands of DNA abnormally bind or ligate together, thereby halting the process of DNA replication. ICL can be caused by the accumulation of toxic substances produced by the body or by treatment with certain cancer therapeutic agents. Eight proteins associated with Fanconi anemia group together to form a complex known as the FA core complex. The FA core complex activates two proteins called FANCD2 and FANCI. Activation of these two proteins brings DNA repair proteins into the area of ​​the ICL so that the cross-links can be removed and DNA replication can proceed. FA core complex. More specifically, the FA core complex is a nuclear multiprotein complex consisting of FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3 ubiquitin ligase, and is composed of FANCD2 and FANCI. mediates activation of the ID complex, a heterodimer that When the FA core complex is monoubiquitinated, it interacts with canonical tumor suppressors downstream of the FA pathway, including FANCD1 / BRCA2, FANCN / PALB2, FANCJ / BRIP1, and FANCO / Rad51C, leading to homologous recombination. Contributes to DNA repair by (HR). Eighty to ninety percent of FA cases are due to mutations in one of three genes, FANCA, FANCC, and FANCG. These genes provide instructions for the production of components of the FA core complex. Mutation of such genes associated with the FA core complex can render the complex non-functional and disrupt the entire FA pathway. As a result, DNA damage is not repaired efficiently and ICLs accumulate over time. Geiselhart, “Review Article,Disrupted Signaling through the Fanconi Anemia Pathway Leads in FA and in vivo An animal study involving intrafemoral injection of a lentivirus encoding the FANCC gene, which results in correction of HSCs in papilloma, was discussed. From this disclosure and knowledge in the art, CRISPR-Cas9 systems that target one or more of the mutations associated with FA, e.g., target one or more of the FANCA, FANCC, or FANCG mutations that give rise to FA. A CRISPR-Cas9 system having one or more sgRNAs and one or more HDR templates that provide modified expression of one or more of FANCA, FANCC or FANCG, respectively, can be used.

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

[0081] The rationale for genome editing for HIV therapy is that individuals homozygous for a loss-of-function mutation in CCR5, the cellular co-receptor for the virus, are highly resistant to infection and otherwise healthy. It is suggested that mimicking this mutation by genome editing could be a safe and effective therapeutic strategy [Liu, R., et al. Cell 86, 367-377 (1996)]. The idea was that when HIV-infected patients received allogeneic bone marrow transplantation from donors homozygous for the loss-of-function CCR5 mutation, undetectable levels of HIV and restoration of normal CD4 T-cell counts were obtained. Clinically validated [Hutter, G., et al. The New England journal of medicine 360, 692-698 (2009)]. Although bone marrow transplantation is not a viable treatment strategy for many HIV patients because of cost and potential graft-versus-host disease, HIV therapies that alter the patient's own T cells are feasible.

[0082] Early studies using ZFNs and NHEJ to knock out CCR5 in a humanized mouse model of HIV showed that transplantation of CCR5-edited CD4 T cells improved viral load and CD4 T cell numbers [Perez, E.E., et al. Nature biotechnology 26, 808-816 (2008)]. Importantly, these models also show that HIV infection leads to selection of CCR5-null cells, suggesting that editing may confer a fitness advantage to produce therapeutic effects in fewer edited cells. It was suggested.

[0083] Following this and other promising preclinical studies, a genome-editing therapy that knocks out CCR5 in patient T cells is now being tested in humans [Holt, N., et al. Nature biotechnology 28, 839-847 (2010). Li, L., et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase I clinical trial, CD4+ T cells from HIV patients were removed, edited with ZFNs designed to knock out the CCR5 gene, and autologously returned to the patient [Tebas, P., et al. The New England journal of medicine 370,901-910 (2014)]. Initial results from this trial suggest that genome editing with ZFNs of the CCR5 locus is safe, but the short follow-up time precludes a full understanding of treatment risks and efficacy.

[0084] Recently, ex vivo editing therapy has been expanded to include gene correction strategies. The ex vivo HDR barrier has been overcome in a recent paper by Genovese and co-workers, who showed that mutant IL2RG in hematopoietic stem cells (HSCs) from patients with SCID-X1 Gene correction of the gene was achieved [Genovese, P., et al. Nature 510, 235-240 (2014)]. Genovese et.al. used a multidisciplinary strategy to achieve gene correction in HSCs. First, an integration deficient lentivirus containing an HDR template encoding a therapeutic cDNA for IL2RG was used to transduce HSCs. After transduction, cells were electroporated with mRNAs encoding ZFNs that target mutational hotspots in IL2RG to stimulate HDR-based gene correction. To increase the HDR rate, we optimized culture conditions with small molecules to promote HSC division. With optimized culture conditions, nucleases and HDR templates, gene-corrected HSCs from SCID-X1 patients were obtained in culture at therapeutically meaningful rates. HSCs from unaffected individuals who underwent the same gene correction procedure were able to maintain long-term hematopoiesis, the gold standard of HSC function, in mice. HSCs have the ability to give rise to all hematopoietic cell types and can be autografted, making HSCs an extremely useful cell population for all hematopoietic genetic disorders [Weissman, I.L. & Shizuru, J.A. Blood 112, 3543-3553. (2008)]. Gene-modified HSCs could in principle be used to treat a wide range of genetic blood disorders, making this trial an exciting breakthrough in therapeutic genome editing.

[0085] In vivo editing therapy In vivo editing therapies face similar challenges as ex vivo strategies and are also limited by the paucity of efficient delivery systems. Inefficient modification of the target locus is exacerbated by delivery inefficiencies, making it particularly difficult to treat tissues lacking robust delivery platforms with this therapy. However, there are already some exciting preclinical therapeutic successes for organ systems where delivery is efficient.

[0086] The first successful in vivo editing therapy was demonstrated in a mouse model of hemophilia B [Li, H., et al. Nature 475, 217-221 (2011)]. As previously mentioned, hemophilia B is an X-linked recessive disorder caused by loss-of-function mutations in the gene encoding factor IX, a key component of the coagulation cascade. When factor IX activity is restored to above 1% of its level in severely affected individuals, the disease can shift significantly to a milder form, suggesting that recombinant factor IX should be given prophylactically to such patients from an early age. Achieving such levels by injecting into the body is generally associated with improved clinical complications [Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)]. Therefore, only low levels of HDR gene correction may be required to alter patient clinical outcomes. In addition, Factor IX is synthesized and secreted by the liver, an organ that can be efficiently transduced by viral vectors encoding editing systems. Based on the knowledge in the art and the teachings of this disclosure, one of ordinary skill in the art will recognize that mutations (X-linked recessive disorders caused by loss-of-function mutations in the gene encoding factor IX) are associated with hemophilia B. Targeted and modified CRISPR-Cas9 systems (e.g., containing a suitable HDR template that delivers the coding sequence for Factor IX) can be used to modify HSCs; Mutations that give rise to disease B can be targeted and HDR can lead to coding of proper factor IX expression.

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

[0088] Building on this study, other groups have recently used in vivo genome editing of the liver with CRISPR-Cas9 to treat a mouse model of hereditary tyrosinemia and to develop mutations that provide protection from cardiovascular disease. successfully created. These two different applications demonstrate the versatility of this technique for disorders involving liver dysfunction [Yin, H., et al. Nature biotechnology 32, 551-553 (2014); Ding, Q., et al. al. Circulation research 115, 488-492 (2014)]. Application of in vivo editing to other organ systems is needed to prove that this strategy is broadly applicable. Efforts are now underway to optimize both viral and non-viral vectors to expand the range of disorders that can be treated with this therapy [Kotterman, M.A. & Schaffer, D.V. Nature reviews.Genetics 15, 445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555 (2014)].

[0089] Editing nuclease specificity The specificity of genome editing tools is one of the major safety concerns for clinical application. Genetic alterations are permanent and deleterious off-target mutations can create cells with oncogenic potential and other unwanted side effects. Furthermore, oncogenic mutations caused by off-target editing can lead to expansion of edited cells, and thus even low levels of off-target mutagenesis can have serious consequences.

[0090] Two issues remain open: evaluation and reduction of off-target effects. A number of studies have attempted to assess the targeting specificity of ZFNs, TALENs, and Cas9 nucleases. ZFN [Pattanayak, V., et al. Nature methods 8,765-770 (2011); Gabriel, R., et al. Nature biotechnology 29, 816-823 (2011)] and TALEN [Guilinger, J.P., et al. 435 (2014)] have only highlighted the challenges of detecting ZFN and TALEN off-target activity. Of note, although these two independent studies attempted to characterize the off-target profiles of the same pair of CCR5-targeted ZFNs, they reported distinct, non-overlapping off-target sites, which It highlights the challenges associated with analysis of nuclease specificity.

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

[0092] Many groups have rapidly improved the targeting specificity of Cas9 to reduce the frequency of off-target effects. For example, transforming Cas9 into a single-stranded DNA nickase that functions as a forced heterodimer dramatically reduces off-target indel formation at computationally predicted off-target sites [Mali, P., et al. Nature biotechnology 31, 833-838 (2013); Ran, F.A., et al. Cell 154, 1380-1389 (2013)]. In addition, guide RNAs based on fusions between catalytically inactive Cas9 and FokI nuclease domains as well as RNA-guided truncations of FokI nucleases can also achieve enhanced levels of targeting specificity [ Fu, Y., et al. Nature biotechnology 32, 279-284 (2014); Guilinger, J.P., et al. Nature biotechnology 32, 577-582 (2014); Tsai, S.Q., et al. These and future improved nuclease strategies are considered for therapeutic applications whenever it is.

[0093] Crispr-Cas systems and compositions for therapeutic applications such as genome editing In general, in addition to the discussion of the CRISPR-Cas system or CRISPR system throughout this specification, the CRISPR-Cas system or CRISPR system is described herein, e.g. Application PCT / US2013 / 074667), these systems collectively refer to transcripts and other elements involved in inducing the expression of CRISPR-associated (“Cas”) genes or their activity, Cas genes , tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences ("direct repeats" in the context of the endogenous CRISPR system and tracrRNA-processed partial direct repeats), guide sequences (also called “spacers” in the context of the endogenous CRISPR system), or the term “RNA” as used herein (e.g., RNA that guides Cas9, e.g., CRISPR RNA , and transactivating (tracr) RNA, or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from the CRISPR locus. In general, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of target sequences (also called protospacers in the context of endogenous CRISPR systems). In the context of forming a CRISPR complex, "target sequence" refers to a sequence to which the guide sequence is designed to have complementarity, such that hybridization between the target sequence and the guide sequence results in formation of the CRISPR complex. Facilitate. A target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located within the nucleus or cytoplasm of the cell. In some embodiments, direct repeats are found on the following criteria: 1. found within 2 Kb of genomic sequence flanking type II CRISPR loci; 2. spanning 20-50 bp; 3. spaced between 20-50 bp can be identified in silico by searching for repeat motifs that satisfy any or all of In some embodiments, two of these criteria can be used, eg, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used. In some embodiments, in the CRISPR complex, the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length: , 10-30 nucleotides in length, and the CRISPR / Cas enzyme will preferably be a type II Cas9 enzyme. In embodiments of the present invention, the terms guide sequence and guide RNA are used interchangeably with the documents mentioned above, eg WO2014 / 093622 (International Application PCT / US2013 / 074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence so as to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence when optimally aligned using an appropriate alignment algorithm is about 50%, about 60%, about 75%. %, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 99%, or more, or more than about 50%, about 60%, about 75% , greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 97.5%, greater than about 99%, or higher. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such suitable algorithms include Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burrows -Algorithms based on Wheeler Transform (e.g. Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (soap .genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 75, or more, or about 5 greater than about 10 greater than about 11 greater than about 12 greater than about 13 greater than about 14 greater than about 15 greater than about 16 greater than about 17 greater than about 18 greater than about 19 Greater than about 20 Greater than about 21 Greater than about 22 Greater than about 23 Greater than about 24 Greater than about 25 Greater than about 26 Greater than about 27 Greater than about 28 Greater than about 29 Greater than, greater than about 30, greater than about 35, greater than about 40, greater than about 45, greater than about 50, greater than about 75, or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, less than about 12 , or less nucleotides in length. Preferably, the guide sequence is 10-30 nucleotides long. The ability of a guide sequence to induce sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, the components of the CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, have the corresponding target sequences, e.g., by transfection with vectors encoding the components of the CRISPR sequences. Introduction into host cells can be followed by assessment of preferential cleavage within the target sequence, eg, by Surveyor assays as described herein. Similarly, cleavage of a target polynucleotide sequence provides components of a CRISPR complex comprising a target sequence, a guide sequence to be tested and a control guide sequence different from the test guide sequence, and binding or cleavage rates at the target sequence can be assessed in vitro by comparing between test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art. Guide sequences can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of the cell. Exemplary target sequences include unique sequences in the target genome. For example, for S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG, where N is A, G, T, or C and X can be any; W is A or T) has a single occurrence in the genome. A unique target sequence in the genome can include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, where N is A, G, T, or C; may be) have a single occurrence in the genome. In the case of S. thermophilus CRISPR1 Cas9, the unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where N is A, G, T, or C. ; X can be any; W is A or T) has a single occurrence in the genome. A unique target sequence in the genome can include a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where N is A, G, T, or C; There may be; W is A or T) has a single occurrence in the genome. In the case of S. pyogenes Cas9, a unique target sequence in the genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXGGXG, where N is A, G, T, or C. ; and X can be any) has a single occurrence in the genome. A unique target sequence in the genome can include S. pyogenes Cas9 of the form MMMMMMMMMMMNNNNNNNNNNNXGGXG, where N is A, G, T, or C; may) have a single occurrence in the genome. In each of these sequences, "M" can be A, G, T, or C and need not be considered when considering a sequence unique. In some embodiments, guide sequences are selected to reduce the degree of secondary structure within the guide sequences. In some embodiments, about 75%, about 50%, about 40%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1 of the nucleotides of the guide sequence % or less, or less than about 75%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, about 5 Less than 1%, less than about 1%, or less are involved in self-complementary base-pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148).Another example of a folding algorithm is a centroid structure prediction algorithm (e.g. A.R. Gruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151 -62) using the online web server RNAfold developed at the Institute for Theoretical Chemistry of the University of Vienna.

[0094] In general, tracr mate sequences are: (1) excision of guide sequences flanked by tracr mate sequences in cells containing corresponding tracr sequences; and (2) targeting of CRISPR complexes containing tracr mate sequences that hybridize to the tracr mate sequences. Any sequence having sufficient complementarity with the tracr sequence to facilitate one or more of the formation of a sequence. Generally, the degree of complementarity is for optimal alignment of the tracr mate and tracr sequences along the shorter length of these sequences. Optimal alignment can be determined by any suitable alignment algorithm and can additionally take into account secondary structure, eg, self-complementarity within either the tracr or tracr mate sequences. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter length of these sequences when optimally aligned is about 25%, about 30%, about 40%. %, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97.5%, about 99%, or more, or more than about 25%, about 30% more than about 40% more than about 50% more than about 60% more than about 70% more than about 80% more than about 90% more than about 95% more than about 97.5% , greater than or higher than about 99%. In some embodiments, the tracr sequence is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, about 50, or more, or more than about 5, about 6, about 7, about 8, about 9 greater than about 10 greater than about 11 greater than about 12 greater than about 13 greater than about 14 greater than about 15 greater than about 16 greater than about 17 greater than about 18 greater than about 19 Greater than, greater than about 20, greater than about 25, greater than about 30, greater than about 40, greater than about 50 or more nucleotides in length. In some embodiments, the tracr and tracr mate sequences are contained within a single transcript such that hybridization between these sequences forms a transcript with secondary structure, eg, a hairpin. In one embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has 2, 3, 4, or 5 hairpins. In a further embodiment of the invention the transcript has at most 5 hairpins. In the hairpin structure, the portion of the sequence 5' to the last "N" upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3' to the loop corresponds to the tracr sequence. A further non-limiting example of a single polynucleotide comprising a guide sequence, a tracr mate sequence, and a tracr sequence is as follows (listed 5′ to 3′), where “N” is the guide sequence the first block in lower case represents the tracr mate sequence, the second block in lower case represents the tracr sequence, and the final poly T sequence represents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtttttcgttatttaaTTTTTT; ttatggcagggtgttttcgttatttaaTTTTTT;(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT;(4)NNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggc accgagtcggtgcTTTTTT; (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTTT; and (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT. In some embodiments, sequences (1)-(3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4)-(6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript containing the tracr mate sequence.

[0095] In some embodiments, candidate tracrRNAs can be subsequently predicted by sequences that satisfy any or all of the following criteria: 1. Repeat-inducing sequence homology (of motifs in Geneious with mismatches of up to 18 bp). search); 2. Presence of putative Rho-dependent transcriptional terminators in the direction of transcription; and 3. Stable hairpin secondary structures between tracrRNA and direct repeats. In some embodiments, two of these criteria can be used, eg, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used.

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

[0097] It is important to control the concentration of delivered CRISPR enzyme mRNA and guide RNA to minimize toxicity and off-target effects. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in cellular or non-human eukaryotic animal models and using deep sequencing to analyze the extent of alterations in potential off-target genomic loci. can be determined by For example, for a guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′ in the EMX1 gene of the human genome, deep sequencing was used to identify the following two off-target loci, 1:5′-GAGTCCTAGCAGGAGAAGAA-3′. and 2:5'-GAGTCTAAGCAGAAGAAGAA-3' can be assessed. Concentrations that give the lowest level of off-target alteration and the highest level of on-target alteration should be chosen for in vivo delivery. Alternatively, to minimize the level of toxicity and off-target effects, the CRISPR enzyme nickase mRNA (e.g., S. pyogenes Cas9 with the D10A mutation) is targeted to the site of interest. can be delivered in pairs of guide RNAs. The two guide RNAs should be spaced as follows: Guide sequences and strategies to minimize toxicity and off-target effects can be similar to WO2014 / 093622 (International Application PCT / US2013 / 074667).

[0098] The CRISPR system is advantageously delivered from a type II CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism that contains an endogenous CRISPR system, eg, Streptococcus pyogenes. In a preferred embodiment of the invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel , Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, C sx1 , Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof.

[0099] In some embodiments, the unmodified CRISPR enzyme, eg, Cas9, has DNA cleaving activity. In some embodiments, the CRISPR enzyme induces cleavage of one or both strands at a target sequence location, eg, within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about induce breaks in one or both strands within 8, about 9, about 10, about 15, about 20, about 25, about 50, about 100, about 200, about 500, or more base pairs . In some embodiments, the vector contains CRISPR mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme loses the ability to cleave one or both strands of a target polynucleotide containing the target sequence. encodes an enzyme. For example, an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes transforms Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). Convert to Other examples of mutations that render Cas9 a nickase include, but are not limited to H840A, N854A, and N863A. As a further example, two or more of the catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III, or the HNH domain) are mutated to create a mutant Cas9 that has substantially lost all DNA-cleaving activity. can be made In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that has substantially lost all DNA-cleaving activity. In some embodiments, the CRISPR enzyme is such that the DNA-cleaving activity of the mutant enzyme is no more than about 25%, no more than about 10%, no more than about 5%, no more than about 1% of the DNA-cleaving activity of the unmutated enzyme. , about 0.1% or less, about 0.01% or less, or less, is considered to have substantially lost all DNA-cleaving activity; or when negligible compared to the non-mutated form. If the enzyme is not SpCas9, mutations can be made at some or all residues corresponding to positions 10, 762, 840, 854, 863 and / or 986 of SpCas9 ( can be confirmed, for example, by standard sequence comparison tools). In particular, some or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A, and / or D986A; also conservative substitutions of any of the substituted amino acids are contemplated. The same (or conservative) substitution of these mutations at the corresponding positions in other Cas9s is also preferred. D10 and H840 in SpCas9 are particularly preferred. However, for other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred. For example in Sa Cas9 mutations at N580, eg N580A are advantageous. Orthologues of SpCas9 can be used in the practice of the invention. The Cas enzyme may be Cas9 identified as it may refer to a general class of enzymes that share homology with the largest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from or derived from spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9). StCas9" refers to wild-type Cas9 from S. thermophilus, the protein sequence of which is present in the SwissProt database under the accession number G3ECR1. Similarly, S. pyogenes Cas9 or spCas9 , which is housed in the SwissProt database under accession number Q99ZW2.Derivation means that, in our view, the derived enzyme is based largely on the wild-type enzyme in that it has a high degree of sequence homology with the wild-type enzyme. but is mutated (modified) in any way described herein.The terms Cas and CRISPR enzymes are generally used herein unless explicitly stated It should be understood that they are used interchangeably.As noted above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it should be understood that the present invention includes more Cas9 from other species of microorganisms, such as SpCas9, SaCa9, and St1Cas9, Cas9 from Streptococcus pyogenes or any Enzymatic action by the closely related Cas9 effects a double-strand break at a target site sequence that hybridizes to 20 nucleotides of the guide sequence, which is followed by a protospacer-adjacent motif (PAM ) sequence (examples include NGG / NRG or PAM, which can be determined as described herein. CRISPR activity by Cas9 for site-specific DNA recognition and cleavage is determined by the guide sequence, determined by the tracr sequence partially hybridizing to this guide sequence, and the PAM sequence Further aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15;37(1):7 The type II CRISPR locus from Streptococcus pyogenes SF370 is a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, and two It contains non-coding RNA elements, tracrRNA, and a characteristic array of repetitive sequences (direct repeats) interspersed with short lengths of non-repetitive sequences (spacers, approximately 30 bp each). In this system, targeted DNA double-strand breaks (DSBs) are performed in four sequential steps. First, two non-coding RNAs, a pre-crRNA array and a tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to direct repeats of pre-crRNA, and this hybridized pre-crRNA is then processed into mature crRNA containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to a DNA target consisting of the protospacer and the corresponding PAM by heteroduplex formation between the spacer region of crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to generate DSBs within the protospacer. Pre-crRNA arrays consisting of a single spacer flanked by two direct repeats (DR) are also encompassed by the term "tracr-mate sequence". In certain embodiments, Cas9 may be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization can be used to develop new functions that can enhance function or create chimeric Cas9 proteins. And Cas9 can be used as a general DNA-binding protein.

[0100] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence that hybridizes to the target sequence to form a complex with one or more Cas proteins) results in Nearby (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pair stretches from the target sequence) inner) are cleaved on one or both strands. Without wishing to be bound by theory, the tracr sequence includes all or part of the wild-type tracr sequence, or all or part thereof (e.g., about 20, about 26, about 32, about 45, about 48, about 54, about 63, about 67, about 85, or more, or greater than about 20, greater than about 26, greater than about 32, greater than about 45, greater than about 48; more than about 54, more than about 63, more than about 67, more than about 85, or more nucleotides) and functional to the guide sequence, e.g., along at least a portion of the tracr sequence Part of the CRISPR complex can also be formed by hybridization to all or part of the tracr mate sequence linked to the .

[0101] An example of a codon-optimized sequence is, in this case, a sequence optimized for eukaryotic, e.g. human expression (i.e. optimized for human expression), or a sequence as described herein. is another eukaryotic, animal or mammalian expression optimized sequence; e.g. See the standardization array. While this is preferred, it should be understood that other examples are possible and that codon optimization for other host species other than humans, or codon optimization for specific organisms is known. In some embodiments, the enzyme coding sequence that encodes the CRISPR enzyme is codon optimized for expression in a particular cell, eg, a eukaryotic cell. Eukaryotic cells may be derived from certain organisms, e.g., mammals, including but not limited to humans, or non-human eukaryotes, animals, or mammals as described herein, e.g., mice, rats, rabbits. , dogs, domestic animals, or non-human mammals or primates, or may be derived therefrom. In some embodiments, processes that alter human germline genetic identities and / or animals that have no substantial medical benefit to humans or animals and are likely to afflict humans or animals. Processes that alter genetic identity, as well as animals resulting from such processes, can also be excluded. Generally, codon optimization involves at least one codon (e.g., about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 50 or more, or about more than 1, about 2, about 3, about 4, about 5, about 10, about 15, about codons greater than 20, greater than about 25, greater than about 50, or more) with the more or most frequently used codons used in the genes of the host cell, while Refers to the process of modifying a nucleic acid sequence to facilitate expression in the intended host cell by maintaining the native amino acid sequence. Different species exhibit particular biases towards certain codons of particular amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which determines, among other things, the characteristics of the codons translated and the specific transfer RNA (tRNA) molecule. Considered dependent on availability. The predominance of the tRNA chosen in the cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases:status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, eg, Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more) of a sequence encoding a CRISPR enzyme most or all codons) correspond to the most frequently used codons for a particular amino acid.

[0102] In some embodiments, the vector includes one or more nuclear localization sequences (NLS), e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or more than about 1, about 2, about 3, about 4, about 5 , encodes a CRISPR enzyme comprising more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, or more NLSs. In some embodiments, the CRISPR enzyme has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more, or about more than 1, about 2, about 3, about 4, about 5, about 6, about greater than 7, greater than about 8, greater than about 9, greater than about 10, or more NLS, about 1, about 2, about 3, about 4 at or near the carboxy terminus , about 5, about 6, about 7, about 8, about 9, about 10, or more, or about more than 1, about 2, about 3, about more than 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, or more NLS, or any of these Combinations (eg, zero or at least one or more NLSs at the amino terminus and zero or more NLSs at the carboxy terminus) are included. When more than one NLS is present, each one or more present in one or more copies and / or independently from the other such that a single NLS can be present in two or more copies. can be selected in combination with other NLS. In one preferred embodiment of the invention, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, the NLS is such that the nearest amino acids of this NLS are about 1, 2, 3, 4, 5, 10, 15, It is considered near the N-terminus or C-terminus if it is within 20, 25, 30, 40, 50 or more amino acids. Non-limiting examples of NLSs include the NLS of the SV40 viral large T antigen having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); the amino acid sequences PAAKRVKLD or RQRRNELKRSP. hRNPA1 M9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of IBB domain from importin alpha; sequences VSRKRPRP and PPPKKARED of fibroid T protein; sequence POPKKKPL of human p53; KKKMAP; Influenza derived from the sequences DLRRR and PKQKKRK of the virus NS1; the sequence RKLKKKIKKL of the hepatitis virus delta antigen; the sequence REKKKFLKRR of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; NLS sequences are included. In general, one or more NLSs are strong enough to accumulate detectable amounts of CRISPR enzyme in the nucleus of eukaryotic cells. In general, the strength of nuclear localization activity can result from the number of NLSs in the CRISPR enzyme, the particular NLS used, or a combination of these factors. Detection of nuclear accumulation can be performed by any suitable technique. For example, a detectable marker can be fused to a CRISPR enzyme, thereby linking its intracellular location, for example, with a means of detecting nuclear location (e.g., a nuclear-specific stain, e.g., DAPI). It can be visualized in combination. Cell nuclei can also be isolated from cells and their contents can then be analyzed by any suitable process that detects proteins, such as immunohistochemical analysis, Western blot, or enzyme activity assay. Accumulation in the nucleus is assayed, for example, for the effect of CRISPR complex formation (e.g., assayed for DNA breaks or mutations in target sequences, or affected by CRISPR complex formation and / or CRISPR enzymatic activity, assay for altered gene expression activity) compared to controls exposed to neither the CRISPR enzyme nor the complex, or to the CRISPR enzyme lacking one or more NLSs, indirectly can also decide.

[0103] Aspects of the invention are directed to reducing the expression of the gene product, or template polynucleotide that is further introduced into the DNA molecule encoding the gene product, or precisely cleaving by allowing reannealing and joining of the two 5' overhangs. intervening sequences that are altered, or activity or function of the gene product altered, or increased expression of the gene product. In one embodiment of the invention the gene product is a protein. Only sgRNA pairs that formed a 5' overhang with less than 8 bp of overlap between the guide sequences (offset greater than -8 bp) were able to mediate the development of detectable indels. Importantly, each of the guides used in these assays was able to efficiently induce indels when paired with wild-type Cas9, and the relative position of the guide pair determined the double-nicking activity. This suggests that it is the most important parameter in the prediction of Since Cas9n and Cas9H840A nick opposite strands of DNA, replacement of Cas9n with Cas9H840A with a given sgRNA pair should be the opposite of the overhang type; was not observed, suggesting that Cas9H840A is a CRISPR enzyme that is substantially devoid of all DNA-cleaving activity (this suggests that the DNA-cleaving activity of the mutant enzyme is greater than that of the non-mutant enzyme). less than about 25%, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, less than about 0.01%, or less; If the mutant has zero or negligible DNA-cleaving activity compared to the type, e.g. is not observed). However, pairs of sgRNAs that form 5' overhangs at Cas9n should, in principle, form corresponding 3' overhangs and double nicks instead. Therefore, sgRNA pairs that lead to the formation of 3' overhangs at Cas9n can be used with another mutated Cas9 to form 5' overhangs and double nicking. Accordingly, in some embodiments, recombinant templates are also provided. The recombination template can be a component of another vector contained in a separate vector or provided as a separate polynucleotide as described herein. In some embodiments, the recombination template is designed to serve as a template for homologous recombination within or near a target sequence that is nicked or cleaved by a CRISPR enzyme, e.g., as part of a CRISPR complex. be. Template polynucleotides can be of any suitable length, e.g. or greater than about 10, greater than about 15, greater than about 20, greater than about 25, greater than about 50, greater than about 75, greater than about 100, greater than about 150, greater than about 200, about 500 , about 1000 or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide comprising the target sequence. When optimally aligned, the template polynucleotide has one or more nucleotides of the target sequence (e.g., about 1, about 5, about 10, about 15, about 20, or more, or more than about 1, more than about 5, more than about 10, more than about 15, more than about 20, or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotides of the template polynucleotide are within about 1, within about 5, within about 10 of the target sequence. , within about 15, within about 20, within about 25, within about 50, within about 75, within about 100, within about 200, within about 300, within about 400, within about 500, within about 1000, within about 5000, about Within 10000 or more nucleotides.

[0104] In some embodiments, one or more vectors driving expression of one or more elements of the CRISPR system are used such that expression of the elements of the CRISPR system induces formation of CRISPR complexes at one or more target sites. It is introduced into the host cell as follows. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements in separate vectors. Alternatively, the CRISPR-based RNA is transferred to a transgenic Cas9 animal or mammal, e.g., an animal or mammal that constitutively, or inducibly, or conditionally expresses Cas9; Prior administration of one or more vectors encoding and expressing Cas9 in vivo can be delivered to an animal or mammal having cells expressing or containing Cas9. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector with one or more additions providing all components of the CRISPR system not contained in this first vector. vectors can be combined. The elements of the CRISPR system that are combined in a single vector may be in any suitable orientation, e.g., one element positioned 5' to a second element (upstream of the second element), or 3 It can be placed on the ' side (downstream of the second element). The coding sequence of one element can be placed on the same or opposite strand of the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter is integrated within a transcript encoding a CRISPR enzyme as well as one or more intron sequences (e.g., two or more within at least one sequence, each within a different intron). drive expression of one or more of the guide sequence (either within an intron or all within a single intron), the tracr mate sequence (optionally operably linked to the guide sequence), and the tracr sequence. In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for the expression of one or more elements of the CRISPR system are described in the above-mentioned documents, e.g. / US2013 / 074667). In some embodiments, vectors contain one or more insertion sites, eg, restriction endonuclease recognition sequences (also called “cloning” sites). In some embodiments, one or more insertion sites (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 1, about 10, or more, or more than about 1, about 2, about 3, about 4, about 5, about 6, about 7 insertion sites) upstream and / or downstream of one or more sequence elements in one or more vectors. positioned. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and, optionally, an insertion site downstream of a regulatory element operably linked to the tracr mate sequence, thereby allowing insertion of the guide sequence. Upon expression after insertion into the site, the guide sequence directs sequence-specific binding of the CRISPR complex to its target sequence in eukaryotic cells. In some embodiments, the vector comprises two or more insertion sites, each insertion site located between two tracr mate sequences to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations thereof. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20 or more, or more than about 1, about 2, about 3, about 4, about 5, about 6, about 7 , can include more than about 8, more than about 9, more than about 10, more than about 15, more than about 20, or more guide sequences. In some embodiments, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more many or more than about 1, more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8 , vectors containing more than about nine, more than about ten, or more such guide sequences can be provided and optionally delivered to cells. In some embodiments, the vector includes a regulatory element operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, eg, a Cas protein. The CRISPR enzyme, or the CRISPR enzyme mRNA, or the CRISPR guide RNA or RNA can be delivered separately; and advantageously at least one of these is delivered by the nanoparticle complex. The CRISPR enzyme mRNA can be delivered before the guide RNA to allow time for the CRISPR enzyme to express. CRISPR enzyme mRNA may be administered 1 to 12 hours (preferably about 2 to 6 hours) before administration of guide RNA. Alternatively, 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 CRISPR enzyme mRNA plus guide RNA. Additional administration of CRISPR enzyme mRNA and / or guide RNA may be useful to achieve the most efficient level of genome modification.

[0105] In one aspect, the invention provides methods for using one or more elements of the CRISPR system. The CRISPR complexes of the invention provide effective means for modifying target polynucleotides. The CRISPR complexes of the invention have a wide variety of utilities, including modifying (eg, deleting, inserting, translocating, inactivating, activating) target polynucleotides in multiple cell types. Accordingly, the CRISPR complexes of the present invention have a wide range of applications, eg, in gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within a target polynucleotide. The guide sequence is linked to the tracr mate sequence, which hybridizes to the tracr mate sequence. In one embodiment, the invention provides a method of cleaving a target polynucleotide. The method includes modifying a target polynucleotide with a CRISPR complex that binds to and cleaves said target polynucleotide. Typically, the CRISPR complexes of the invention form breaks (eg, single- or double-stranded breaks) in genomic sequences when introduced into a cell. For example, this method can be used to cut disease genes in cells. Breaks formed by CRISPR complexes can be repaired by repair processes such as the miss-repair-prone non-homologous end joining (NHEJ) pathway or high-fidelity homologous recombination repair (HDR). During these repair processes, exogenous polynucleotide templates can be introduced into genomic sequences. In some methods, the HDR process is used to modify the genomic sequence. For example, an exogenous polynucleotide template is introduced into the cell that contains sequences to be integrated flanked by upstream and downstream sequences. The upstream and downstream sequences share sequence similarity on either side of the integration site within the chromosome. If desired, the donor nucleotide is DNA, e.g., DNA plasmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), viral vectors, linear fragments of DNA, PCR fragments, naked nucleic acids, or delivery vehicles, e.g. , a nucleic acid complexed with a liposome or a poloxamer. An exogenous polynucleotide template contains a sequence (eg, a mutated gene) to be incorporated. The sequences for integration may be endogenous or exogenous to the cell. Examples of sequences to be incorporated include polynucleotides encoding proteins or non-coding RNAs (eg, microRNAs). Thus, the sequences for integration can be operably linked to one or more appropriate control sequences. Alternatively, the sequences to be incorporated can provide control functions. The upstream and downstream sequences in the exogenous polynucleotide template are chosen to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. An upstream sequence is a nucleic acid sequence that shares sequence similarity with genomic sequences upstream of the target site for integration. Likewise, downstream sequences are nucleic acid sequences that share sequence similarity with chromosomal sequences downstream of the target site of integration. The upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target genomic sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity with the target genomic sequence. . In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or about 100% sequence identity with the target genomic sequence. Upstream or downstream sequences are from about 20bp to about 2500bp, such as about 50bp, about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp. , about 1200bp, about 1300bp, about 1400bp, about 1500bp, about 1600bp, about 1700bp, about 1800bp, about 1900bp, about 2000bp, about 2100bp, about 2200bp, about 2300bp, about 2400bp, or about 2500bp. In some methods, exemplary upstream or downstream sequences have from about 200bp to about 2000bp, from about 600bp to about 1000bp, or especially from 700bp to about 1000bp. In some methods, an exogenous polynucleotide template can further comprise a marker. Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. Exogenous polynucleotide templates of the invention can be produced using recombinant techniques (see, eg, Sambrook et al., 2001 and Ausubel et al., 1996). In a method of modifying a target polynucleotide by incorporating an exogenous polynucleotide template, a double-stranded break is introduced into the genomic sequence by the CRISPR complex, which break allows the exogenous polynucleotide template to be integrated into the genome. As such, it is repaired by homologous recombination of this template. The presence of a double-strand break facilitates template integration. In other embodiments, the invention provides methods of altering the expression of polynucleotides in eukaryotic cells. The method involves increasing or decreasing expression of a target polynucleotide through the use of a CRISPR enzyme that binds to the target polynucleotide. In some methods, the target polynucleotide can be inactivated to alter its expression within the cell. For example, when a CRISPR complex binds to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is no longer transcribed, the encoded protein is no longer produced, or the sequence does not function like the wild-type sequence. no longer. For example, a protein or microRNA coding sequence can be inactivated such that transcription of this protein or microRN or pre-microRNA does not occur. In some methods, a control sequence can be inactivated so that it no longer functions as a control sequence. As used herein, the term "control sequence" refers to any nucleic acid sequence that effectuates transcription, translation, or accessibility of a nucleic acid sequence. Examples of regulatory sequences include promoters, transcription terminators, and enhancers. The target polynucleotide of the CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, a target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. Target polynucleotides can be sequences that encode gene products (eg, proteins) or non-coding sequences (eg, regulatory polynucleotides or junk DNA). Examples of target polynucleotides include sequences associated with signaling biochemical pathways, eg, signaling biochemical pathway-associated genes or polynucleotides. Examples of target polynucleotides include disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide produces a transcript or translation product at an aberrant level or in an aberrant morphology in cells derived from disease-affected tissue compared to non-disease control tissue or cells Refers to any gene or polynucleotide. A disease-associated gene can be a gene that becomes expressed at an abnormally high level; a disease-associated gene can be a gene that becomes expressed at an abnormally low level, the altered expression of which is associated with disease. Correlate with onset and / or progression. A disease-associated gene also refers to a gene that has a mutation or genetic variation that is directly involved in disease pathogenesis or that is in linkage disequilibrium with a gene involved in disease pathogenesis. Transcription or translation products may be known or unknown, and may be at normal or abnormal levels. The target polynucleotide of the CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, a target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. Target polynucleotides can be sequences that encode gene products (eg, proteins) or non-coding sequences (eg, regulatory polynucleotides or junk DNA). Without wishing to be bound by theory, it is believed that the target sequence must be associated with a PAM (protospacer adjacent motif); ie, a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for PAMs vary depending on the CRISPR enzyme used, but PAMs are typically sequences of 2-5 base pairs adjacent to the protospacer (i.e., the target sequence). . Examples of PAM sequences are provided in the Examples section below, and one skilled in the art will be able to identify additional PAM sequences for use with a given CRISPR enzyme. In some embodiments, the method comprises allowing the CRISPR complex to bind to a target polynucleotide resulting in cleavage of said 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 in said target polynucleotide, said guide sequence linked to a tracr mate sequence, said tracr mate sequence hybridizing to a tracr sequence . In one aspect, the invention provides a method of altering expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing binding of the CRISPR complex to a polynucleotide, wherein said binding increases or decreases expression of said polynucleotide; It comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in nucleotides, said guide sequence linked to a tracr mate sequence, which hybridizes to the tracr sequence. Similar considerations and conditions apply to methods of altering target polynucleotides as described above. In fact, these sampling, culturing, and reintroduction options apply to all aspects of the present invention. In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell, which method can be performed in vivo, ex vivo, or in vitro.In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal and modifying the one or more cells. Culturing can be performed ex vivo at all stages. One or more cells can even be reintroduced into a non-human animal or plant. For reintroduced cells, it is particularly preferred that the cells are stem cells.

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

[0107] The present invention relates to the engineering and optimization of systems, methods, and compositions used to control gene expression involving sequence targeting, e.g., genome perturbation or genome editing, related to the CRISPR-Cas system and its components. is connected with. In an advantageous embodiment the Cas enzyme is Cas9. An advantage of the methods of the invention is that the CRISPR system minimizes or avoids off-target binding and its side effects. This is accomplished using a system arranged to have a high degree of sequence specificity for target DNA.

[0108] self-inactivating system After the intended change has been introduced, such as by editing the intended copy of the gene in the cell's genome, there is no further need for CRISRP / Cas9 expression to continue in the cell. In fact, sustained expression may be undesirable in certain cases, such as off-target effects at unintended genomic sites. Timed expression may therefore be useful. Inducible expression provides one approach, but applicants have also engineered a self-inactivating CRISPR-Cas9 system that relies on the use of non-coding guide target sequences within the CRISPR vector itself. Thus, after the onset of expression, the CRISPR system may bring about its own disruption, but before disruption is complete, it may have time to edit the genomic copy of the target gene (the target gene is the normal gene in diploid cells). Point mutations require at most two edits). Simply, the self-inactivating CRISPR-Cas system targets the coding sequence of the CRISPR enzyme itself, or one or more non-coding guide target sequences complementary to unique sequences present in one or more of the following: (i.e., guide RNA) that targets: (a) within the promoter driving expression of the non-coding RNA element, (b) within the promoter driving expression of the Cas9 gene, (c) the Cas9 coding Within 100 bp of the ATG translation start codon in the sequence, (d) within the inverted terminal repeat (iTR) of the viral delivery vector, eg in the AAV genome.

[0109] HDR efficiency Although the amount of genomic modification in the target cell population required to produce a therapeutic effect varies by disease, the efficacy of many editing therapies improves with increased editing rates. As previously mentioned, the editing rate is controlled by the activity of the DSB repair pathway and the efficiency of delivery to cells of interest. Improving either one of these factors is therefore likely to improve the efficacy of editing therapy.

[0110] Attempts to increase the rate of activity of the DSB repair pathway have generally focused on HDR, as the challenges of cell cycle regulation and delivery of HDR templates with nucleases make strategies using this pathway less efficient than NHEJ. This is because it is relatively low. Cell cycle regulation is now somewhat circumvented by stimulating mitosis with pharmacological agents ex vivo for slow-cycling cell types [Kormann, M.S., et al. Nature biotechnology 29, 154-157. (2011)]. However, true postmitotic cells do not appear to be suitable for such manipulations, limiting the applicability of this strategy. Attempts have been made to circumvent the need for HDR entirely by directly ligating a DNA template containing the therapeutic transgene into the target DSB. Such ligation events have been observed, but at too low a rate to be therapeutically useful [Ran, F.A., et al. Cell 154, 1380-1389 (2013); Orlando, S.J., et al. Nucleic acids research 38, e152( 2010)]. Perhaps dramatically new approaches are needed to improve HDR efficiency and increase the therapeutic efficacy of strategies that require precise genome modification.

[0111] Genome editing offers an interesting opportunity to tackle a number of intractable diseases. Nevertheless, this technology is still in its early stages and requires many iterations to systematically optimize its efficacy, safety and specificity. In addition, despite the tremendous excitement surrounding genome editing, strategic planning and rigorous but feasible controls are required to ensure the successful development of this class of potentially life-changing medicines. A process is required.

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

[0113] For in vivo applications, the most promising delivery systems are viral vectors, especially adeno-associated virus (AAV) vectors, which have recently been approved for clinical use [Wirth, T., et al. (2013)]. AAV has many serotypes and has been shown to have high delivery efficacy in different tissue types, including eye, brain, liver, and muscle [Samulski, R.J. & Muzyczka, N. Annual Review of Virology 1,427. -451 (2014)]. However, AAV vectors have relatively poor packaging capacity, which poses several challenges for nuclease delivery. ZFNs are relatively small and dimeric ZFN pairs can be packaged into a single AAV, whereas dimeric TALEN pairs are much larger and may have to be packaged into two separate AAV vectors. expensive. As for Cas9, short orthologs can be packaged into a single AAV with guide RNA. So far, AAV-mediated nuclease expression has been successfully demonstrated in several tissue types, including liver and brain [Li, H., et al. Nature 475, 217-221 (2011); Swiech, L., et al. Nature biotechnology (2014)].

[0114] Despite the potential of AAV-mediated in vivo nuclease expression, there are several challenges that require further development. First, AAV-mediated nuclease expression is often constitutive and it would be more desirable if nuclease expression could be blocked after a successful genome editing event in the target cell. Second, patients who have already been naturally exposed to AAV are likely to have developed immunity to a particular serotype. AAV may therefore not be a suitable delivery vehicle for these patients. To overcome these challenges faced with viral vectors, nanoparticle- and lipid-based in vivo mRNA or protein delivery systems may offer attractive alternatives [Zuris, J.A., et al.Nature biotechnology (2014) Kormann, M.S., et al. Nature biotechnology 29, 154-157 (2011)].

[0115] Using this disclosure and knowledge in the art, it is possible that the delivery systems described herein, both in general and in detail, can be used to deliver CRISPR-Cas systems, or components thereof or nucleic acid molecules thereof (e.g., HDR templates). or a nucleic acid molecule that encodes or provides a component thereof.

[0116] Vector delivery, e.g. plasmid, viral delivery: CRISPR enzymes, e.g. Cas9, and / or any subject RNA, e.g. guide RNA, in any suitable vector, e.g. plasmid or viral vector, e.g. AAV), lentiviral, adenoviral, or viral vectors of other species, or combinations thereof. Cas9 and one or more guide RNAs can be packaged in one or more vectors, eg, plasmid or viral vectors. In some embodiments, the vector, e.g., plasmid or viral vector, is delivered to the tissue of interest, e.g., by intramuscular injection, otherwise delivery is intravenous, transdermal, intranasal, buccal, mucosal, or by other delivery methods. Such delivery may be single dose or multiple dose. One skilled in the art will appreciate that the actual dose delivered herein will depend on a variety of factors, such as the choice of vector, the target cell, organism or tissue, the general health of the subject to be treated, the transformation / transformation sought. It will be appreciated that the extent of modification, route of administration, mode of administration, type of transformation / modification sought, etc., can vary widely.

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

[0118] In one embodiment herein the delivery is by adenovirus and the delivery is at least 1 x 10 5 of particles (also called particle units, pu) of the adenoviral vector. In one embodiment herein, this dose is preferably at least about 1 x 10 6 of particles (e.g., about 1 × 10 6 ~1×10 12 particles), more preferably at least about 1 x 10 7 of particles, more preferably at least about 1 x 10 8 of particles (e.g., about 1 × 10 8 ~1×10 11 of particles or about 1 x 10 8 ~1×10 12 particles), and most preferably at least about 1 x 10 0 of particles (e.g., about 1 × 10 9 ~1×10 10 of particles or about 1 x 10 9 ~1×10 12 particles), or even at least about 1 x 10 10 of particles (e.g., about 1 × 10 10 ~1×10 12 particles) adenoviral vectors. Alternatively, the dose is about 1 x 10 14 less particles, preferably about 1 x 10 13 the following particles, even more preferably about 1 x 10 12 the following particles, even more preferably about 1 x 10 11 less particles, and most preferably about 1 x 10 10 less particles (e.g., about 1 x 10 9 particles below). Therefore, the dose is, for example, about 1 x 10 6 of particle units (pu), about 2 × 10 6 pu, about 4×10 6 pu, about 1×10 7 pu, about 2×10 7 pu, about 4×10 7 pu, about 1×10 8 pu, about 2×10 8 pu, about 4×10 8 pu, about 1×10 9 pu, about 2×10 9 pu, about 4×10 9 pu, about 1×10 10 pu, about 2×10 10 pu, about 4×10 10 pu, about 1×10 11 pu, about 2 x 10 11 pu, about 4×10 11 pu, about 1×10 12 pu, about 2×10 12 pu, or about 4 x 10 12 A single dose of adenoviral vector, including pu adenoviral vector, may be included. See, for example, the adenoviral vectors of U.S. Pat. No. 8,454,972 B2, issued June 4, 2013 to Nabel et al., which is incorporated herein by reference; See line 58. In one embodiment herein, the adenovirus is delivered by multiple administrations.

[0119] In one embodiment herein, delivery is by AAV. A therapeutically effective dose for in vivo delivery of AAV to humans is approximately 1×10 10 ~ about 1 x 10 10 of a functional AAV / ml solution of about 20 to about 50 ml of saline. Dosage may be adjusted to balance therapeutic effect against any side effects. In one embodiment herein, the dose of AAV is generally about 1 x 10 5 ~1×10 50 genome of AAV, approximately 1 x 10 8 ~1×10 20 genome of AAV, approximately 1 x 10 10 ~ about 1 x 10 16 genome, or approximately 1 x 10 11 ~ about 1 x 10 16 is the concentration range of genomic AAV. The human dose is approximately 1 x 10 13 can be a genomic AAV of Such concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. Other effective doses can be readily established by those skilled in the art through routine trials establishing dose-response curves. See, for example, paragraph 27, lines 45-60 of US Pat. No. 8,404,658 B2, issued March 26, 2013 to Hajjar et al.

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

[0121] The doses herein are based on an average 70 kg person. The frequency of administration is within the purview of the medical or veterinary practitioner (eg, physician, veterinarian) or one of ordinary skill in the art. Also note that mice used for experiments are typically around 20 g, and experiments in mice can be scaled up to 70 kg humans.

[0122] In some embodiments, the RNA molecules of the invention are delivered, such as in liposomes or lipofection formulations, and can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, incorporated herein by reference. Delivery systems have been developed specifically for the delivery of improved siRNAs to mammalian cells (see, for example, 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. 2002, 32:107-108 and Simeoni et al., NAR 2003, 31, 11:2717-2724) can be applied to the present invention. siRNAs have recently been used successfully in silencing gene expression in primates (see, eg, Tolentino et al., Retina 24(4):660, which may also be applied to the present invention).

[0123] Indeed, delivery of RNA is a useful method of in vivo delivery. Liposomes or nanoparticles can be used to deliver Cas9 and gRNA (and, for example, HR repair templates) into cells. Thus, delivery of CRISPR enzymes, such as Cas9, and / or delivery of RNAs of the invention can be performed in RNA form via microvesicles, liposomes, or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged within liposomal particles for delivery in vivo. Liposomal transfection reagents, such as Life Technologies' Lipofectamine and other commercially available reagents, can effectively deliver RNA molecules to the liver.

[0124] The means of delivery of RNA is also preferably nanoparticulate delivery of RNA (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S. ., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010) or delivery by 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). Indeed, exosomes should be particularly useful for delivery of siRNA, a system that has some similarities to the CRISPR system. For example, El-Andaloussi S, et al. Nov 15.) describe how exosomes are promising tools for drug delivery across various biological barriers and can be used to deliver siRNA in vtiro and in vivo. In this approach, targeted exosomes are generated by transfection of expression vectors containing exosomal proteins fused to peptide ligands. Exosomes are then purified and characterized from transfected cell supernatants, and RNA is then introduced into the exosomes. Delivery or administration according to the present invention, particularly but not exclusively to the brain, can be performed using exosomes. Conjugation of Vitamin E (alpha-tocopherol) to CRISPR Cas to deliver short interfering RNA (siRNA) to the brain, for example by Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) It can be delivered to the brain along with high density lipoprotein (HDL) in a similar manner. Mice were infused by osmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE / HDL and connected to the Brain Infusion Kit 3 (Alzet). did. A brain injection cannula was placed approximately 0.5 mm posterior to bregma in the midline for injection into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA containing HDL was able to produce comparable target reduction with the same ICV injection method. A similar dose of CRISPR Cas conjugated to α-tocopherol co-administered with HDL to target the brain may be contemplated in humans in the present invention, e.g. Cas can be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April 2011)) described a method for lentiviral-mediated delivery of short hairpin RNAs targeting PKCγ for in vivo gene silencing in the rat spinal cord. Zou et al. 9 Approximately 10 μl of recombinant lentivirus with a titer of transducing units (TU) / ml was administered via an intrathecal catheter. Similar amounts of CRISPR Cas expressed in brain-targeted lentiviral vectors may be contemplated in humans in the present invention, e.g. 9 Approximately 10-50 ml of brain-targeted CRISPR Cas with a lentivirus having a titer of 100 transducing units (TU) / ml can be envisioned.

[0125] For local delivery to the brain, this can be achieved in various ways. For example, substances can be delivered into the striatum, eg, by injection. Injection can be performed stereotactically via a craniotomy.

[0126] In some embodiments, the invention provides one or more polynucleotides, eg, or one or more vectors, one or more transcripts thereof, and / or transcribed therefrom, as described herein. A method is provided comprising delivering an entity or protein to a host cell. In some aspects, the invention further provides cells produced by such methods, and animals comprising or produced from such cells. In some embodiments, the CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. Conventional viral-based and non-viral-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues.

[0127] Using such methods, nucleic acids encoding components of the CRISPR system can be administered to cells in culture or to cells in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to cells. For a review of gene therapy procedures, Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167. -175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); , British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Boehm(eds) (1995); and Yu et al., Gene Therapy 1:13-. See 26 (1994). Non-viral methods of delivery of nucleic acids include lipofection, microinjection, microprojectile bombardment, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355), and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin ( trademark)). Cationic and neutral lipids suitable for efficient receptor-recognizing lipofection of polynucleotides include those of Felgner, WO91 / 17424; WO91 / 16024. Delivery may be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration). Preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, are well known to those of skill in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787). Delivery of nucleic acids using RNA or DNA virus-based systems utilizes highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors may be administered directly to the patient (in vivo) or may be used to treat cells in vitro and optionally the modified cells may be administered to the patient (ex vivo). Conventional viral-based systems can include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors for gene transfer. Retroviral, lentiviral, and adeno-associated viral gene transfer methods are capable of integration in the host genome, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. Retroviral tropism can be altered by the incorporation of foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system may therefore depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats capable of packaging up to 6-10 kb of foreign sequences. A minimal cis-acting LTR is sufficient for vector replication and packaging, which is then used to integrate the therapeutic gene into target cells, resulting in permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. (e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176 :58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); See specification). In another embodiment, Cocal vecyclovirus envelope pseudotyped retroviral vector particles are contemplated (e.g., US Patent Application Publication No. 20120164118, assigned to Fred Hutchinson Cancer Research Center). (see specification). Cocal virus belongs to the Vesiculovirus genus and is the causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from ticks in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)) and has been isolated from insects and cattle in Trinidad, Brazil, and Argentina. , and horses. Many of the vesicloviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculovirus are common in people living in rural areas, where the virus is endemic and infectious in the laboratory; infection in humans usually results in flu-like symptoms. The cocal virus envelope glycoprotein shares 71.5% identity with VSV-G Indiana at the amino acid level, and a phylogenetic comparison of the envelope genes of vesiculoviruses shows that among the vesiculoviruses, cocal virus is VSV- Although serologically different from the G Indiana strain, it is shown to be most closely related. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006 (1984). Caucasian vecyclovirus envelope-pseudotyped retroviral vector particles include, for example, lentiviruses, alpharetroviruses, beta, which may contain retroviral Gag, Pol, and / or one or more accessory proteins and cocalveciclovirus envelope proteins. Retrovirus, gammaretrovirus, deltaretrovirus, and epsilon retrovirus vector particles may be included. Within certain aspects of these embodiments, the Gag, Pol, and accessory proteins are lentiviral and / or gammaretroviral. Adenovirus-based systems can be used for applications where transient expression is preferred. Adenoviral-based vectors can exhibit extremely high transduction efficiencies in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors can also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (e.g. , West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); 94:1351 (1994) Construction of recombinant AAV vectors has been published in numerous publications, including US Pat. No. 5,173,414; 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Virol.63:03822-3828 (1989) Packaging cells are typically used to form virus particles capable of infecting host cells. 293 cells, which package virus, and Ψ2 cells or PA317 cells, which package retrovirus.Viral vectors used in gene therapy are usually made by creating cell lines that package the nucleic acid vector into viral particles. Vectors typically contain the minimal viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences replaced by an expression cassette for the polynucleotide to be expressed. Defective viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used for gene therapy typically only have ITR sequences from the AAV genome that are necessary for packaging and integration into the host genome. Viral DNA is packaged into a cell line containing a helper plasmid that encodes other AAV genes, namely rep and cap, but lacks the ITR sequences. This cell line can also be infected with adenovirus as a helper. The helper virus facilitates replication of the AAV vector and expression of the AAV genes from the helper plasmid. Since the helper plasmid lacks the ITR sequences, it is not packaged in large quantities. Contamination with adenovirus can be reduced, for example, by heat treatment to which adenovirus is more susceptible than AAV. AAV is therefore considered an ideal candidate for use as a transduction vector. Such AAV transduction vectors may contain sufficient cis-acting functions to replicate in the presence of adenoviral or herpesviral or poxviral (eg, vaccinia virus) helper functions provided in trans. Recombinant AAV (rAAV). can be used to carry exogenous genes into cells of various lineages. In these vectors, the AAV cap and / or rep genes have been deleted from the viral genome and replaced with DNA segments of choice. Current AAV vectors can accommodate inserts of up to 4300 bases. There are a number of methods for making rAAV, and the present invention provides rAAV and methods for preparing rAAV. For example, one or more plasmids containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells. Additionally, a second or additional helper plasmid is co-transfected into these cells to provide the AAV rep and / or cap genes essential for replication and packaging of the recombinant viral construct. Under these conditions, AAV rep and / or cap proteins act in trans to stimulate replication and packaging of rAAV constructs. Harvest rAAV 2-3 days after transfection. Conventionally, rAAV is recovered from cells along with adenovirus. Contaminating adenovirus is then inactivated by heat treatment. In the present invention rAAV is advantageously recovered from the cell supernatant rather than from the cells themselves. Thus, in a first aspect, the present invention provides for preparing rAAV, and in addition to the foregoing, rAAV can be prepared by a method comprising or consisting essentially of: exogenous DNA containing DNA for expression; Infecting susceptible cells with rAAV containing DNA and a helper virus (e.g., a poxvirus such as adenovirus, herpesvirus, vaccinia virus), wherein the rAAV lacks functional cap and / or rep. (and a helper virus (e.g., a poxvirus such as adenovirus, herpesvirus, vaccinia virus) provides cap and / or rev functions that rAAV lacks); infecting a susceptible cell with the rAAV containing rAAV, wherein the recombinant lacks functional cap and / or rep; transfecting said cells; or infecting susceptible cells with rAAV containing exogenous DNA, including DNA for expression, wherein said recombinant lacks functional cap and / or rep; providing the cell with cap and / or rep functions deficient in the recombinant; transfecting a susceptible cell with plasmids for inserting exogenous DNA into the recombinant and for providing rep and / or cap functions, thus transfecting functional cap and / or or rep-deficient rAAV containing exogenous DNA, including DNA for expression. The rAAV may be derived from AAV as described herein, advantageously rAAV1, rAAV2, AAV5 or rAAV with a hybrid or capsid that may comprise AAV1, AAV2, AAV5 or any combination thereof. could be. The AAV of the rAAV can be selected in relation to the cells to which the rAAV targets; or any combination thereof can be selected; and AAV4 can be selected for targeting cardiac tissue. In addition to 293 cells, other cells that can be used in the practice of the invention and the relative infectivity of specific AAV serotypes in vitro on those cells (Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) are as follows:

[0128] [Table 2]

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

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

[0131] General packaging and promoter A method for packaging a Cas9-encoding nucleic acid molecule, e.g., DNA, into a vector, e.g., a viral vector, to mediate genome modification in vivo is: To achieve NHEJ-mediated gene knockout: Single viral vectors: A vector 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 vector size limit): Double viral vectors: Vector 1 containing one expression cassette driving the expression of Cas9: promoter-Cas9-encoding nucleic acid molecule-terminator: Vector 2 containing one or more expression cassettes driving expression of one or more guide RNAs: ·Promoter-gRNA1-Terminator: Promoter-gRNA(N)-Terminator: (up to vector size limit): Mediates homology-dependent repair: · In addition to the single and double viral vector approaches described above, additional vectors are used to deliver homology-dependent repair templates.

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

[0133] Promoters used to drive guide RNAs can include: Pol III promoter, such as U6 or H1 Use of Pol II promoter and intron cassette to express gRNA.

[0134] Adeno-associated virus (AAV) Cas9 and one or more guide RNAs can be isolated using adeno-associated virus (AAV), lentivirus, adenovirus, or other types of plasmid or viral vectors, particularly, for example, US Pat. No. 8,454,972 (adenovirus from US Pat. It can be delivered using formulations and doses from publications on clinical trials on adenovirus. For example, for AAV, the route of administration, formulation, and dosage can be similar to US Pat. No. 8,454,972 and clinical trials for AAV. For adenovirus, the route of administration, formulation, and dosage can be similar to US Pat. No. 8,404,658 and clinical trials for adenovirus. For plasmid delivery, the route of administration, formulation, and dosage can be similar to US Pat. No. 5,846,946 and clinical studies with plasmids. Dosages can be based on or extrapolated from an average 70 kg person (eg, an adult male human) and can be adjusted for different weights and species of patients, subjects, mammals. The frequency of administration is within the realm of the medical or veterinary practitioner (e.g., physician, veterinarian), age, sex, general health, other conditions of the patient or subject, and the particular condition being addressed. or depending on usual factors, including symptoms. Viral vectors can be injected into the tissue of interest. For cell-type specific genomic modifications, Cas9 expression can be driven by cell-type specific promoters. For example, liver-specific expression can use the albumin promoter, and neuron-specific expression (eg, when targeting CNS disorders) can use the Synapsin I promoter. With regard to in vivo delivery, AAV is superior to other viral vectors for a few reasons: low toxicity, which is obtained by purification methods that do not require ultracentrifugation of cell particles that can activate immune responses. Advantageous.

[0135] Since AAV does not integrate into the host genome, it is less likely to cause insertional mutagenesis.

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

[0137] [Table 3]

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

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

[0140] [Table 4]

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

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

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

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

[0145] In another embodiment, a self-inactivating lentiviral vector (e.g., , DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and / or adapted to the CRISPR-Cas system of the present invention. A minimum of 2.5 x 106 CD34+ cells per kg of patient body weight were collected and treated with 2 µmol / L-glutamine, stem cell factor (100 ng / ml), Flt-3 ligand (Flt-3L) (100 ng / ml), and thrombopoietin (100 ng / ml). 10 ng / ml) (CellGenix) in X-VIVO15 medium (Lonza) at a concentration of 2×10 6 cells / ml for 16-20 hours. Prestimulated cells can be transduced with lentivirus in 5-fold infection in fibronectin-coated 75 cm2 tissue culture flasks (25 mg / cm2) (RetroNectin, Takara Bio Inc.) for 16-24 hours. .

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

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

[0148] CRISPR enzyme coding sequence and / or guide RNA to include one or more modified nucleotides, e.g., with pseudo-U or 5-methyl-C, to facilitate expression and reduce possible toxicity. can be modified.

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

[0150] Although much clinical research on RNA delivery has focused on RNAi or antisense, these systems can be applied to deliver RNA for the practice of the present invention. The following references on RNAi and others should be read as appropriate.

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

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

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

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

[0155] nanoparticles CRISPR enzyme mRNA and guide RNA can be co-delivered using nanoparticles or lipid envelopes.

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

[0157] In one embodiment, nanoparticles based on self-assembled bioadhesive polymers are contemplated, which are applied for oral delivery of peptides, intravenous delivery of peptides, and nasal delivery of peptides, all of which are delivery to the brain. can do. Other embodiments are also contemplated, such as oral absorption and ocular delivery of hydrophobic drugs. Molecular envelope technology involves engineered polymeric envelopes delivered to protected disease sites (e.g., Mazza, M. et al. ACS Nano, 2013.7(2):1016-1026; Siew, A., et al. Mol Pharm Lalatsa, A., et al. J Contr Rel, 2012.161(2): 523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6): 1665- 80;Lalatsa,A.,et al.Mol Pharm,2012.9(6):1764-74;Garrett,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,I.F.Expert Opin Drug Deliv,2006.3(5):629-40;Qu, X., et al. Biomacromolecules, 2006.7(12):3452-9 and Uchegbu, I.F., et al. Int J Pharm, 2001.224:185-199). A dose of about 5 mg / kg is contemplated, with single or multiple doses depending on the target tissue.

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

[0159] US Patent Application Publication No. 20110293703 relates to lipid compounds that are also particularly useful for the administration of polynucleotides, which lipid compounds can be applied for delivery of the CRISPR Cas system of the invention. In one aspect, an aminoalcohol lipid compound is combined with an agent to be delivered to a cell or subject to form a microparticle, nanoparticle, liposome, or micelle. Agents to be delivered by particles, liposomes, or micelles can be in gas, liquid, or solid form, and the agents can be polynucleotides, proteins, peptides, or small molecules. The aminoalcohol lipid compounds can be combined with other aminoalcohol lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles can then optionally be combined with pharmaceutical excipients to form pharmaceutical compositions.

[0160] US Patent Application Publication No. 20110293703 also provides methods of preparing aminoalcohol lipid compounds. One or more equivalents of an amine are reacted with one or more equivalents of an epoxide-terminated compound under suitable conditions to form the aminoalcohol lipid compounds of the invention. In certain embodiments, all amino groups of the amine are sufficiently reacted with the epoxide-terminated compound to form a tertiary amine. In other embodiments, not all amino groups of the amine react sufficiently with the epoxide-terminated compound to form tertiary amines, thus leaving primary or secondary amines in the aminoalcohol lipid compound. is formed. These primary or secondary amines can remain intact or react with another electrophile, eg, a different epoxide-terminated compound. As will be appreciated by those skilled in the art, reacting an amine with less than an excess of epoxide-terminated compounds will result in a number of different aminoalcohol lipid compounds with varying 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 can contain 1, 2, 3, or 4 epoxide-derived compound tails separate from the various amino portions of the molecule, primary amines, secondary amines, and tertiary An amine is formed. In certain embodiments, all amino groups are completely unfunctionalized. In certain embodiments, two identical types of epoxide-terminated compounds are used. In other embodiments, two or more different epoxide-terminated compounds are used. Synthesis of aminoalcohol lipid compounds is carried out with or without a solvent, and this synthesis can be carried out at elevated temperatures of 30-100°C, preferably about 50-90°C. The prepared aminoalcohol lipid compound can optionally be purified. For example, a mixture of aminoalcohol lipid compounds can be purified to obtain aminoalcohol lipid compounds with a particular number of epoxide-derived compound tails. Alternatively, the mixture can be purified to obtain specific stereoisomers or regioisomers. Aminoalcohol lipid compounds can also be alkylated and / or acylated using alkyl halides (eg, methyl iodide) or other alkylating agents.

[0161] US Patent Application Publication No. 20110293703 also provides a library of aminoalcohol lipid compounds prepared by the methods of the invention. These aminoalcohol lipid compounds can be prepared and / or screened using high throughput techniques including liquid handlers, robots, microtiter plates, computers, and the like. In certain embodiments, aminoalcohol lipid compounds are screened for their ability to transfect polynucleotides or other agents (eg, proteins, peptides, small molecules) into cells.

[0162] US Patent Application Publication No. 20130302401 relates to a class of poly(β-aminoalcohol)s (PBAAs) prepared using combinatorial polymerization. The PBAAs of the present invention are useful in biotechnology as coatings (e.g., thin film or multi-layer thin film coatings for medical devices or implants), additives, materials, excipients, non-bioadhesive agents, micropatterning agents, and cell encapsulating agents. and can be used for medical applications. When used as surface coatings, these PBAAs caused different levels of inflammation both in vitro and in vivo due to their chemical structure. The wide chemical diversity of this class of materials has allowed us to identify polymer coatings that inhibit macrophage activity in vitro. In addition, these coatings reduce inflammatory cell recruitment and alleviate fibrosis after subcutaneous injection of carboxylated polystyrene microparticles. These polymers can be used to form polyelectrolyte composite capsules for cell encapsulation. The invention may also have many other biological applications such as, for example, antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Application Publication No. 20130302401 can be applied to the CRISPR Cas system of the present invention.

[0163] In another embodiment, lipid nanoparticles (LNPs) are also contemplated. Anti-transthyretin short interfering RNA has been delivered to humans encapsulated within lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29) and such systems can be adapted and applied to the CRISPR Cas system of the present invention. A dose of about 0.01 to about 1 mg / kg of body weight administered intravenously is contemplated. Agents that reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine. Multiple doses of about 0.3 mg / kg every 4 weeks for a total of 5 doses are also contemplated.

[0164] LNPs have been shown to be highly effective in delivering siRNA to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No.4, pages 363-470), Delivery of RNA encoding CRISPR Cas to the liver is therefore contemplated. About 4 doses of 6 mg / kg of LNP every 2 weeks may be contemplated. Tabernero et al. observed tumor regression after the first 2 cycles of 0.7 mg / kg LNP, and by the end of 6 cycles, patients showed complete regression of lymph node metastases and substantial shrinkage of liver tumors. It was demonstrated that a partial reaction was achieved. A complete response was obtained after 40 doses in this patient who remained in remission and terminated treatment after 26 months of dosing. Two patients with extrahepatic sites of disease involving kidney, lung, and lymph nodes and RCC who progressed after prior treatment with VEGF pathway inhibitors had stable disease at all sites for approximately 8-12 months. Patients with PNETs and liver metastasis continued on an extension study for 18 months (36 doses) with stable disease.

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

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

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

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

[0169] References available in connection with the teachings herein include: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2012 5:6962-6970, Cutler et al., J.Am.Chem.Soc.2012 134:1376-1391, Young et al., Nano Lett.2012 12:3867-71 USA.2012 109:11975-80,Mirkin, Nanomedicine 2012 7:635-638 Zhang et al.,J.Am.Chem.Soc.2012 134:16488- 1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. (2013), and Mirkin, et al., Small, 10:186-192.

[0170] Self-assembled nanoparticles containing RNA can be formed using polyethyleneimine (PEI) PEGylated with Arg-Gly-Asp (RGD) peptide ligands attached to the distal end of polyethylene glycol (PEG). . This system, for example, targets tumor neovasculature expressing integrins and delivers siRNAs that suppress the expression of vascular endothelial growth factor receptor-2 (VEGF R2) as a means of achieving tumor angiogenesis. used (see, eg, Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymers and nucleic acids to provide a net molar excess of ionized nitrogen (polymer) to phosphate (nucleic acid) in the range of 2-6. . Electrostatic interactions between cationic polymers and nucleic acids form polyplexes with an average particle size distribution of about 100 nm, hence the term nanoplexes herein. A dose of approximately 100-200 mg of CRISPR Cas is contemplated for delivery in the self-assembled nanoparticles of Schiffelers et al.

[0171] The nanoplexes of Bartlett et al. (PNAS, September 25, 2007, vol.104, no.39) can also be applied to the present invention. Bartlett et al.'s nanoplexes are prepared by mixing equal volumes of aqueous solutions of cationic polymers and nucleic acids and adding a net molar excess of ionized nitrogen (polymer) to phosphate (nucleic acid) in the range of 2-6. . Electrostatic interactions between cationic polymers and nucleic acids form polyplexes with an average particle size distribution of about 100 nm, hence the term nanoplexes herein. Bartlett et al.'s DOTA-siRNA was synthesized as follows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA-NHSester). was ordered from Macrocyclics (Dallas, TX). Amine-modified RNA sense strand and 100-fold molar excess of DOTA-NHSester in carbonate buffer (pH 9) were added to a microcentrifuge tube. The contents were allowed to react by stirring at room temperature for 4 hours. The DOTA-RNA sense strand conjugate was ethanol precipitated, resuspended in water, and annealed to the unmodified antisense strand to yield DOTA-siRNA. All liquids were treated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove trace metal contaminants. Tf-targeted and Tf-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. 1% adamantane-PEG molecules on the surface of target nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (wt / vol) glucose carrier solution for injection.

[0172] Davis et al. (Nature, Vol 464, 15 April 2010) will conduct an RNA clinical trial using a targeted nanoparticle delivery system (Clinical Trial Registry Number NCT00689065). Patients with solid tumors refractory to standard cancer therapy are administered targeted nanoparticles by 30-minute intravenous injection on days 1, 3, 8, and 10 of a 21-day cycle. The nanoparticles target: (1) a linear cyclodextrin-based polymer (CDP), (2) ligands presented on the outer surface of the nanoparticles to bind to TF receptors (TFR) on the surface of cancer cells. human transferrin protein (TF), (3) a hydrophilic polymer (polyethylene glycol (PEG) used to improve the stability of nanoparticles in biological fluids), and (4) RRM2. It consists of a synthetic delivery system containing an siRNA designed to (the sequence used in the clinic has already been designated as siR2B+5). TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anticancer target. These nanoparticles (clinical form designated as CALAA-01) have been shown to be well tolerated in multiple dose studies in non-human primates. A single patient with chronic myelogenous leukemia was given siRNA by liposomal delivery, but the Davis et al. is. To confirm that the targeted delivery system could effectively deliver functional siRNA to human tumors, Davis et al. studied three patients, each comprising three different dosing cohorts: each with metastatic melanoma; 18, 24, and 30mg / m respectively 2 Biopsies from patients A, B, and C who received CALAA-01 at doses of . Similar doses may be contemplated for the CRISPR Cas system of the invention. The delivery of the present invention uses a linear cyclodextrin-based polymer (CDP), a human transferrin protein that targets ligands presented on the outer surface of the nanoparticles to bind to TF receptors (TFR) on the surface of cancer cells. TF), and / or nanoparticles containing hydrophilic polymers (eg, polyethylene glycol (PEG), which is used to improve the stability of nanoparticles in biological fluids).

[0173] For the present invention, one or more components of the CRISPR complex, such as CRISPR enzyme or mRNA or guide RNA or sgRNA or HDR template if present, are delivered using one or more particles or nanoparticles or lipid envelopes. It is preferable to obtain Other delivery systems or vectors can be used in conjunction with the nanoparticle aspect of the invention.

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

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

[0176] Semi-solid nanoparticles and flexible nanoparticles have been produced and are within the scope of the present invention. The semi-solid prototypical nanoparticles are liposomes. Various types of liposomal nanoparticles are currently in clinical use as delivery systems for anticancer drugs and vaccines. Nanoparticles that are half hydrophilic and half hydrophobic are called Janus particles and are particularly effective in stabilizing emulsions. The nanoparticles can self-assemble at the water / oil interface and function as solid surfactants.

[0177] US Pat. No. 8,709,843, incorporated herein by reference, provides drug delivery systems for targeted delivery of particles containing therapeutic agents to tissues, cells, and subcellular compartments. The invention provides targeted particles comprising a surfactant, a hydrophilic polymer, or a polymer conjugated to a lipid. U.S. Pat. No. 6,007,845, incorporated herein by reference, describes a multi-block copolymer core formed by covalent bonding of a polyfunctional compound with one or more hydrophobic polymers and one or more hydrophilic polymers. and providing a particle comprising a biologically active material. U.S. Pat. No. 5,855,913, which is incorporated herein by reference, discloses aerodynamically light particles having a tap density of less than 0.4 g / cm and an average diameter of 5 μm to 30 μm, and pulmonary system particles on the surface thereof. Provided is a microparticle composition comprising a surfactant for drug delivery to cells. U.S. Pat. No. 5,985,309, incorporated herein by reference, describes the use of surfactants and / or positively or negatively charged therapeutic or diagnostic agents with charged molecules of opposite charge for delivery to the pulmonary system. Particles containing hydrophilic or hydrophobic complexes are provided. U.S. Pat. No. 5,543,158, which is incorporated herein by reference, describes biodegradable injectable nanoparticles having a biodegradable solid core comprising biologically active materials on the surface and poly(alkylene glycol) moieties. I will provide a. WO2012135025 (also published as U.S. Patent Application Publication No. 20120251560), incorporated herein by reference, describes conjugated polyethyleneimine (PEI) polymers and conjugated azamacrocycles (summarizing (referred to as "conjugated lipomers" or "lipomers"). In certain embodiments, such methods and materials described herein, e.g., conjugated lipomers, can be used in vivo, ex vivo, and to modulate gene expression, including modulating protein expression. It can be envisioned that it can be used in conjunction with the CRISPR-Cas system to achieve genomic perturbation in vitro.

[0178] In one embodiment, the nanoparticles may be epoxide modified lipid polymers, advantageously 7C1 (e.g. James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online 11 May 2014, doi:10.1038 / nnano .2014.84). C71 was synthesized by reacting C15 epoxide-terminated lipids with PEI600 in a 14:1 molar ratio, and C14PEG2000 was used to formulate nanoparticles (35–60 nm diameter) stable in PBS solution for at least 40 days. did. Although epoxide-modified lipid-polymers can be utilized to deliver the CRISPR-Cas system of the present invention to pulmonary, cardiovascular, or renal cells, those of skill in the art will appreciate other methods for delivery to other target organs. This system could be adapted. A dose of about 0.05 to about 0.6 mg / kg is contemplated. Administration over several days or weeks with a total dose of about 2 mg / kg is also contemplated.

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

[0180] To obtain a pool of immunologically inactive exosomes, Alvarez-Erviti et al. harvested bone marrow from inbred C57BL / 6 mice of the allogeneic major histocompatibility complex (MHC) haplotype. Because immature dendritic cells produce large amounts of exosomes that are free of T cell activators such as MHC-II and CD86, Alvarez-Erviti et al. CSF) was used to select dendritic cells. The next day, exosomes were purified from the culture supernatant using a well-established ultracentrifugation protocol. The exosomes obtained were physically homogeneous, with a size distribution peaking at a diameter of 80 nm as determined by nanoparticle tracking analysis (NTA) and electron microscopy. Alvarez-Erviti et al. 6 We obtained 6-12 μg of exosomes per cell (measured based on protein concentration).

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

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

[0183] Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomes induced immune responses in vivo by assessing serum levels of IL-6, IP-10, TNFα, and IFN-α. . After exosome treatment, insignificant changes in all cytokines were recorded as well as siRNA transfection reagent treatment in contrast to siRNA-RVG-9R, which strongly stimulates IL-6 secretion, demonstrating the immunology of exosome treatment. A relatively inactive profile was confirmed. Given that exosomes encapsulate only 20% of the siRNA, delivery with RVG exosomes showed that comparable mRNA knockdown and larger protein knockdown was achieved with 1 / 5 the siRNA without corresponding levels of immune stimulation. Therefore, it appears to be more efficient than RVG-9R delivery. This experiment demonstrates the therapeutic potential of RVG exosome technology, which may be suitable for long-term silencing of genes associated with neurodegenerative diseases. Alvarez-Erviti et al.'s exosome delivery system can be applied to deliver therapeutic targets of the CRISPR-Cas system of the present invention, particularly to neurodegenerative diseases. A dose of about 100-1000 mg CRISPR Cas encapsulated in about 100-1000 mg RVG exosomes may be contemplated in the present invention.

[0184] El-Andaloussi et al. (Nature Protocols 7, 2112-2126 (2012)) disclose how exosomes from cultured cells can be used to deliver RNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes by transfection of expression vectors containing exosomal proteins fused to peptide ligands. Next, El-Andaloussi et al. describe a method for purification and characterization of exosomes from supernatants of transfected cells. El-Andaloussi et al. then detail the key steps to introduce RNA into exosomes. Finally, El-Andaloussi et al. review how exosomes are used to efficiently deliver RNA to the mouse brain in vitro and in vivo. Examples of prospective results in which exosome-mediated RNA delivery is assessed by functional assays and imaging are also shown. The entire protocol takes approximately 3 weeks. Delivery or administration according to the present invention can be performed using exosomes produced from autologous dendritic cells. The teachings herein can be used to implement the present invention.

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

[0186] Exosomes from plasma were separated by buffy coat centrifugation at 900 g for 20 min and cell supernatant was collected, cells were removed by centrifugation at 300 g for 10 min, and 16500 g for 30 min. It can be prepared by separating and then filtering through a 0.22 mm filter. Pellet the exosomes by ultracentrifugation at 120000 g for 70 minutes. Chemical transfection of siRNA into exosomes is performed according to the manufacturer's instructions for the RNAi Human / Mouse Starter Kit (Qiagen, Hilden, Germany). siRNA is added to 100 ml PBS to a final concentration of 2 mmol / ml. After addition of HiPerFect transfection reagent, the mixture is incubated for 10 min at RT. Re-isolate the exosomes using aldehyde / sulphate latex beads to remove excess micelles. Chemical transfection of CRISPR Cas into exosomes can be performed similarly to siRNA. Exosomes can be cultured with monocytes and lymphocytes isolated from peripheral blood of healthy donors. Therefore, it can be envisioned that exosomes containing CRISPR Cas can be introduced into monocytes and lymphocytes and self-reintroduced into humans. Thus, delivery or administration according to the present invention can be performed using plasma exosomes.

[0187] Liposome Delivery or administration according to the present invention can be done with liposomes. Liposomes are spherical vesicle structures composed of a unilamellar or multilamellar lipid bilayer surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and allow their cargo to cross biological membranes. It has attracted considerable attention as a drug delivery vehicle (e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011.doi:10.1155). / 2011 / 46967 (for reference). Liposomes can be formed from several different types of lipids; phospholipids are most commonly used as drug carriers to form liposomes. Liposome formation occurs spontaneously when lipid films are mixed with aqueous solutions, but can also be facilitated by applying force in the form of agitation by using a homogenizer, sonicator, or extruder (e.g. , Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011.doi:10.1155 / 2011 / 469679 (for reference).

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

[0189] In certain advantageous embodiments, Trojan Horse liposomes (also known as molecular Trojan horses) are preferred and the protocol can be found at http: / / cshprotocols.cshlp.org / content / 2010 / 4 / pdb. You can check it with prot5407.long. These particles are capable of delivering transgenes throughout the brain after vascular injection. It is believed, but not limited to, that neutral lipid particles with specific antibodies conjugated to their surface can cross the blood-brain barrier by endocytosis. Applicants hypothesize that Trojan horse liposomes will be utilized to deliver the CRISPR family of nucleases to the brain by vascular injection, allowing whole-brain transgenic animals without fetal manipulation. In vivo administration of about 1-5 g of DNA or RNA in liposomes can be contemplated.

[0190] In another embodiment, the CRISPR Cas system or components thereof can be administered in liposomes, such as stable nucleic acid lipid particles (SNALPs) (see Morrissey et al., Nature Biotechnology, Vol. 23, No. 8). , August 2005). Daily intravenous injections of about 1 mg / kg / day, 3 mg / kg / day, or 5 mg / kg / day of the specific CRISPR Cas targeted in SNALP are contemplated. Daily treatment can be for about 3 days, followed by once-a-week dosing for 5 weeks. In another embodiment, certain CRISPR Cas encapsulated in SNALP administered by intravenous injection at doses of about 1 mg / kg or 2.5 mg / kg are also contemplated (eg, Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation contains the lipid 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N , N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in mole percent ratios of 2:40:10:48 (e.g. , Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

[0191] In another embodiment, stable nucleic acid lipid particles (SNALPs) demonstrated effective delivery of molecules in highly vascularized HepG2-derived liver tumors, but poorly vascularized HCT-116-derived liver tumors. (see, eg, Li, Gene Therapy (2012) 19, 775-780). SNALP liposomes contained D-Lin-DMA and PEG-C- DMA can be prepared by formulating with distearoylphosphatidylcholine (DSPC), cholesterol, and siRNA. The resulting SNALP liposomes are about 80-100 nm in size. In yet another embodiment, SNALP is synthetic cholesterol (Sigma-Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly( ethylene glycol) 2000) carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic 1,2-dilinoleoyloxy-3-N,N dimethylaminopropane (see, for example, Geisbert et al., Lancet 2010;375:1896-905). For example, a dose of about 2 mg / kg total CRISPR Cas per dose may be contemplated as a bolus intravenous infusion.

[0192] In yet another embodiment, the SNALP is synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2 -dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, eg, Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for in vivo studies may have a final lipid / RNA mass ratio of approximately 9:1. The safety profile of RNAi nanopharmaceuticals has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, eg, Advanced Drug Delivery Reviews 64 (2012) 1730-1737). Stable nucleic acid lipid particles (SNALPs) are composed of four different lipids - a low pH cationic ionic lipid (DLinDMA), a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. The particles are approximately 80 nm in diameter and are neutrally charged at physiological pH. In the formulation, ionic lipids serve to condense lipids with anionic RNA during particle formation. When positively charged under increasingly acidic endosomal conditions, ionic lipids also mediate the fusion of SNALP with the endosomal membrane, allowing release of RNA into the cytoplasm. The PEG-lipid stabilizes the particles and reduces aggregation in the formulation and subsequently provides a neutral, hydrophilic exterior that improves pharmacokinetic properties.

[0193] To date, two clinical programs have been initiated with SNALP formulations containing RNA. Tekmira Pharmaceuticals recently completed a Phase 1 single-dose trial of SNALP-ApoB in adult volunteers with high LDL cholesterol. ApoB is mainly 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 (dose escalating at seven dose levels). No liver toxicity (expected as a potential dose-limiting toxicity based on preclinical studies) was observed. One subject (out of two) at the highest dose exhibited flu-like symptoms consistent with stimulation of the immune system and the decision was made to conclude the study.

[0194] Alnylam Pharmaceuticals has advanced ALN-TTR01 as well. ALN-TTR01 utilizes the SNALP technology described above and targets hepatocyte production of both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR symptoms have been described: familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC)-both caused by autosomal dominant mutations in TTR; and senility caused by wild-type TTR. Systemic amyloidosis (SSA). Recently, a placebo-controlled single-dose escalation phase 1 trial of ALN-TTR01 was completed in patients with ATR. ALN-TTR01 was administered as a 15-minute intravenous infusion to 31 patients (23 on study drug and 8 on placebo) at a dose range of 0.01-1.0 mg / kg (based on siRNA). Treatment was well tolerated with no significant increase in liver function tests. Injection-related reactions were seen in 3 of 23 patients at 0.4 mg / kg and above; all responded to slowing the infusion rate and all continued on study. Minimal and transient elevations of serum cytokines IL-6, IP-10, and IL-1ra were seen in two patients at the highest dose of 1 mg / kg (predicted from preclinical and NHP studies). was done). The expected pharmacodynamic effect of ALN-TTR01 was observed at 1 mg / kg by lowering serum TTR.

[0195] In yet another embodiment, SNALP is performed by solubilizing the cationic lipid, DSPC, cholesterol, and PEG-lipid, respectively, in a molar ratio of 40:10:40:10, for example, with ethanol. (see Semple et al., Nature Niotechnology, Volume 28 Number 2 February 2010, pp. 172-177). This lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) and mixed to a final ethanol and lipid concentration of 30% (vol / vol) and 6.1 mg / ml, respectively, and stored at 22°C prior to extrusion. for 2 minutes. The hydrated lipids were applied to a double layer filter (Nuclepore) with a pore size of 80 nm at 22 °C using a Lipex Extruder (Northern Lipids) until a vesicle diameter of 70-90 nm was obtained as determined by dynamic light scattering analysis. extruded through. This generally requires 1-3 passes. siRNA (solubilized in an aqueous solution of 50 mM citrate, pH 4 containing 30% ethanol) was added to pre-equilibrated (35° C.) vesicles at a rate of approximately 5 ml / min with mixing. Once the final target siRNA / lipid ratio of 0.06 (wt / wt) was reached, the mixture was incubated at 35°C for an additional 30 minutes to allow vesicle reconstitution and siRNA encapsulation. The ethanol is then removed and the external buffer is removed to PBS (155 mM NaCl, 3 mM NaCl) by dialysis or tangential flow diafiltration. 2 HPO 4 , 1 mM KH 2 P.O. 4 , pH 7.5). siRNA was encapsulated in SNALP using a controlled serial dilution process. The lipid components of KC2-SNALP were DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma), used in a molar ratio of 57.1:7.1:34.3:1.4. and PEG-C-DMA. Once encapsulated particles were formed, SNALP was dialyzed against PBS and passed through a 0.2 μm filter to sterilize prior to use. The average particle size was 75-85 nm and 90-95% of the siRNA was encapsulated within the lipid particles. The final si...

Claims

[Claim 1] The invention described in the specification.

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