Materials and methods for treatment of pain-related disorders

Abstract

To provide safe and effective treatments involving SCN9A-related indications.SOLUTION: The present application provides materials and methods for treating a patient having one or more conditions associated with SCN9A regardless of being ex vivo or in vivo. In addition, the present application provides materials and methods for editing and / or modulating the expression of the SCN9A gene in a cell by genome editing.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to the field of gene editing, and specifically to alterations in the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene.

[0002] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 358,763, filed July 6, 2016, and U.S. Provisional Patent Application No. 62 / 461,874, filed February 22, 2017, both of which are incorporated herein by reference in their entireties.

[0003] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING

[0001] This application contains a sequence listing in computer-readable form [filenames: 170152PCT (SCN9A) sequence listing (Part 1): 18,643,508 bytes - ASCII text file; created June 28, 2017; 170152PCT (SCN9A) sequence listing (Part 2): 16,350,005 bytes - ASCII text file; created June 28, 2017; and 170152PCT (SCN9A) sequence listing (Part 3): 14,922,048 bytes], which are incorporated by reference in their entireties and form part of this disclosure. [Background technology]

[0004] Genome engineering refers to strategies and techniques for targeted modification of an organism's genetic information (genome). Genome engineering is a highly active field of research due to its broad applicability, particularly in the field of human health. For example, genome engineering can be used to alter (e.g., correct or knock out) genes harboring harmful mutations or to explore gene function. Early techniques developed for inserting transgenes into living cells were often limited by the random insertion of new sequences into the genome. Random insertion into the genome can disrupt the normal regulation of neighboring genes, leading to serious and undesirable effects. Furthermore, random insertion techniques exhibit little reproducibility because there is no guarantee that the sequence will be inserted in the same location in two different cells. Recent genome engineering strategies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (HEs), and MegaTALs, allow for the modification of specific regions of DNA, thereby increasing the precision of the changes compared to earlier techniques. Although these new platforms demonstrate a fairly high degree of reproducibility, they still have limitations.

[0005] Despite the efforts of researchers and medical professionals worldwide to address genetic disorders, and despite the promise of genome engineering approaches, there remains a critical need to develop safe and effective treatments for SCN9A-related indications.

[0006] By using genome engineering tools that can effect permanent changes in the genome and address SCN9A-associated disorders or conditions with as little as one treatment, the resulting treatments may completely cure certain SCN9A-associated indications and / or diseases. Summary of the Invention

[0007] Provided herein are cellular ex vivo and in vivo methods for effecting permanent genomic changes by genome editing, introducing one or more insertions, deletions, or mutations of at least one nucleotide into or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene, thereby reducing or eliminating the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene product, which can be used to treat pain. Also provided are components and compositions, as well as vectors, for carrying out such methods.

[0008] Provided herein are methods for editing the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene in a cell by genome editing, comprising introducing into the cell one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-stranded (SSB) or double-stranded breaks (DSB) within or near the SCN9A gene or an SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0009] Also provided herein are ex vivo methods for treating a patient with an SCN9A-associated condition or disorder, comprising editing patient-specific induced pluripotent stem cells (iPSCs) within or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene; differentiating the edited iPSCs into neurons of the peripheral nervous system; and administering the neurons of the peripheral nervous system to the patient.

[0010] In some aspects, the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSCs to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory elements, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0011] Also provided herein is an ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising editing mesenchymal stem cells within or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene; differentiating the edited mesenchymal stem cells into neurons of the peripheral nervous system; and administering the neurons of the peripheral nervous system to the patient.

[0012] In some aspects, the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the mesenchymal stem cells to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory elements, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0013] Also provided herein is an in vivo method for treating a patient with an SCN9A-associated disorder, comprising editing the sodium channel voltage-gated alpha subunit 9 (SCN9A) gene in the patient's cells.

[0014] In some aspects, the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the cell to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0015] In some embodiments, the cells are neurons of the peripheral nervous system. In some embodiments, one or more deoxyribonucleic acid (DNA) endonucleases are delivered to neurons of the peripheral nervous system via direct intraganglionic or intrathecal injection, or intrathecal delivery.

[0016] Also provided herein is a method for changing the contiguous genomic sequence of SCN9A gene in a cell, comprising contacting the cell with one or more deoxyribonucleic acid (DNA) endonucleases to make one or more single-strand breaks (SSB) or double-strand breaks (DSB).In some embodiments, the change in the contiguous genomic sequence occurs in one or more exons of the SCN9A gene.

[0017] In some aspects, the one or more deoxyribonucleic acid (DNA) endonucleases are selected from any of SEQ ID NOs: 1-620 and variants having at least 90% homology to any of the sequences listed in SEQ ID NOs: 1-620.

[0018] In some embodiments, the one or more deoxyribonucleic acid (DNA) endonucleases are one or more proteins or polypeptides. In some embodiments, the one or more deoxyribonucleic acid (DNA) endonucleases are one or more polynucleotides encoding one or more DNA endonucleases. In some embodiments, the one or more deoxyribonucleic acid (DNA) endonucleases are one or more ribonucleic acids (RNAs) encoding one or more DNA endonucleases. In some embodiments, the one or more ribonucleic acids (RNAs) are one or more chemically modified RNAs. In some embodiments, the one or more ribonucleic acids (RNAs) are chemically modified in the coding region. In some embodiments, the one or more polynucleotides or the one or more ribonucleic acids (RNAs) are codon-optimized.

[0019] In some embodiments, the method further comprises introducing one or more gRNAs or one or more sgRNAs into the cell. In some embodiments, the one or more gRNAs or one or more sgRNAs comprise a spacer sequence complementary to a DNA sequence within or near the SCN9A gene. In some embodiments, the one or more gRNAs or one or more sgRNAs are chemically modified. In some embodiments, the one or more gRNAs or one or more sgRNAs are pre-complexed with one or more deoxyribonucleic acid (DNA) endonucleases. In some embodiments, the pre-complexing involves covalent binding of the one or more gRNAs or one or more sgRNAs to one or more deoxyribonucleic acid (DNA) endonucleases.

[0020] In some embodiments, one or more deoxyribonucleic acid (DNA) endonucleases are formulated into liposomes or lipid nanoparticles. In some embodiments, one or more deoxyribonucleic acid (DNA) endonucleases are formulated into liposomes or lipid nanoparticles that also contain one or more gRNAs or one or more sgRNAs.

[0021] In some aspects, one or more deoxyribonucleic acid (DNA) endonucleases are encoded within the AAV vector particle. In some aspects, one or more gRNAs or one or more sgRNAs are encoded within the AAV vector particle. In some aspects, one or more deoxyribonucleic acid (DNA) endonucleases are encoded within an AAV vector particle that also encodes one or more gRNAs or one or more sgRNAs. In some aspects, the AAV vector particle is selected from any of those disclosed in SEQ ID NOs: 4734-5302 and Table 2.

[0022] Also provided herein is a single guide RNA comprising at least one spacer sequence that is an RNA sequence corresponding to any of SEQ ID NOs: 5305-125469. In some embodiments, the single guide RNA further comprises a spacer extension region. In some embodiments, the single guide RNA further comprises a tracrRNA extension region. In some embodiments, the single guide RNA is chemically modified.

[0023] In some embodiments, the single guide RNA is pre-complexed with a site-directed polypeptide. In some embodiments, the site-directed polypeptide is a DNA endonuclease. In some embodiments, the DNA endonuclease is Cas9 or CPf1 endonuclease. In some embodiments, the Cas9 or Cpf1 endonuclease is selected from the group consisting of S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpf1, and Acidaminococcus sp. BV3L6 Cpf1, and variants having at least 90% homology to these endonucleases. In some embodiments, the Cas9 or Cpf1 endonuclease comprises one or more nuclear localization signals (NLS). In some embodiments, at least one NLS is at or within 50 amino acids of the amino terminus of the Cas9 or Cpfl endonuclease, and / or at least one NLS is at or within 50 amino acids of the carboxy terminus of the Cas9 or Cpfl endonuclease.

[0024] Also provided herein are RNAs that encode the single molecule guide polynucleotides described herein.

[0025] Also provided herein are RNAs encoding the CRISPR / Cas systems described herein.

[0026] Also provided herein are DNAs encoding the single guide RNA molecules described herein.

[0027] Also provided herein are DNAs encoding the CRISPR / Cas systems described herein.

[0028] Also provided herein is a vector comprising the DNA encoding single-molecule guide RNA or CRISPR / Cas system.In some embodiments, the vector is a plasmid.In some embodiments, the vector is an AAV vector particle, and wherein the AAV vector serotype is selected from SEQ ID NOs: 4734-5302 or those listed in Table 2.

[0029] Various aspects of the materials and methods disclosed and described herein can be better understood by reference to the accompanying drawings. [Brief explanation of the drawings]

[0030] [Figure 1] Figures A-B show type II CRISPR / Cas systems. A is a diagram of a type II CRISPR / Cas system including a gRNA. B is another diagram of a type II CRISPR / Cas system including an sgRNA. [Figure 2A] We show cleavage efficiencies ranging from 93.3 to 98.5% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2B] We show cleavage efficiencies ranging from 86.1 to 93% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2C] We show cleavage efficiencies ranging from 78.8 to 86% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2D] We show cleavage efficiencies ranging from 67.2 to 78.8% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2E] We show cleavage efficiencies ranging from 50.7 to 66.8% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2F]We show cleavage efficiencies ranging from 23.4 to 49.8% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 2G] We show cleavage efficiencies ranging from 4.1 to 22.1% for S. pyogenes gRNAs selected via in-vitro transcribed (IVT) gRNA screening. [Figure 3A] We demonstrate cleavage efficiencies ranging from 80.9 to 98.5% for S. pyogenes gRNAs in HEK293T cells targeting the SCN9A gene. [Figure 3B] We show a cleavage efficiency ranging from 50.7 to 80.9% for S. pyogenes gRNAs in HEK293T cells targeting the SCN9A gene. [Figure 3C] We show a range of cleavage efficiencies of 4.1–49.8% for S. pyogenes gRNAs in HEK293T cells targeting the SCN9A gene. DETAILED DESCRIPTION OF THE INVENTION

[0031] Brief description of the sequence listing SEQ ID NOs: 1 to 620 are Cas endonuclease ortholog sequences.

[0032] SEQ ID NOs: 621 to 631 do not include the sequence.

[0033] SEQ ID NOs: 632 to 4715 are microRNA sequences.

[0034] SEQ ID NOs: 4716 to 4733 do not include the sequence.

[0035] SEQ ID NOs: 4734 to 5302 are AAV serotype sequences.

[0036] SEQ ID NO: 5303 is the SCN9A nucleotide sequence.

[0037] SEQ ID NO: 5304 is a gene sequence including 1 to 5 kilobase pairs upstream and / or downstream of the SCN9A gene.

[0038] SEQ ID NOs: 5305-6250 are 20 bp spacer sequences for targeting the T. denticola Cas9 endonuclease within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0039] SEQ ID NOs: 6251-8561 are 20 bp spacer sequences for targeting the S. thermophilus Cas9 endonuclease within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0040] SEQ ID NOs: 8562-13614 are 20 bp spacer sequences for targeting the S. aureus Cas9 endonuclease within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0041] SEQ ID NOs: 13615-18988 are 20 bp spacer sequences for targeting the N. meningitides Cas9 endonuclease within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0042] SEQ ID NOs: 18989-56863 are 20 bp spacer sequences for targeting the S. pyogenes Cas9 endonuclease within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0043] SEQ ID NOs: 56864-125469 are 20 bp spacer sequences for targeting Acidaminococcus, a Lachnospiraceae, and Franciscella Novicida Cpf1 endonucleases within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0044] SEQ ID NOs: 125470 to 125499 do not include the sequence.

[0045] SEQ ID NO: 125500 is a sample guide RNA (gRNA) for S. pyogenes Cas9 endonuclease.

[0046] SEQ ID NOs: 125501 to 125503 show sample sgRNA sequences.

[0047] Detailed Description I. Introduction Genome editing The present disclosure provides strategies and techniques for targeted alteration of an organism's genetic information (genome). As used herein, the term "alteration" or "alteration of genetic information" refers to any change in a cell's genome. In the context of treating a genetic disorder, alterations can include, but are not limited to, insertions, deletions, and modifications. As used herein, the term "insertion" refers to the addition of one or more nucleotides in a DNA sequence. Insertions can range from small insertions of a few nucleotides to the insertion of larger segments, such as cDNAs or genes. The term "deletion" refers to the loss or removal of one or more nucleotides in a DNA sequence or the loss or removal of gene function. In some cases, deletions can include, for example, the loss of several nucleotides, exons, introns, gene segments, or the entire sequence of a gene. In some cases, gene deletion refers to the removal or reduction of gene function or expression or its gene product. This can result not only from the deletion of sequences within or near a gene, but also from other events (e.g., insertions, nonsense mutations) that disrupt gene expression. The term "correction," as used herein, refers to a change in one or more nucleotides in the genome of a cell, whether by insertion, deletion, or substitution. Such a correction, whether in structure or function, can result in a more favorable genotypic or phenotypic outcome for the corrected genomic site. One non-limiting example of "correction" includes the correction of a mutation or defective sequence to a wild-type sequence, restoring structure or function to a gene or its gene product(s). Depending on the nature of the mutation, correction can be achieved by various strategies disclosed herein. In one non-limiting example, a missense mutation can be corrected by replacing the region containing the mutation with its wild-type counterpart. As another example, a duplication mutation in a gene (e.g., a repeat expansion) can be corrected by removing the extra sequence.

[0048] In some embodiments, alterations may also include gene knock-in, knock-out, or knock-down. As used herein, the term "knock-in" refers to the addition of a DNA sequence, or a fragment thereof, to a genome. Such knocked-in DNA sequences may include an entire gene or multiple genes, and may include regulatory sequences associated with the gene or any portion or fragment thereof. For example, a cDNA encoding a wild-type protein may be inserted into the genome of a cell carrying a mutant gene. A knock-in strategy need not replace a defective gene in whole or in part. In some cases, a knock-in strategy may further involve replacing an existing sequence with a prepared sequence, e.g., replacing a mutant allele with a wild-type copy. On the other hand, the term "knock-out" refers to the elimination of a gene or gene expression. For example, a gene can be knocked out by deleting or adding a nucleotide sequence that disrupts the reading frame. As another example, a gene may be knocked out by replacing a portion of the gene with a related sequence. Finally, the term "knock-down" as used herein refers to the reduction of expression of a gene or its gene product(s). As a result of gene knockdown, the activity or function of the protein may be attenuated or protein levels may be reduced or eliminated.

[0049] Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably in a precise or predetermined manner. Examples of genome editing methods described herein include methods that use site-specific nucleases to cleave deoxyribonucleic acid (DNA) at precise target locations within the genome, thereby creating single- or double-stranded DNA breaks at specific locations within the genome. Such breaks can be, or are usually, repaired by natural endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), as reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015). These two major DNA repair processes comprise a family of distinct pathways. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes accompanied by the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR utilizes a homologous, or donor, sequence as a template for inserting a specific DNA sequence at the breakpoint. The homologous sequence can be in the endogenous genome, for example, in a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus, which has a region of high homology with the nuclease cleavage locus, but can also contain sequence changes, including additional sequences or deletions, that can be integrated into the cleavage target locus. The third repair mechanism can be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ", in which the genetic outcome is similar to NHEJ, in which small deletions and insertions can occur at the cleavage site.MMEJ can utilize several base pairs of homologous sequences adjacent to the DNA break site to promote a more favorable DNA end-joining repair outcome, and recent reports have further clarified the molecular mechanism of this process; see, for example, Cho and Greenberg, Nature 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online doi:10.1038 / nsmb.2961 (2015); Mateos-Gomez et al., Nature 518, 254-57 (2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0050] Each of these genome editing mechanisms can be used to create the desired genome change. A step in the genome editing process can be to create one or two DNA breaks (the latter as a double-strand break or two single-strand breaks) at the target locus near the site of the intended mutation. This can be achieved by using site-specific polypeptides, as described and exemplified herein.

[0051] CRISPR endonuclease system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a type of immune system that helps defend prokaryotes against foreign invaders, such as viruses and phages. CRISPR loci function in three stages: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of the foreign invader nucleic acid. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

[0052] CRISPR loci contain several short, repetitive sequences called "repeats." When expressed, repeats can form secondary structures (e.g., hairpins) and / or contain unstructured single-stranded sequences. Repeats usually occur in clusters and can diverge frequently between species. The repeats are regularly spaced with unique intervening sequences called "spacers," resulting in a repeat-spacer-repeat locus structure. The spacers are identical to or highly homologous to known foreign invader sequences. The spacer-repeat units encode crisprRNAs (crRNAs), which are processed into the mature form of spacer-repeat units. The crRNA contains a "seed" or spacer sequence responsible for targeting the target nucleic acid (in its naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0053] CRISPR loci also contain polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes encode endonucleases involved in the biosynthesis and interference steps of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0054] Type II CRISPR systems In natural type II CRISPR systems, crRNA biogenesis requires a trans-activating CRISPR RNA (tracrRNA). Non-limiting examples of type II CRISPR systems are shown in Figures 1A and 1B. The tracrRNA can be modified by endogenous RNase III and then hybridize to crRNA repeats in the pre-crRNA array. Endogenous RNase III can be recruited to cleave the pre-crRNA. The cleaved crRNA can be subjected to exoribonuclease trimming to generate mature crRNA (e.g., 5' trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-specific polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex can guide the complex to the target nucleic acid (to which the crRNA can hybridize). Hybridization of the crRNA to the target nucleic acid can activate Cas9 for target nucleic acid cleavage. The target nucleic acid in type II CRISPR systems is called a protospacer adjacent motif (PAM). In nature, the PAM is essential for facilitating the binding of a site-specific polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also known as Nmeni or CASS4) are further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) demonstrate that the CRISPR / Cas9 system is useful for RNA-programmable genome editing, and International Patent Application Publication No. WO2013 / 176772 provides numerous examples and applications of CRISPR / Cas endonuclease systems for site-specific gene editing.

[0055] V-type CRISPR system Type V CRISPR systems have several key differences from type II systems. For example, Cpf1, in contrast to type II systems, is a single RNA-guided endonuclease without a tracrRNA. Indeed, Cpf1-associated CRISPR arrays can be processed into mature crRNAs without the need for an additional transactivating tracrRNA. Type V CRISPR arrays can be processed into short mature crRNAs, 42–44 nucleotides in length, each of which begins with a 19-nucleotide direct repeat followed by a 23–25-nucleotide spacer sequence. In contrast, mature crRNAs in type II systems begin with a 20–24-nucleotide spacer sequence followed by approximately 22 nucleotides of direct repeats. Furthermore, Cpf1 can utilize a T-rich protospacer adjacent motif, allowing the Cpf1-crRNA complex to efficiently cleave target DNA preceded by a short T-rich PAM, as opposed to the G-rich PAM that follows the target DNA in type II systems. Thus, type V systems cleave at points distal to the PAM, while type II systems cleave at points adjacent to the PAM. Additionally, in contrast to type II systems, Cpf1 cleaves DNA with 4- or 5-nucleotide 5' overhangs via staggered DNA double-strand breaks. Type II systems cleave via blunt double-strand breaks. Like type II systems, Cpf1 contains a predicted RuvC-like endonuclease domain but lacks the second HNH endonuclease domain, which is in contrast to type II systems.

[0056] Cas gene / polypeptide and protospacer adjacent motifs Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptide as published in Fonfara et al., Nucleic Acids Research, 42:2577-2590 (2014). The CRISPR / Cas gene nomenclature system has undergone extensive rewrites since the discovery of Cas genes. Fonfara et al. also provide PAM sequences in Cas9 polypeptides from various species (see also Table 1 above).

[0057] II. Compositions and Methods of the Present Disclosure Provided herein are cell ex vivo and in vivo methods for using genome manipulation tools to create permanent changes in the genome by deleting or mutating the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene. Such methods use endonucleases, such as CRISPR-related (Cas9, Cpf1, etc.) nucleases, to permanently edit the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene within or near the genomic locus. In this way, the examples described in this disclosure can be useful for reducing or eliminating expression of the SCN9A gene with just one treatment (rather than delivering a potential treatment over the patient's lifetime).

[0058] Site-specific polypeptides (endonucleases, enzymes) The site-specific polypeptide is a nuclease used in genome editing to cleave DNA. The site-specific polypeptide can be administered to a cell or patient as one or more polypeptides or one or more mRNAs encoding the polypeptide. Any of the enzymes or orthologs listed in SEQ ID NOS: 1-620 or disclosed herein can be used in the methods described herein. A single guide RNA can be pre-complexed with the site-specific polypeptide. The site-specific polypeptide can be any of the DNA endonucleases disclosed herein.

[0059] In the context of the CRISPR / Cas9 or CRISPR / Cpf1 system, the site-specific polypeptide can bind to a guide RNA, which then specifies a site within the target DNA to which the polypeptide is directed. In the CRISPR / Cas9 or CRISPR / Cpf1 system disclosed herein, the site-specific polypeptide can be an endonuclease, for example, a DNA endonuclease.

[0060] The site-directed polypeptide may comprise multiple nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains may be linked together by a linker. For example, the linker may comprise a flexible linker. The linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length.

[0061] The naturally occurring wild-type Cas9 enzyme contains two nuclease domains, an HNH nuclease domain and a RuvC domain. As used herein, the term "Cas9" refers to both naturally occurring and recombinant Cas9. Cas9 enzymes contemplated herein can contain an HNH or HNH-like nuclease domain, and / or a RuvC or RuvC-like nuclease domain.

[0062] The HNH or HNH-like domain contains an McrA-like fold. The HNH or HNH-like domain contains two antiparallel β-strands and an α-helix. The HNH or HNH-like domain contains a metal binding site (e.g., a divalent cation binding site). The HNH or HNH-like domain can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA target strand).

[0063] The RuvC or RuvC-like domain contains an RNase H or RNase H-like fold. The RuvC / RNase H domain is involved in a diverse set of nucleic acid-based functions, including action on both RNA and DNA. The RNase H domain contains five β-strands surrounded by multiple α-helices. The RuvC / RNase H or RuvC / RNase H-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The RuvC / RNase H or RuvC / RNase H-like domain can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0064] The site-specific polypeptide can introduce double-strand or single-strand breaks into nucleic acids, such as genomic DNA. The double-strand break can stimulate the cell's endogenous DNA repair pathway (e.g., homology-dependent repair (HDR), NHEJ, alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining (MMEJ)). NHEJ can repair the cut target nucleic acid without the need for a homologous template. This can sometimes result in a small deletion or insertion (indel) at the cut site in the target nucleic acid, and can lead to disruption or alteration of gene expression. HDR can occur when a homologous repair template or donor is available. The homologous donor template can contain a sequence that is homologous to the sequence adjacent to the target nucleic acid cut site. Sister chromatids can be used as repair templates by cells. However, for genome editing purposes, the repair template can be provided as an exogenous nucleic acid, such as a plasmid, a double-stranded oligonucleotide, a single-stranded oligonucleotide, or a viral nucleic acid. Using an exogenous donor template, additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) can be introduced between adjacent regions of homology, so that additional or altered nucleic acid sequences are also integrated into the target locus. MMEJ can produce genetic results similar to NHEJ, where small deletions and insertions can occur at the break site. To promote favorable end-joining DNA repair results, MMEJ can utilize several base pairs of homologous sequences adjacent to the break site. In some cases, it may be possible to predict the likely repair outcome based on analysis of possible microhomologies in the nuclease target region.

[0065] Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. The exogenous polynucleotide sequence is referred to herein as a "donor polynucleotide" (or donor or donor sequence). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

[0066] Modification of target DNA by NHEJ and / or HDR can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation. The processes of deleting genomic DNA and integrating non-naturally occurring nucleic acids into genomic DNA are examples of genome editing.

[0067] The site-directed polypeptide can comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, US2014 / 0068797, SEQ ID NO: 8 or Sapranauskas et al., Nucleic Acids Res, 39(21):9275-9282(2011)), and various other site-directed polypeptides. The site-directed polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. A site-directed polypeptide may comprise at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 consecutive amino acids. A site-directed polypeptide may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 consecutive amino acids within the HNH nuclease domain of the site-directed polypeptide. A site-directed polypeptide may comprise at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 consecutive amino acids within the HNH nuclease domain of the site-directed polypeptide. The site-directed polypeptide can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the RuvC nuclease domain of the site-directed polypeptide.The site-directed polypeptide can comprise at most 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the RuvC nuclease domain of the site-directed polypeptide.

[0068] The site-directed polypeptide may include a modified form of a wild-type exemplary site-directed polypeptide. The modified form of a wild-type exemplary site-directed polypeptide may include a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. The modified form of a wild-type exemplary site-directed polypeptide may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form of the site-directed polypeptide may not have substantial nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to as "enzymatically inactive" in the present invention.

[0069] Modified forms of site-directed polypeptides can include mutations that enable them to induce single-strand breaks (SSBs) in a target nucleic acid (e.g., by cleaving only one of the sugar-phosphate backbones of a double-stranded target nucleic acid). In some embodiments, the mutations can result in less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of one or more of the multiple nucleic acid cleavage domains of a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, mutations can occur in one or more of the multiple nucleic acid cleavage domains to maintain the ability to cleave the complementary strand of a target nucleic acid, but reduce its ability to cleave the non-complementary strand of a target nucleic acid. Mutations can be made in one or more of the nucleic acid cleavage domains to maintain the ability to cleave non-complementary strands of target nucleic acids, but reduce their ability to cleave complementary strands of target nucleic acids. For example, residues of a wild-type exemplary S. pyogenes Cas9 polypeptide, such as AsplO, His840, Asn854, and Asn856, can be mutated to inactivate one or more of the nucleic acid cleavage domains (e.g., nuclease domains). The mutated residues can correspond to residues AsplO, His840, Asn854, and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A, or N856A. One of skill in the art will recognize that mutations other than alanine substitutions can be suitable.

[0070] In some embodiments, the D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. The H840A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. The N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. The N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to generate a site-directed polypeptide that substantially lacks DNA cleavage activity. A site-directed polypeptide that includes one substantially inactive nuclease domain is referred to as a "nickase."

[0071] RNA-guided endonucleases, such as Cas9 nickase variants, can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is typically guided by a single guide RNA designed to hybridize with a specific sequence of about 20 nucleotides within a target sequence (such as an endogenous genomic locus). However, some mismatches can be tolerated between the guide RNA and the target locus, which effectively reduces the required homology length within the target site to as little as 13 nt of homology, thereby increasing the possibility of CRISPR / Cas9 complex binding and double-stranded nucleic acid cleavage elsewhere within the target genome, also known as off-target cleavage. Because Cas9 nickase variants only cleave one strand each, to produce a double-stranded cleavage, a pair of nickases must bind in close proximity to each other on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is equivalent to a double-stranded cleavage. This requires two separate guide RNAs (one for each nickase) to bind in close proximity and on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology required to create a double-strand break, thereby reducing the likelihood of a double-strand break event occurring elsewhere in the genome, where the two guide RNA sites (if present) are unlikely to be close enough to each other to allow the formation of a double-strand break. As described in the art, nickases can also be used to promote HDR in contrast to NHEJ. HDR can be used to introduce selected changes into target sites in the genome by using specific donor sequences that effectively mediate the desired changes.

[0072] Contemplated mutations may include substitutions, additions, and deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to an unnatural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid to an amino acid mimetic (e.g., a phosphomimetic). The mutation may be conservative. For example, the mutation converts the mutated amino acid to an amino acid similar in size, shape, charge, polarity, or configuration, and / or a rotamer of the mutated amino acid (e.g., a cysteine / serine mutation, a lysine / asparagine mutation, a histidine / phenylalanine mutation). Mutations may result in a shift in the reading frame and / or the creation of a premature stop codon. Mutations may alter the regulatory region of a gene or locus, affecting the expression of one or more genes.

[0073] Site-directed polypeptides (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-directed polypeptides) can target nucleic acids. Site-directed polypeptides (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonucleases) can target DNA. Site-directed polypeptides (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonucleases) can target RNA.

[0074] The site-directed polypeptide may include one or more non-native sequences (eg, the site-directed polypeptide is a fusion protein).

[0075] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

[0076] The site-directed polypeptide can comprise an amino acid sequence that comprises at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

[0077] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains, wherein one or both of the nucleic acid cleavage domains comprises at least 50% amino acid identity to the nuclease domain from Cas9 from a bacterium (e.g., S. pyogenes).

[0078] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or a linker connecting the site-directed polypeptide to the non-native sequence.

[0079] The site-directed polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein the site-directed polypeptide comprises a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

[0080] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein one of the nuclease domains comprises a mutation of aspartic acid 10 and / or one of the nuclease domains comprises a mutation of histidine 840, and wherein the mutation reduces the cleavage activity of the nuclease domain(s) by at least 50%.

[0081] The one or more site-specific polypeptides, e.g., DNA endonucleases, can include two nickases that together make one double-strand break at a specific locus in the genome, or four nickases that together make or result in two double-strand breaks at a specific locus in the genome. Alternatively, one site-specific polypeptide, e.g., a DNA endonuclease, can make or result in one double-strand break at a specific locus in the genome.

[0082] The active ingredient of the Cas9 strain is Acaryochloris marina MBIC11017;Acetohalobium arabaticum DSM 5501;Acidithiobacillus caldus;Acidithiobacillus ferrooxidans ATCC 23270;Alicyclobacillus acidocaldarius LAA1;Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446;Allochromatium vinosum DSM 180;Ammonifex degensii KC4;Anabaena variabilis ATCC 29413;Arthrospira maxima CS-328;Arthrospira platensis str.Paraca;Arthrospira sp.PCC 8005;Bacillus pseudomycoides DSM 12442;Bacillus selenitireducens MLS10;Burkholderiales bacterium 1_1_47;Caldicellulosiruptor becscii DSM 6725;Candidatus Desulforudis audaxviator MP104C;Caldicellulosiruptor hydrothermalis_108;Clostridium phage c-st;Clostridium botulinum A3 str.Loch Maree;Clostridium botulinum Ba4 str.657;Clostridium difficile QCD-63q42;Crocosphaera watsonii WH 8501;Cyanothece sp.ATCC 51142;Cyanothece sp.CCY0110;Cyanothece sp.PCC 7424;Cyanothece sp.PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp.bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. sp.ELB17;Methanohalobium evestigatum Z-7303;Microcystis phage Ma-LMM01;Microcystis aeruginosa NIES-843;Microscilla marina ATCC 23134;Microcoleus chthonoplastes PCC 7420;Neisseria meningitidis;Nitrosococcus halophilus Nc4;Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111;Nodularia spumigena CCY9414; Nostoc sp.PCC 7120;Oscillatoria sp.PCC 6506;Pelotomaculum_thermopropionicum_SI;Petrotoga mobilis SJ95;Polaromonas naphthalenivorans CJ2;Polaromonas sp.JS666;Pseudoalteromonas haloplanktis TAC125;Streptomyces pristinaespiralis ATCC 25486;Streptomyces pristinaespiralis ATCC 25486;Streptococcus thermophilus;Streptomyces viridochromogenes DSM 40736;Streptosporangium roseum DSM 43021;Synechococcus sp.PCC 7335; and the Cas protein identified in Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013;10(5):726-737).

[0083] In addition to Cas9 orthologs, other Cas9 variants, such as inactive dCas9 and fusion proteins of effector domains with different functions, can serve as platforms for genetic modification. Any of the above enzymes can be useful in the present disclosure.

[0084] Further examples of endonucleases that may be utilized in the present disclosure are set forth in SEQ ID NOs: 1-620. These proteins may be modified before use or may be encoded in nucleic acid sequences, such as DNA, RNA, or mRNA, or in vector constructs, such as the plasmids or AAV vectors taught herein. Furthermore, they may be codon-optimized.

[0085] SEQ ID NOs: 1-620 disclose a non-exclusive list of endonuclease sequences.

[0086] Genome-targeting nucleic acids The present disclosure provides a genome-targeting nucleic acid that can direct the activity of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be RNA. The genome-targeting RNA is referred to herein as a "guide RNA" or "gRNA." The guide RNA can include a target nucleic acid sequence of interest and at least one spacer sequence that hybridizes with a CRISPR repeat sequence. In type II systems, the gRNA also includes a second RNA called a tracrRNA sequence. In type II guide RNAs (gRNAs), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In type V guide RNAs (gRNAs), the crRNA forms a duplex. In both systems, the duplex can bind to the site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. The genome-targeting nucleic acid can confer target specificity to the complex through its association with the site-directed polypeptide. Thus, the genome-targeting nucleic acid can direct the activity of the site-directed polypeptide.

[0087] Exemplary guide RNAs include the spacer sequences in SEQ ID NOS: 5305-125469 of the Sequence Listing. As will be understood by those of ordinary skill in the art, each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. For example, each of the spacer sequences in SEQ ID NOS: 5305-125469 of the Sequence Listing can be incorporated into a single RNA chimera or crRNA (together with the corresponding tracrRNA). See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0088] The genome-targeting nucleic acid can be a dual-molecule guide RNA. The genome-targeting nucleic acid can be a single-molecule guide RNA.

[0089] A dual-molecule guide RNA may comprise two strands of RNA. The first strand comprises, from 5' to 3', an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0090] In Type II systems, the single guide RNA (sgRNA) may comprise, from 5' to 3', an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that confer additional functionality (e.g., stability) to the guide RNA. The single guide linker may link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

[0091] The sgRNA may include a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA may include a spacer sequence of various lengths, including 17 to 30 nucleotides, at the 5' end of the sgRNA sequence (see Table 1).

[0092] The sgRNA may not include a uracil at the 3' end of the sgRNA sequence, such as in SEQ ID NO: 125502 in Table 1. The sgRNA may include one or more uracils at the 3' end of the sgRNA sequence, such as in SEQ ID NO: 125503 in Table 1. For example, the sgRNA may include one uracil (U) at the 3' end of the sgRNA sequence. The sgRNA may include two uracils (UU) at the 3' end of the sgRNA sequence. The sgRNA may include three uracils (UUU) at the 3' end of the sgRNA sequence. The sgRNA may include four uracils (UUUU) at the 3' end of the sgRNA sequence. The sgRNA may include five uracils (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA may include six uracils (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain seven uracils (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA may contain eight uracils (UUUUUUUU) at the 3' end of the sgRNA sequence.

[0093] The sgRNA may be unmodified or modified. For example, a modified sgRNA may contain one or more 2'-O-methyl phosphorothioate nucleotides. [Table 1]

[0094] The single guide RNA (sgRNA) in the V-type system may comprise, from 5' to 3', a minimum CRISPR repeat sequence and a spacer sequence.

[0095] For example, guide RNAs or other smaller RNAs used in the CRISPR / Cas9 or CRISPR / Cpf1 systems can be easily synthesized by chemical means, as exemplified below and described in the art. Chemical synthesis procedures are constantly expanding, but as polynucleotide lengths increase significantly beyond about 100 nucleotides, purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult. One approach used to generate relatively long RNAs is to generate two or more molecules and ligate them together. Significantly longer RNAs, such as those encoding Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. As described in the art, various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic production of RNA, such as modifications that increase stability, reduce the likelihood or severity of innate immune responses, and / or enhance other properties.

[0096] Spacer extension sequence In some examples of genome-targeting nucleic acids, the spacer extension sequence can modify activity, provide stability, and / or provide a location for modifying the genome-targeting nucleic acid. The spacer extension sequence can modify on-target or off-target activity or specificity. In some examples, a spacer extension sequence can be added. The spacer extension sequence can have a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or more than 7000 nucleotides. The spacer extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. The spacer extension sequence may be less than 10 nucleotides in length. The spacer extension sequence may be between 10 and 30 nucleotides in length. The spacer extension sequence may be between 30 and 70 nucleotides in length.

[0097] The spacer extension sequence may include another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). The moiety may decrease or increase the stability of the nucleic acid targeting nucleic acid. The moiety may be a transcription terminator segment (i.e., a transcription termination sequence). The moiety may function in eukaryotic cells. The moiety may function in prokaryotic cells. The moiety may function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)), a riboswitch sequence (e.g., to allow for controlled stability and / or controlled accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that directs the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.), a modification or sequence that allows for tracking (e.g., direct conjugation with a fluorescent molecule, conjugation with a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.).

[0098] Spacer sequence The spacer sequence hybridizes with the sequence in the target nucleic acid of interest. The spacer of the genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0099] In the CRISPR / Cas system described herein, a spacer sequence can be designed to hybridize with the target nucleic acid located 5' to the PAM of the Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have a mismatch. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, S. pyogenes recognizes a PAM in the target nucleic acid that includes the sequence 5'-NRG-3', where R includes either A or G, and N is any nucleotide, and N is located immediately 3' to the target nucleic acid sequence that the spacer sequence targets.

[0100] The target nucleic acid sequence may contain 20 nucleotides. The target nucleic acid may contain fewer than 20 nucleotides. The target nucleic acid may contain more than 20 nucleotides. The target nucleic acid may contain at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid may contain at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid sequence may contain 20 bases immediately 5' to the first nucleotide of the PAM. For example, in a sequence containing 5'-NNNNNNNNNNNNNNNNNNNNNRG-3' (SEQ ID NO: 125500), the target nucleic acid may contain a sequence corresponding to any nucleotide N, where the underlined NRG sequence is the S. pyogenes PAM. This target nucleic acid sequence is often referred to as the PAM strand, and the complementary nucleic acid sequence is often referred to as the non-PAM strand. One of skill in the art will recognize that the spacer sequence hybridizes to the non-PAM strand of the target nucleic acid (Figure 1A and B).

[0101] The spacer sequence that hybridizes with the target nucleic acid may have a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, or from about 1 nt to about 25 nt. The spacer sequence may be 0 nt to about 40 nt, about 10 nt to about 35 nt, about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 19 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt. In some examples, the spacer sequence may contain 20 nucleotides. In some examples, the spacer may contain 19 nucleotides. In some examples, the spacer may contain 18 nucleotides. In some examples, the spacer may comprise 22 nucleotides.

[0102] In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some instances, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid may be at least 60% over about 20 contiguous nucleotides. The lengths of the spacer sequence and the target nucleic acid may differ by 1 to 6 nucleotides, which may be considered a bulge or multiple bulges.

[0103] Spacer sequence can be designed or selected using computer program.Computer program can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genome context, chromatin accessibility, %GC, (for example, the frequency of genomic occurrence of identical or similar sequences, but different in one or more spots as a result of mismatch, insertion or deletion), methylation status, the existence of SNP, etc.

[0104] Minimum CRISPR repeat sequence In some embodiments, a minimum CRISPR repeat sequence is a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

[0105] In some embodiments, the minimum CRISPR repeat sequence comprises nucleotides capable of hybridizing with the minimum tracrRNA sequence in cells. The minimum CRISPR repeat sequence and the minimum tracrRNA sequence can form a duplex, i.e., a base-paired double-stranded structure. The minimum CRISPR repeat sequence and the minimum tracrRNA sequence can bind together to a site-specific polypeptide. At least a portion of the minimum CRISPR repeat sequence can hybridize with the minimum tracrRNA sequence. At least a portion of the minimum CRISPR repeat sequence can comprise at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the minimum tracrRNA sequence. In some embodiments, at least a portion of the minimum CRISPR repeat sequence comprises at most about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the minimum tracrRNA sequence.

[0106] The minimum CRISPR repeat sequence can be about 7 nucleotides to about 100 nucleotides in length. For example, the length of the minimum CRISPR repeat sequence is about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some embodiments, the minimum CRISPR repeat sequence is about 9 nucleotides in length. In some embodiments, the minimum CRISPR repeat sequence is about 12 nucleotides in length.

[0107] The minimum CRISPR repeat sequence can be at least about 60% identical to a reference minimum CRISPR repeat sequence (e.g., a wild-type crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8 consecutive nucleotides. For example, the minimum CRISPR repeat sequence can be at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the reference minimum CRISPR repeat sequence over a stretch of at least 6, 7, or 8 consecutive nucleotides.

[0108] Minimum tracrRNA sequence A minimum tracrRNA sequence can be a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from S. pyogenes).

[0109] The minimum tracrRNA sequence may contain nucleotides that hybridize with the minimum CRISPR repeat sequence in cells. The minimum tracrRNA sequence and the minimum CRISPR repeat sequence form a duplex, i.e., a base-paired double-stranded structure. The minimum tracrRNA sequence and the minimum CRISPR repeat may bind together to a site-specific polypeptide. At least a portion of the minimum tracrRNA sequence may hybridize with the minimum CRISPR repeat sequence. The minimum tracrRNA sequence may be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum CRISPR repeat sequence.

[0110] The minimum tracrRNA sequence can be from about 7 nucleotides to about 100 nucleotides in length. For example, the minimum tracrRNA sequence can be about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt in length. The minimum tracrRNA sequence can be about 9 nucleotides in length. The minimum tracrRNA sequence can be about 12 nucleotides in length. The minimum tracrRNA can consist of tracrRNA nt 23 to 48 as described in Jinek et al. (supra).

[0111] The minimum tracrRNA sequence can be at least about 60% identical to a reference minimum tracrRNA (e.g., a wild-type tracrRNA from S. pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimum tracrRNA sequence can be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the reference minimum tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0112] The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise a double helix. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0113] A duplex may contain mismatches (i.e., the two strands of the duplex are not 100% complementary). A duplex may contain at least about 1, 2, 3, 4, or 5 or mismatches. A duplex may contain at most about 1, 2, 3, 4, or 5 or mismatches. A duplex may contain no more than two mismatches.

[0114] bulge In some cases, a "bulge" may exist in the duplex between the minimum CRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region of nucleotides within the duplex. The bulge may contribute to the binding of the duplex to the site-specific polypeptide. The bulge may include an unpaired 5'-XXXY-3' (where X is any purine and Y includes a nucleotide that can form a wobble pair with a nucleotide on the opposite strand) on one side of the duplex and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on the two sides of the duplex may be different.

[0115] In one example, the bulge may comprise an unpaired purine (e.g., adenine) on the minimum CRISPR repeat strand of the bulge. In some examples, the bulge may comprise an unpaired 5'-AAGY-3' on the minimum tracrRNA sequence strand of the bulge, where Y comprises a nucleotide that can form a wobble pair with a nucleotide on the minimum CRISPR repeat strand.

[0116] The bulge on the side of the duplex toward the minimum CRISPR repeat may contain at least 1, 2, 3, 4, or 5 or more unpaired nucleotides. The bulge on the side of the duplex toward the minimum CRISPR repeat may contain at most 1, 2, 3, 4, or 5 or more unpaired nucleotides. The bulge on the side of the duplex toward the minimum CRISPR repeat may contain one unpaired nucleotide.

[0117] A bulge on the side of the duplex facing the minimum tracrRNA sequence can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. A bulge on the side of the duplex facing the minimum tracrRNA sequence can contain at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. A bulge on the second side of the duplex (e.g., facing the minimum tracrRNA sequence) can contain four unpaired nucleotides.

[0118] The bulge may include at least one wobble pairing. In some examples, the bulge may include at most one wobble pairing. The bulge may include at least one purine nucleotide. The bulge may include at least three purine nucleotides. The bulge sequence may include at least five purine nucleotides. The bulge sequence may include at least one guanine nucleotide. In some examples, the bulge sequence may include at least one adenine nucleotide.

[0119] hairpin In various examples, one or more hairpins can be located 3' of the minimum tracrRNA within the 3' tracrRNA sequence.

[0120] The hairpin can begin at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides 3' from the last paired nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpin can begin at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides 3' from the last paired nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex.

[0121] A hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more contiguous nucleotides. A hairpin can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more contiguous nucleotides.

[0122] The hairpin may contain a CC dinucleotide (ie, two consecutive cytosine nucleotides).

[0123] The hairpin may include duplex nucleotides (e.g., nucleotides within the hairpin that hybridize together). For example, the hairpin may include a CC dinucleotide that hybridizes with a GG dinucleotide within the hairpin duplex of the 3' tracrRNA sequence.

[0124] One or more of the hairpins may interact with the guide RNA-interacting region of the site-directed polypeptide.

[0125] In some instances, there are two or more hairpins, and in other instances, there are three or more hairpins.

[0126] 3'tracrRNA sequence The 3' tracrRNA sequence can include a sequence having at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

[0127] The 3' tracrRNA sequence can be about 6 nucleotides to about 100 nucleotides in length. For example, the 3' tracrRNA sequence can be about 6 nucleotides (nt) to about 50 nt, about 6 nt to about 40 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. The 3' tracrRNA sequence can be about 14 nucleotides in length.

[0128] The 3' tracrRNA sequence can be at least about 60% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the 3' tracrRNA sequence can be at least about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0129] The 3' tracrRNA sequence may contain more than one duplex region (e.g., hairpin, hybridized region). The 3' tracrRNA sequence may contain two duplex regions.

[0130] The 3' tracrRNA sequence may comprise a stem-loop structure. The stem-loop structure within the 3' tracrRNA may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides. The stem-loop structure within the 3' tracrRNA may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The stem-loop structure may comprise a functional moiety. For example, the stem-loop structure may comprise an aptamer, a ribozyme, a protein-interacting hairpin, a CRISPR array, an intron, or an exon. The stem-loop structure may comprise at least about 1, 2, 3, 4, or 5 or more functional moieties. The stem-loop structure may comprise at most about 1, 2, 3, 4, or 5 or more functional moieties.

[0131] The hairpin in the 3' tracrRNA sequence can include a P-domain. In some instances, the P-domain can include a double-stranded region in the hairpin.

[0132] tracrRNA extension sequence A tracrRNA extension sequence may be provided regardless of whether the tracrRNA is in the context of a single-molecule guide or a dual-molecule guide. The tracrRNA extension sequence may be from about 1 nucleotide to about 400 nucleotides in length. The tracrRNA extension sequence may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or greater than 400 nucleotides in length. The tracrRNA extension sequence may be from about 20 to about 5000 or more nucleotides in length. The tracrRNA extension sequence may be greater than 1000 nucleotides in length. The tracrRNA extension sequence can be less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or more nucleotides in length. The tracrRNA extension sequence can be less than 1000 nucleotides in length. The tracrRNA extension sequence can comprise less than 10 nucleotides in length. The tracrRNA extension sequence can be 10-30 nucleotides in length. The tracrRNA extension sequence can be 30-70 nucleotides in length.

[0133] The tracrRNA extension sequence may contain functional moieties (e.g., stability control sequences, ribozymes, endoribonuclease binding sequences). The functional moieties may include transcription terminator segments (i.e., transcription termination sequences). The functional moieties may have a total length of about 10 nucleotides (nt) to about 100 nucleotides, about 10 nt to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. The functional moieties may function in eukaryotic cells. The functional moieties may function in prokaryotic cells. The functional moiety can function in both eukaryotic and prokaryotic cells.

[0134] Non-limiting examples of suitable tracrRNA extension functional portions include a 3' poly-adenylation tail, a riboswitch sequence (e.g., to allow for controlled stability and / or controlled accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that directs the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.), a modification or sequence that allows tracking (e.g., direct conjugation with a fluorescent molecule, conjugation with a moiety that facilitates fluorescent detection, a sequence that allows fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, transcriptional repressor, DNA methyltransferase, DNA demethylase, histone acetyltransferase, histone deacetylase, etc.). The tracrRNA extension sequence may include a primer binding site or a molecular index (e.g., a barcode sequence). The tracrRNA extension sequence may include one or more affinity tags.

[0135] Single molecule guide linker sequence The linker sequence of a single-molecule guide nucleic acid can be about 3 nucleotides to about 100 nucleotides in length. Jinek et al. (supra) used, for example, a simple four-nucleotide "tetraloop" (-GAAA-) (Science, 337(6096):816-821(2012)). Illustrative linker lengths are about 3 nucleotides (nt) to about 90 nt, about 3 nt to about 80 nt, about 3 nt to about 70 nt, about 3 nt to about 60 nt, about 3 nt to about 50 nt, about 3 nt to about 40 nt, about 3 nt to about 30 nt, about 3 nt to about 20 nt, and about 3 nt to about 10 nt. For example, the linker may have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. The linker of the single-molecule guide nucleic acid may be between 4 and 40 nucleotides. A linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. A linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0136] The linker can comprise any of a variety of sequences, but in some instances, the linker does not comprise a sequence with extensive regions of homology to other portions of the guide RNA, which may result in intramolecular binding that may interfere with other functional regions of the guide. Jinek et al. (supra) used the simple four-nucleotide sequence -GAAA- (Science, 337(6096):816-821(2012)), but many other sequences, including longer sequences, can be used as well.

[0137] Linker sequence can comprise functional moiety.For example, linker sequence can comprise one or more features, including aptamer, ribozyme, protein-interaction hairpin, protein binding site, CRISPR array, intron or exon.Linker sequence can comprise at least about 1, 2, 3, 4, or 5 or more functional moiety.In some examples, linker sequence can comprise at most about 1, 2, 3, 4, or 5 or more functional moiety.

[0138] Nucleic acid modifications (chemical and structural modifications) In some aspects, as further described herein and as known in the art, polynucleotides introduced into cells may contain one or more modifications that can be used individually or in combination to, for example, enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or otherwise enhance.

[0139] In certain examples, modified polynucleotides can be used in CRISPR / Cas9 or CRISPR / Cpf1 systems, where the guide RNA (either a single-molecule guide or a dual-molecule guide) and / or the DNA or RNA encoding the Cas9 or Cpf1 endonuclease introduced into a cell can be modified, as described and exemplified below. Such modified polynucleotides can be used in CRISPR / Cas9 or CRISPR / Cpf1 systems to edit any one or more genomic loci.

[0140] For the purpose of non-limiting illustration of such use, when using the CRISPR / Cas9 or CRISPR / Cpf1 system, modification of the guide RNA can be used to enhance the formation or stability of a CRISPR / Cas9 or CRISPR / Cpf1 genome editing complex, which includes a guide RNA, which may be a single guide molecule or a double guide molecule, and a Cas9 or Cpf1 endonuclease. Modification of the guide RNA can also or alternatively be used to enhance the initiation, stability, or reaction rate of the interaction of the genome editing complex with the target sequence in the genome, which can be used, for example, to enhance on-target activity. Modification of the guide RNA can also or alternatively be used to enhance specificity, for example, the relative rate of genome editing at the on-target site compared to the effect at other (off-target) sites.

[0141] Modifications can also, or alternatively, be used to increase the stability of the guide RNA, for example, by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby increasing its half-life in the cell. Modifications that increase guide RNA half-life can be particularly useful in embodiments in which the Cas9 or Cpfl endonuclease is introduced into the cell to be edited via an RNA that requires translation to produce the endonuclease, since increasing the half-life of the guide RNA that is introduced simultaneously with the RNA encoding the endonuclease can be used to increase the time that the guide RNA and the encoded Cas9 or Cpfl endonuclease are present together in the cell.

[0142] Modifications can also, or alternatively, be used to reduce the likelihood or extent to which RNA introduced into a cell will induce an innate immune response. Such responses, which have been well characterized in the context of RNA interference (RNAi), including small interfering RNA (siRNA), as described below and in the art, tend to be associated with a decrease in the half-life of the RNA and / or the induction of cytokines or other factors associated with the immune response.

[0143] One or more types of modifications can also be made to the RNA encoding the endonuclease that is introduced into a cell, including, but not limited to, modifications that enhance the stability of the RNA (such as by increasing its degradation by RNases present in the cell), modifications that enhance translation of the resulting product (i.e., the endonuclease), and / or modifications that reduce the likelihood or extent to which the introduced RNA will induce a natural immune response.

[0144] Combinations of such modifications can also be used, for example, in the case of CRISPR / Cas9 or CRISPR / Cpfl, one or more types of modifications can be made to the guide RNA (including those exemplified above) and / or one or more types of modifications can be made to the RNA encoding the Cas endonuclease (including those exemplified above).

[0145] For example, guide RNAs or other smaller RNAs used in the CRISPR / Cas9 or CRISPR / Cpf1 systems can be easily synthesized by chemical means and can easily incorporate several modifications, as exemplified below and described in the art. Chemical synthesis procedures are constantly expanding, but as polynucleotide lengths increase significantly beyond about 100 nucleotides, purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult. One approach that can be used to generate chemically modified RNAs of relatively large lengths is to generate two or more molecules and ligate them together. Much longer RNAs, such as those encoding Cas9 endonuclease, are more easily generated enzymatically. While fewer types of modifications can be used in enzyme-generated RNAs, there are still modifications that can be used to enhance stability, reduce the likelihood or severity of innate immune responses, and / or enhance other properties, as described further below and in the art; new types of modifications are regularly developed.

[0146] As examples of various types of modifications, particularly those often used with relatively small chemically synthesized RNAs, modifications can include one or more nucleotides modified at the 2' position of the sugar, in some embodiments, 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotides. In some examples, RNA modifications include 2'-fluoro, 2'-amino, or 2'-O-methyl modifications at the ribose of pyrimidines, abasic residues, or inverted bases at the 3' end of RNA. Such modifications are routinely incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher Tm (i.e., higher target binding affinity) for a given target than 2'-deoxyoligonucleotides.

[0147] Some nucleotide and nucleoside modifications have been shown to render incorporated oligonucleotides more resistant to nuclease digestion than native oligonucleotides; these modified oligonucleotides remain intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. Some oligonucleotides have phosphorothioate backbones and heteroatom backbones, particularly CH2-NH-O-CH2, CH, -N(CH3)-O-CH2 (also known as methylene(methylimino) or MMI backbones), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones (where the natural phosphodiester backbone is represented as OPO-CH); amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbones (where the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are attached directly or indirectly to the aza nitrogen atoms of the polyamide backbone). Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and inverted polarity nucleoside linkages in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Polarity); U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676 See US Pat. Nos. 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0148] Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14):4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97:9591-9596 (2000); and U.S. Patent No. 5,034,506, issued July 23, 1991.

[0149] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122:8595-8602 (2000).

[0150] Modified oligonucleotide backbones that do not contain a phosphorus atom have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (some formed from the sugar portion of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH2 moieties; U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235 ,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; No. 5,470,967; No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602, See Nos. 240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0151] One or more substituted sugar moieties, such as any of the following at the 2' position: OH, SH, SCH3, F, OCN, OCHOCH3, OCHO(CH2)nCH3, O(CH2)nNH2, or O(CH2)nCH3 (where n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. In some aspects, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al., HeIv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of pentofuranosyl groups.

[0152] In some cases, both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide can be replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases are maintained and can be linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings on PNA compounds can be found in Nielsen et al., Science, 254:1497-1500 (1991).

[0153] Guide RNAs may also, or alternatively, include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only rarely or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also called 5-methyl-2'deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, Y.S., in Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are examples of base substitutions.

[0154] Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and These may include thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0155] Additionally, nucleobases can include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science and Engineering," pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, YS, in Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B. ea., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., in Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are also an example of a base substitution, particularly when combined with a 2'-O-methoxyethyl sugar modification.Modified nucleobases are disclosed in U.S. Patent Nos. 3,687,808, as well as U.S. Patent Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; Nos. 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication No. 2003 / 0158403.

[0156] Thus, the term "modification" refers to an unnatural sugar, phosphate, or base incorporated into the guide RNA, the endonuclease, or both the guide RNA and the endonuclease. Not all positions within a given oligonucleotide need be uniformly modified, and in fact more than one of the above modifications can be incorporated into a single oligonucleotide, or even into a single nucleoside within an oligonucleotide.

[0157] The guide RNA and / or the mRNA (or DNA) encoding the endonuclease may be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.Such moieties include, but are not limited to, lipid moieties, e.g., cholesterol moieties [Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4:1053-1060 (1994)]; thioethers, e.g., hexyl-S-tritylthiol [Manoharan et al., Ann. NY Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20:533-538 (1992)]; aliphatic chains, such as dodecanediol or undecyl residues [Kabanov et al., FEBS Lett., 259:327-330 (1990) and Svinarchuk et al., Biochimie, 75:49-54 (1993)]; phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18:3777-3783 (1990)]; polyamine or polyethylene glycol chains [Mancharan et al., Nucleosides & Nucleotides, 14:969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264:229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t-oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937 (1996)].Also, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414 ,077;No.5,486,603;No.5,512,439;No.5,578,718;No.5,608,046;No.4,587,044;No.4,605,735;No.4,667,025; Same No. 4,762,779; Same No. 4,789,737; Same No. 4,824,941; Same No. 4,835,263; Same No. 4,876,335; Same No. 4,904,582; Same No. 4,958,013; Same No. 5,082 , No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; Same No. 5,258,506; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241; Same No. 5,391,723; Same No. 5,416 See Nos. 5,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0158] Sugars and other moieties can be used to target complexes containing proteins and nucleotides, such as cationic polysomes and liposomes, to specific sites. For example, hepatocyte-directed delivery can be mediated via the asialoglycoprotein receptor (ASGPR); see, e.g., Hu, et al., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known in the art and continually being developed can be used to target biomolecules and / or their complexes to specific target cells of interest.

[0159] These targeting moieties or conjugates may contain a conjugate group covalently attached to a functional group, such as a primary or secondary hydroxyl group. Conjugate groups of the present disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties, in the context of the present disclosure, include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties, in the context of the present disclosure, include groups that improve uptake, distribution, metabolism, or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application PCT / US92 / 09196, filed October 23, 1992 (published as WO1993007883), and U.S. Patent No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.See, for example, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414, 077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082 , No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; Same No. 5,258,506; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241; Same No. 5,391,723; Same No. 5,41 See Nos. 6,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0160] Relatively long polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can also be modified by various means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other moieties at the 5' or 3' end of the molecule, and other modifications. As an illustration, mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. For example, modifications to mRNA can be applied to increase its translation or stability (such as by increasing its resistance to degradation in cells) or to reduce the tendency of the RNA to induce an innate immune response, which is often observed in cells after the introduction of exogenous RNA, particularly relatively long RNAs such as those encoding Cas9.

[0161] Many such modifications have been described in the art, such as polyA tails, 5' cap analogs (e.g., anti-reverse cap analog (ARCA) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), use of modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with phosphatase to remove the 5' terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are regularly developed.

[0162] Numerous commercial suppliers of modified RNAs exist, including, for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon, and many others. As described by TriLink, for example, 5-methyl-CTP can be used to confer desirable characteristics, such as increased nuclease stability, increased translation, or reduced interaction of in vitro transcribed RNA with innate immune receptors. As explained in the publications by Kormann et al. and Warren et al., referenced below, 5-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, and even pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation.

[0163] It has been shown that chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic efficacy; see, e.g., Kormann et al., Nature Biotechnology 29, 154-157 (2011). For example, such modifications can be used to increase the stability and / or reduce immunogenicity of RNA molecules. Using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U, and 5-methyl-C, it was found that replacing exactly one-quarter of uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, resulted in a significant decrease in toll-like receptor (TLR)-mediated recognition of mRNA in mice. By reducing activation of the innate immune system, these modifications can be used to effectively increase the stability and longevity of mRNA in vivo; see, e.g., Kormann et al. (supra).

[0164] It has also been shown that repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass the innate immune antiviral response can reprogram differentiated human cells to pluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs, acting as primary reprogramming proteins, can be an efficient means of reprogramming multiple human cell types. Such cells are called induced pluripotent stem cells (iPSCs), and it has been found that enzymatically synthesized RNAs incorporating 5-methyl-CTP, pseudo-UTP, and anti-reverse cap analog (ARCA) can be used to effectively circumvent cellular antiviral responses; see, e.g., Warren et al. (supra).

[0165] Other modifications of polynucleotides described in the art include, for example, the use of a polyA tail, the addition of a 5' cap analog (e.g., m7G(5')ppp(5')G (mCAP)), modification of the 5' or 3' untranslated region (UTR), or treatment with phosphatase to remove the 5' terminal phosphate, and new approaches are regularly developed.

[0166] Some compositions and techniques applicable to the generation of modified RNA for use herein have been developed in connection with the modification of RNA interference (RNAi), including small interfering RNA (siRNA). siRNA presents a particular challenge in vivo, because their effect on gene silencing via mRNA interference is generally transient and may require repeated administration. In addition, siRNA is double-stranded RNA (dsRNA), and mammalian cells have developed immune responses to detect and neutralize dsRNA, which is often a by-product of viral infection. Thus, there are mammalian enzymes, such as PKR (dsRNA-responsive kinase) and potentially retinoic acid-inducible gene I (RIG-I), that can mediate cellular responses to dsRNA, as well as Toll-like receptors (e.g., TLR3, TLR7, and TLR8) that can initiate cytokine induction in response to such molecules; see, e.g., Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3):513-524 (2012); Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan, Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

[0167] As described herein, numerous and varied modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that may be useful for the introduction of polynucleotides into human cells; see, e.g., Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, 2:77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al., Curr Opin Mol Ther., 12(2):158-67 (2010); Deleaviey et al., Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al., Nucleic Acid Ther 22(3):205-210 (2012); see the review by Bremsen et al., Front Genet 3:154 (2012).

[0168] As mentioned above, there are numerous commercial suppliers of modified RNAs, many of which specialize in modifications designed to improve the efficacy of siRNAs. Various approaches have been proposed based on various findings reported in the literature. For example, as reported by Kole, Nature Reviews Drug Discovery 11:125-140 (2012), Dharmacon notes that replacing non-bridging oxygens with sulfur (phosphorothioate, PS) is widely used to improve the nuclease resistance of siRNAs. Modification of the 2' position of ribose has been reported to improve the nuclease resistance of internucleotide phosphate bonds while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. As reported by Soutschek et al. Nature 432:173-178 (2004), the combination of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydro) is associated with highly stable siRNAs for in vivo applications; as reported by Volkov, Oligonucleotides 19:191-202 (2009), 2'-O-methyl modifications have been reported to be effective in improving stability. With regard to reducing the induction of innate immune responses, modifying specific sequences with 2'-O-methyl, 2'-fluoro, and 2'-hydro has been reported to generally reduce TLR7 / TLR8 interaction while preserving silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine, have also been shown to minimize immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, K. et al., Immunity 23:165-175 (2005).

[0169] As known in the art and commercially available, for use herein, several conjugates can be applied to polynucleotides, e.g., RNA, that can enhance their delivery and / or cellular uptake, including, for example, cholesterol, tocopherol and folate, lipids, peptides, polymers, linkers and aptamers; see, for example, the review by Winkler, Ther. Deliv. 4:791-809 (2013) and the references cited therein.

[0170] Codon optimization Polynucleotides encoding site-directed polypeptides can be codon-optimized according to standard methods in the art for expression in cells containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, it is contemplated that a human codon-optimized polynucleotide encoding Cas9 be used to generate the Cas9 polypeptide.

[0171] Complex of genome-targeting nucleic acid and site-specific polypeptide The genome-targeting nucleic acid interacts with a site-specific polypeptide (e.g., a nucleic acid-guided nuclease, such as Cas9), thereby forming a complex. The genome-targeting nucleic acid directs the site-specific polypeptide to the target nucleic acid.

[0172] Ribonucleoprotein complexes (RNPs) The site-specific polypeptide and the genome-targeting nucleic acid can be administered separately to cells or patients. Alternatively, the site-specific polypeptide can be pre-complexed with one or more guide RNAs or one or more crRNAs together with tracrRNA. The pre-complexed material can then be administered to cells or patients. Such pre-complexed material is known as a ribonucleoprotein particle (RNP). The site-specific polypeptide in the RNP can be, for example, Cas9 endonuclease or Cpfl endonuclease. The site-specific polypeptide can be flanked by one or more nuclear localization signals (NLSs) at the N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, the Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as the SV40 NLS. The weight ratio of the genome-targeting nucleic acid to the site-specific polypeptide in the RNP can be 1:1. For example, the weight ratio of sgRNA to Cas9 endonuclease in the RNP can be 1:1.

[0173] Nucleic acids encoding system components The present disclosure provides nucleic acids comprising nucleotide sequences encoding genome-targeting nucleic acids of the present disclosure, site-directed polypeptides of the present disclosure, and / or any nucleic acid or protein molecules necessary to carry out aspects of the methods of the present disclosure.

[0174] Nucleic acids encoding the genome-targeting nucleic acids of the present disclosure, the site-specific polypeptides of the present disclosure, and / or any nucleic acid or protein molecules necessary to carry out aspects of the methods of the present disclosure can comprise vectors (e.g., recombinant expression vectors).

[0175] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, in which additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are introduced into a host cell and integrated into the genome of the host cell, whereby they are replicated along with the host genome.

[0176] In some instances, vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or more simply, "expression vectors," and serve equivalent functions.

[0177] The term "operably linked" means that the nucleotide sequence of interest is linked to a control sequence(s) in a manner that allows for expression of the nucleotide sequence. The term "control sequence" is intended to include, for example, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such control sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific control sequences). Those skilled in the art will recognize that the design of the expression vector can depend on factors such as the choice of target cell, the desired expression level, and the like.

[0178] Contemplated expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus), and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus, as well as other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors can be used as long as they are compatible with the host cell.

[0179] In some examples, the vector may contain one or more transcriptional and / or translational control elements. Depending on the host / vector system used, any of several suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., can be used in the expression vector. The vector may be a self-inactivating vector that inactivates either viral sequences or components of the CRISPR machinery or other elements.

[0180] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, the human elongation factor-1 promoter (EF1), hybrid constructs containing the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

[0181] For expressing small RNAs, including guide RNAs used in conjunction with Cas endonucleases, various promoters may be advantageous, such as RNA polymerase III promoters, including U6 and H1. Descriptions of the use of such promoters and parameters for enhancing them are known in the art, and additional information and approaches are regularly described; see, for example, Ma, H. et al., Molecular Therapy - Nucleic Acids 3, e161 (2014) doi:10.1038 / mtna.2014.12.

[0182] The expression vector may also contain a ribosome binding site for translation initiation and transcription termination. The expression vector may also contain appropriate sequences for amplifying expression. The expression vector may also contain a nucleotide sequence encoding a non-natural tag (e.g., a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.) fused to the site-specific polypeptide, thus generating a fusion protein.

[0183] The promoter can be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some cases, the promoter can be a spatially restricted and / or temporarily restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.).

[0184] The genome-targeting nucleic acid and / or the nucleic acid encoding the site-specific polypeptide of the present disclosure can be packaged in or on the surface of a delivery vehicle for delivery to cells.Contemplated delivery vehicles include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles.As described in the art, various targeting moieties can be used to enhance the preferential interaction of such vehicles with desired cell types or locations.

[0185] Introduction of the complexes, polypeptides, and nucleic acids of the present disclosure into cells can be achieved by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0186] Treatment approach Provided herein are methods for treating patients with pain. One embodiment of such methods is ex vivo cell-based therapy. For example, a biopsy of a patient's peripheral nerve is performed. Nerve tissue can be isolated from the patient's skin or leg. Cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) are then isolated from the biopsy. The chromosomal DNA of the cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) can then be edited using the materials and methods described herein. Finally, the edited cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) are transplanted into the patient. Cells of any origin or type can be used as progenitor cells.

[0187] Another aspect of such methods is ex vivo cell-based therapy. For example, patient-specific induced pluripotent stem cells (iPSCs) can be generated. The chromosomal DNA of these iPSCs can then be edited using the materials and methods described herein. The genome-edited iPSCs can then be differentiated into cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia). Finally, the differentiated cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) can be transplanted into the patient.

[0188] Yet another aspect of such methods is ex vivo cell-based therapy. For example, mesenchymal stem cells can be isolated from a patient, such as from the patient's bone marrow or peripheral blood. The chromosomal DNA of these mesenchymal stem cells can then be edited using the materials and methods described herein. The genome-edited mesenchymal stem cells can then be differentiated into cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia). Finally, the differentiated cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) are transplanted into the patient.

[0189] One advantage of the ex vivo cell therapy approach is that comprehensive analysis of therapeutic agents can be performed before administration. Nuclease-based therapeutic agents may have some off-target effects. Performing gene editing ex vivo allows for characterization of the edited cell population before transplantation. The present disclosure includes sequencing the entire genome of the edited cells to ensure that any off-target effects are at genomic locations associated with minimal risk to the patient. Furthermore, specific cell populations, including clonal populations, can be isolated before transplantation.

[0190] Another advantage of ex vivo cell therapy relates to genetic modification in iPSCs compared to other primary cell sources. iPSCs are abundant, making it easy to obtain the large numbers of cells needed for cell-based therapies. Furthermore, iPSCs are an ideal cell type for clonal isolation, allowing for precise screening of genomic modifications without compromising viability. In contrast, other primary cells, such as glial cells, are only viable for a few passages and are difficult to clonally expand. Therefore, manipulating iPSCs for pain treatment may be much easier and may shorten the amount of time required to make the desired genetic modifications.

[0191] The methods can also include in vivo-based therapies, in which the chromosomal DNA of a patient's cells is edited using the materials and methods described herein. In some embodiments, the target cells in in vivo-based therapies are neurons of the peripheral nervous system.

[0192] While certain cells present attractive targets for ex vivo treatment and therapy, increased delivery efficiency may allow for direct in vivo delivery to such cells. Ideally, targeting and editing are directed to the relevant cells. Cleavage in other cells can also be prevented by using promoters that are active only in specific cells and / or developmental stages. Additional promoters can be inducible and thus temporally controlled when nucleases are delivered as plasmids. The amount of time delivered RNA and proteins remain within the cell can also be adjusted using treatments or added domains to alter half-life. In vivo treatment eliminates many treatment steps, but lower delivery rates may require higher editing rates. In vivo treatment may eliminate the problems and loss of ex vivo treatment and the engraftment and proper integration of neurons and glial cells into existing brain circuits.

[0193] An advantage of in vivo gene therapy can be the ease of manufacturing and administering the therapeutic. The same therapeutic approach and treatment has the potential to be used to treat multiple patients, for example, multiple patients who share the same or similar genotypes or alleles. In contrast, ex vivo cell therapy typically requires the use of a patient's own cells, which are isolated, manipulated, and then returned to the same patient.

[0194] Also provided herein is a cellular method for editing the SCN9A gene in cells by genome editing. For example, cells can be isolated from patients or animals. Then, the chromosomal DNA of the cells can be edited using the materials and methods described herein.

[0195] The methods provided herein, whether cellular, ex vivo, or in vivo, can involve reducing (knockdown) or eliminating (knockout) expression of the SCN9A gene by introducing one or more insertions, deletions, or mutations within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene.

[0196] For example, a knockdown or knockout strategy may involve disrupting the reading frame within the SCN9A gene by introducing random insertions or deletions (indels) that arise due to an imprecise NHEJ repair pathway. This can be achieved by using one or more CRISPR endonucleases and gRNAs (e.g., crRNA + tracrRNA, or sgRNA) to induce one single- or double-strand break within the SCN9A gene, or by using two or more CRISPR endonucleases and two or more sgRNAs to induce two or more single- or double-strand breaks within the SCN9A gene. This approach may require the development and optimization of sgRNAs for the SCN9A gene.

[0197] Alternatively, knockdown or knockout strategies can also involve the deletion of one or more segments within or near the SCN9A gene or other DNA sequences encoding regulatory elements of the SCN9A gene. This deletion strategy requires at least one pair of gRNAs (e.g., crRNA + tracrRNA, or sgRNA) that can bind to two different sites within or near the SCN9A gene, and one or more CRISPR endonucleases. The CRISPR endonucleases, configured with two gRNAs, induce two double-stranded breaks at the desired locations. After cleavage, the two ends, whether blunt or with overhangs, can be joined by NHEJ, resulting in the deletion of the intervening segment. In certain embodiments, the NHEJ repair pathway can cause insertions, deletions, or mutations at the junctions.

[0198] In addition to the genome editing strategies described above, another strategy involves modulating the expression, function, or activity of SCN9A by editing within the regulatory sequences.

[0199] In addition to the editing options described above, Cas9 or similar proteins can be used to target effector domains to the same target site identified for editing, or to additional target sites within the effector domain's range. Various chromatin-modifying enzymes, methylases or demethylases, can be used to alter the expression of target genes. One possibility is to reduce the expression of SCN9A protein if a mutation results in undesired activity. These types of epigenetic control have several advantages, particularly because they have limited potential off-target effects.

[0200] Many types of genomic target sites may exist in addition to coding and splicing sequences.

[0201] Regulation of transcription and translation involves numerous different classes of sites that interact with cellular proteins or nucleotides. DNA binding sites for transcription factors or other proteins are often targeted by mutation or deletion to study their role, but they can also be targeted to alter gene expression. Sites can be added by direct genome editing using non-homologous end-joining (NHEJ) or homology-directed repair (HDR). The increasing use of genome sequencing, RNA expression, and genome-wide studies of transcription factor binding have enhanced our ability to identify how these sites lead to developmental or temporal gene regulation. These control systems can be direct or involve widespread, coordinated control that may require the integration of activity from multiple enhancers. Transcription factors bind to degenerate DNA sequences, typically 6–12 bp in length. The low level of specificity offered by individual sites suggests that complex interactions and rules are involved in binding and functional outcomes. Less degenerate binding sites may provide simpler means of regulation. Artificial transcription factors can be designed to identify longer sequences with fewer similar sequences in the genome and a lower likelihood of off-target cleavage. Any of these types of binding sites can be mutated, deleted, or even created to allow for changes in gene regulation or expression (Canver, MC et al., Nature (2015)).

[0202] Another class of gene regulatory regions with these characteristics is microRNA (miRNA) binding sites. miRNAs are noncoding RNAs that play an important role in post-transcriptional gene regulation. miRNAs can regulate the expression of 30% of all mammalian protein-coding genes. Specific and potent gene silencing by double-stranded RNA (RNAi) has led to the discovery of additional small noncoding RNAs (Canver, MC et al., Nature (2015)). The largest class of noncoding RNAs important for gene silencing are miRNAs. In mammals, miRNAs are initially transcribed as long RNA transcripts that can be separate transcription units, part of protein introns, or other transcripts. These long transcripts, called primary miRNAs (pri-miRNAs), contain imperfectly base-paired hairpin structures. These pri-miRNAs can be cleaved into one or more shorter precursor miRNAs (pre-miRNAs) by the microprocessor, a nuclear protein complex that includes Drosha.

[0203] Pre-miRNAs are short stem-loops approximately 70 nucleotides in length with a 2-nucleotide 3' overhang that is carried into the mature 19-25 nucleotide miRNA:miRNA* duplex. The miRNA strand with lower base-pairing stability (guide strand) can be loaded into the RNA-induced silencing complex (RISC). The passenger strand (marked *) can function but is usually degraded. Mature miRNAs tether RISC to partially complementary sequence motifs in target mRNAs, primarily found within the 3' untranslated region (UTR), to induce post-transcriptional gene silencing (Bartel, D.P.Cell 136, 215-233 (2009); Saj, A. & Lai, E.C.Curr Opin Genet Dev 21, 504-510 (2011)).

[0204] miRNAs may be important in development, differentiation, cell cycle, and growth control, and in virtually every biological pathway in mammals and other multicellular organisms. miRNAs may also be involved in cell cycle control, apoptosis, and stem cell differentiation, hematopoiesis, hypoxia, muscle development, neurogenesis, insulin secretion, cholesterol metabolism, aging, viral replication, and the immune response.

[0205] A single miRNA can target hundreds of different mRNA transcripts, while individual miRNA transcripts can be targeted by many different miRNAs. The latest release of miRBase (v.21) annotates over 28,645 microRNAs. Some miRNAs can be encoded by multiple loci, some of which can be expressed from tandemly co-transcribed clusters. This allows for complex regulatory networks with multiple pathways and feedback controls. miRNAs can be an integral part of these feedback and regulatory circuits and can help regulate gene expression by keeping protein production within limits (Herranz, H. & Cohen, SM Genes Dev 24, 1339-1344 (2010); Posadas, DM & Carthew, RW Curr Opin Genet Dev 27, 1-6 (2014)).

[0206] miRNA may also be important in many human diseases related to abnormal miRNA expression. This association highlights the importance of the miRNA regulatory pathway. Recent miRNA deletion studies have linked miRNA to the regulation of immune response (Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

[0207] miRNAs are also strongly associated with cancer and may play a role in different types of cancer. miRNAs have been found to be downregulated in many tumors. miRNAs may be important in regulating key cancer-related pathways, such as cell cycle control and DNA damage response, and therefore may be used in diagnosis and clinically targeted. microRNAs may delicately control the balance of angiogenesis, as experiments depleting all microRNAs suppress tumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

[0208] As has been shown for protein-coding genes, miRNA genes may also undergo epigenetic changes that occur with cancer. Many miRNA loci can be associated with CpG islands, which increases their chances of being regulated by DNA methylation (Weber, B., Stresemann, C., Brueckner, B. & Lyko, F. Cell Cycle 6, 1001-1005 (2007)). Most studies have used treatment with chromatin-remodeling drugs to reveal epigenetically silenced miRNAs.

[0209] In addition to their role in RNA silencing, miRNAs can also activate translation (Posadas, D.M. & Carthew, R.W. Curr Opin Genet Dev 27, 1-6 (2014)). Knocking out miRNA sites can decrease expression of target genes, whereas introducing these sites can increase expression.

[0210] Individual miRNAs can be most effectively knocked out by mutating the seed sequence (2-8 bases of the microRNA), which may be important for binding specificity. Cleavage in this region, followed by misrepair by NHEJ, can effectively disable miRNA function by blocking binding to the target site. miRNAs can also be inhibited by specifically targeting a special loop region adjacent to the palindromic sequence. Catalytically inactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. et al., Sci Rep 4, 3943 (2014)). In addition to targeting miRNAs, the binding site can also be targeted and mutated to prevent miRNA-mediated silencing.

[0211] According to the present disclosure, either microRNAs (miRNAs) or their binding sites may be incorporated into the compositions of the present invention.

[0212] The composition may have a region including, but not limited to, a region comprising the sequence of any of the microRNAs set forth in SEQ ID NOs: 632-4715, the reverse complement of a microRNA set forth in SEQ ID NOs: 632-4715, or the microRNA antiseed region of any of the microRNAs set forth in SEQ ID NOs: 632-4715.

[0213] The compositions of the present disclosure may include one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA, such as those taught in U.S. Patent Application Publication Nos. 2005 / 0261218 and 2005 / 0059005. As non-limiting examples, known microRNAs, their sequences, and their binding site sequences in the human genome are listed below in SEQ ID NOS: 632-4715.

[0214] The microRNA sequence comprises a "seed" sequence, i.e., a sequence within the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, the microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), and the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposite the microRNA at position 1. In some embodiments, the microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), and the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposite the microRNA at position 1. See, e.g., Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6;27(1):91-105. The bases of the microRNA seed are perfectly complementary to the target sequence.

[0215] The identification of microRNAs, microRNA target regions, and their expression patterns and roles in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi:10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403).

[0216] For example, if the composition is not intended to be delivered to the liver and ends up there, then miR-122, a liver-enriched microRNA, can inhibit expression of the delivered sequence if one or more target sites for miR-122 are engineered into the polynucleotide encoding that target sequence. Introduction of one or more binding sites for different microRNAs can be engineered to further reduce longevity, stability, and protein translation, thus providing an additional layer of durability.

[0217] As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that "binding" may follow traditional Watson-Crick hybridization rules, or may reflect any stable association between the microRNA and a target sequence at or near the microRNA site.

[0218] Conversely, for purposes of the compositions of the present disclosure, microRNA binding sites can be engineered (i.e., removed) from the sequences in which they naturally occur in order to increase protein expression in specific tissues.

[0219] Specifically, microRNAs are known to be differentially expressed in immune cells (also known as hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, and natural killer cells. Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infection, inflammation, and undesirable immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of hematopoietic cell (immune cell) development, proliferation, differentiation, and apoptosis. For example, miR-142 and miR-146 are exclusively expressed in immune cells, and are particularly abundantly expressed in myeloid dendritic cells. Introduction of a miR-142 binding site into the 3'-UTR of a polypeptide of the present disclosure can selectively suppress gene expression in antigen-presenting cells through miR-142-mediated mRNA degradation, limiting antigen presentation in professional APCs (e.g., dendritic cells), thereby preventing antigen-mediated immune responses after gene delivery (see Annoni A et al., Blood, 2009, 114, 5152-5161).

[0220] In one example, a microRNA binding site known to be expressed in immune cells, particularly antigen-presenting cells, can be engineered into a polynucleotide to suppress expression of the polynucleotide in APCs via microRNA-mediated RNA degradation, thereby suppressing antigen-mediated immune responses, while expression of the polynucleotide is maintained in non-immune cells in which the immune cell-specific microRNA is not expressed.

[0221] Many microRNA expression studies have been conducted and described in the field to outline the differential expression of microRNAs in various cancer cells / tissues and other diseases. Some microRNAs are abnormally overexpressed in certain cancer cells, while others are underexpressed. For example, microRNAs are differentially expressed in cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and disease (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, US83892). 10); Asthma and inflammation (US8415096); Prostate cancer (US2013 / 0053264); Hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 0 54828, US8252538); lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, US2010 / 0323357); skin T cells Lymphoma (WO2013 / 011378); colorectal cancer cells (WO2011 / 0281756, WO2011 / 076142); cancer-positive lymph nodes (WO2009 / 100430, US2009 / 0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263), thyroid cancer (WO2013 / 066678 ); ovarian cancer cells (US2012 / 0309645, WO2011 / 095623); breast cancer cells (WO2008 / 154098, WO2007 / 081740, US2012 / 0214699), leukemia and lymphoma (WO2008 / 073915, US2009 / 0092974, US2012 / 036081, US2012 / 0283310, WO2010 / 018563).

[0222] Non-limiting examples of microRNA sequences and target tissues and / or cells are disclosed in SEQ ID NOs: 632-4715.

[0223] Genome Engineering Strategies In some embodiments, the methods of the present disclosure can include editing one or both alleles. Gene editing to modify an allele(s) has the advantage of permanently modifying the target gene or gene product.

[0224] The steps of the disclosed ex vivo methods may include editing cells of the peripheral nervous system isolated from a patient (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia) using genome engineering. Alternatively, the steps of the disclosed ex vivo methods may include editing patient-specific iPSCs or mesenchymal stem cells. Similarly, the steps of the disclosed in vivo methods may include editing cells of a patient suffering from pain using genome engineering. Similarly, the steps of the disclosed cellular methods may include editing the SCN9A gene in human cells by genome engineering.

[0225] The patient who experiences pain may show a wide range of mutations in SCN9A gene.Therefore, different patients may need different editing strategies.Any CRISPR endonuclease can be used in the method of the present disclosure, and each CRISPR endonuclease has its own associated PAM, which may or may not be disease-specific.

[0226] For example, expression of the SCN9A gene can be disrupted or eliminated by introducing random insertions or deletions (indels) that occur due to an imprecise NHEJ repair pathway. The target region can be the coding sequence (i.e., exon) of the SCN9A gene. Insertion or deletion of nucleotides into the coding sequence of the gene can cause a "frameshift" in which the normal three-letter codon pattern is disrupted. In this way, gene expression, and therefore protein production, can be reduced or eliminated. This approach can also be used to target any intron, intron:exon junction, or regulatory DNA element of the SCN9A gene, where sequence changes may disrupt the expression of the SCN9A gene.

[0227] As another example, NHEJ can also be used to delete segments of genes, either directly or by altering splice donor or acceptor sites through cleavage by one or several gRNAs targeting several positions. This can be useful when small random indels are inefficient at knocking out the target gene. A pair of gRNAs has been used for this type of deletion.

[0228] In the absence of a donor, ends from a DNA break or from different breaks can be joined using several non-homologous repair pathways, in which DNA ends are joined with little or no base pairing at the junction. In addition to canonical NHEJ, there are similar repair mechanisms, such as alt-NHEJ. When there are two breaks, the intervening segment can be deleted or inverted. NHEJ repair pathways can cause insertions, deletions, or mutations at the joint.

[0229] NHEJ can also result in homology-independent targeted integration. For example, the inclusion of a nuclease target site on a donor plasmid can promote integration of a transgene into a chromosomal double-strand break after in vivo nuclease cleavage of both the donor and the chromosome (Cristea., Biotechnol Bioeng. 2013 Mar;110(3):871-80). NHEJ has been used to insert a 15-kb inducible gene expression cassette into a defined locus in a human cell line after nuclease cleavage. (See, e.g., Maresca, M., Lin, VG, Guo, N. & Yang, Y., Genome Res 23, 539-546 (2013); Suzuki et al. Nature, 540, 144-149 (2016)). The integrated sequence can disrupt the reading frame of the SCN9A gene or alter the structure of the gene.

[0230] As a further alternative, homology-directed repair (HDR) can also be used to knock out genes or alter gene function. For example, an HDR knockout strategy can involve disrupting the structure or function of the SCN9A gene by inserting a non-functional or unrelated sequence into the SCN9A gene. This can be achieved by inducing single- or double-stranded breaks in a gene of interest using one or more CRISPR endonucleases and gRNAs (e.g., crRNA + tracrRNA, or sgRNA), or two or more single- or double-stranded breaks, in the presence of an exogenously introduced donor DNA template (the donor DNA template can be a short single-stranded oligonucleotide, a short double-stranded oligonucleotide, or a long single- or double-stranded DNA molecule) to direct the cellular DSB response to homology-directed repair. This approach may require the development and optimization of gRNAs and donor DNA molecules for the SCN9A gene.

[0231] Homology-directed repair (HDR) is essentially an error-free mechanism that uses a homologous DNA sequence supplied as a template during DSB repair. Because the rate of HDR is a function of the distance between the mutation and the break site, it is important to select an overlapping or closest target site. The template may contain additional sequences adjacent to the homologous region or may contain sequences that differ from the genomic sequence, thus enabling sequence editing.

[0232] The most common form of HDR is homologous recombination. There are additional pathways for HDR, including single-strand annealing and alternative HDR. Genome engineering tools allow researchers to manipulate cellular homologous recombination pathways to create site-specific modifications in the genome. It has been discovered that cells can repair double-strand breaks using synthetic donor molecules provided in trans. Thus, by introducing a double-strand break near a specific mutation and providing an appropriate donor, targeted changes can be made in the genome. Specific breaks can be repaired only by receiving a homologous donor.6 In cells, this increases the rate of HDR by more than 1,000-fold over that of normal cells. Because the rate of homology-directed repair (HDR) at a particular nucleotide is a function of the distance to the break site, it is important to select overlapping or closest target sites. Gene editing has an advantage over gene addition because in situ editing leaves the rest of the genome undisturbed.

[0233] Donors supplied for HDR editing vary significantly but can contain the intended sequence with small or large flanking homology arms to allow annealing to genomic DNA. The homology regions flanking the introduced genetic change can be as small as 30 bp or less, or as large as multi-kilobase cassettes that may contain promoters, cDNA, etc. Both single-stranded and double-stranded oligonucleotide donors have been used. While longer ssDNA can also be generated and used, these oligonucleotides range in size from less than 100 nt to over several kb. Double-stranded donors, including PCR amplicons, plasmids, and minicircles, can also be used. Generally, AAV vectors can be a very effective means of delivering donor templates, but the packaging limit for individual donors is <5 kb. Active transcription of donors increases HDR by 3-fold, indicating that the inclusion of a promoter may increase conversion. Conversely, methylation of donor CpGs reduces gene expression and HDR.

[0234] In addition to wild-type endonucleases such as Cas9, there are nickase mutants in which one or the other nuclease domain is inactivated, resulting in cleavage of only one DNA strand. HDR can be directed by an individual Cas nickase or by using a pair of nickases flanking the target region. The donor can be single-stranded, nicked, or dsDNA.

[0235] Donor DNA can be provided together with nuclease or independently by various different methods, such as transfection, nanoparticles, microinjection, or viral transduction.To increase the availability of donor for HDR, a series of tethering options have been proposed.Examples include binding donor to nuclease, binding to nearby DNA binding protein, or binding to proteins involved in DNA end joining or repair.

[0236] The choice of repair pathway can be guided by many culture conditions, such as those affecting the cell cycle, or by targeting DNA repair and related proteins. For example, to increase HDR, key NHEJ molecules such as KU70, KU80, or DNA ligase IV can be inhibited.

[0237] In addition to genome editing by NHEJ or HDR, site-specific gene insertion using both the NHEJ pathway and HDR has been performed. A combined approach may be applicable in certain situations, possibly involving intron / exon boundaries. While NHEJ may prove effective for ligation at introns, error-free HDR may be more suitable for coding regions.

[0238] The SCN9A gene contains multiple exons, as shown in Table 3. Any one or more of these exons or nearby introns can be targeted to create one or more indels that disrupt the reading frame and ultimately eliminate aberrant SCN9A protein activity.

[0239] In some embodiments, the method can provide a gRNA pair that creates a deletion by cutting the gene twice at a position adjacent to the unnecessary sequence. This sequence can include one or more exons, introns, intron:exon junctions, other DNA sequences encoding regulatory elements of the SCN9A gene, or a combination thereof. Cleavage can be achieved by a pair of DNA endonucleases, each of which creates a DSB in the genome, or by multiple nickases that work together to create a DSB in the genome.

[0240] Alternatively, the method can provide a single gRNA to create a double-stranded break within a coding sequence or splicing sequence. The double-stranded break can be created by a single DNA endonuclease or by multiple nickases that work together to create a DSB in the genome.

[0241] Splicing donors and acceptors are generally within 100 base pairs of adjacent introns. In some examples, the method may provide gRNAs that cleave approximately + / - 100 to 3100 bp for each exon / intron junction of interest.

[0242] For any genome editing strategy, gene edits can be confirmed by sequencing or PCR analysis.

[0243] Selection of target sequence Shifting the position of the 5' and / or 3' boundaries relative to a particular reference locus can be used to facilitate or enhance particular applications of gene editing, depending in part on the endonuclease system selected for editing, as further described and illustrated herein.

[0244] In a first non-limiting example of such target sequence selection, many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as the requirement for a PAM sequence motif at a specific position adjacent to the DNA cleavage site in the case of a CRISPR Type II or Type V endonuclease.

[0245] In another non-limiting example of target sequence selection or optimization, for a particular combination of target sequence and gene editing endonuclease, the frequency of off-target activity (i.e., the frequency of DSBs occurring at sites other than the selected target sequence) can be evaluated relative to the frequency of on-target activity. In some cases, cells correctly edited at a desired locus may have a selective advantage compared to other cells. Illustrative, but non-limiting, examples of selective advantages include the acquisition of attributes such as improved replication rate, persistence, resistance to certain conditions, improved in vivo engraftment success or persistence after introduction into a patient, and other attributes associated with the maintenance or increased number or viability of such cells. In other cases, cells correctly edited at a desired locus can be positively selected by one or more screening methods used to identify, sort, or select correctly edited cells. Both selective advantage and directional selection methods can utilize the phenotype associated with the change. In some cases, cells can be edited more than once to generate a second modification that creates a new phenotype, which is used to select or purify the intended cell population. Such a second modification can be made by adding a second gRNA that allows for the expression of a selectable or screenable marker. In some cases, a DNA fragment containing the cDNA and the selectable marker can be used to correctly edit the cell at the desired locus.

[0246] Regardless of whether any selective advantage is applicable or whether any directed selection should be applied in a particular case, target sequence selection can also be guided by consideration of off-target frequency to increase the effectiveness of application and / or reduce the likelihood of undesired modifications at sites other than the desired target. As further described and exemplified herein and in the art, the occurrence of off-target activity can be influenced by numerous factors, including the similarity and dissimilarity between the target site and various off-target sites, as well as the specific endonuclease used. Bioinformatics tools are available to assist in the prediction of off-target activity, and in many cases, such tools can also be used to identify sites with the highest likelihood of off-target activity, which can then be evaluated in an experimental setting to assess the relative frequency of off-target activity versus on-target activity, thereby enabling the selection of sequences with higher relative on-target activity. Illustrative examples of such techniques are provided herein, and others are known in the art.

[0247] Another aspect of target sequence selection relates to homologous recombination events. Sequences sharing regions of homology can serve as a focus for homologous recombination events that result in the deletion of intervening sequences. Such recombination events occur periodically during the normal course of replication of chromosomes and other DNA sequences, and at other times when DNA sequences are synthesized, such as in the repair of double-strand breaks (DSBs), during the normal cell replication cycle, but can be enhanced by the occurrence of various events (such as ultraviolet light and other inducers of DNA breaks) or the presence of specific agents (such as various chemical inducers). Many such inducers indiscriminately generate DSBs in the genome, and DSBs can be induced and repaired regularly in normal cells. During repair, the original sequence can be reconstructed with perfect fidelity, although in some cases small insertions or deletions (called "indels") are introduced at the DSB site.

[0248] In addition, DSB can be specifically induced at a specific location, as in the case of the endonuclease system described herein, which can be used to cause directed or preferential gene modification events at selected chromosomal locations.The tendency of homologous sequences to undergo recombination in the context of DNA repair (and replication) can be utilized in many situations, and this is the basis for one application of gene editing systems such as CRISPR, where homology-directed repair is used to insert the sequence of interest provided by the use of "donor" polynucleotides into desired chromosomal locations.

[0249] Regions of homology between specific sequences, which may be small regions of "microhomology" that may contain as few as 10 base pairs or less, can also be used to create the desired deletion. For example, a single DSB can be introduced at a site that exhibits microhomology with a nearby sequence. During normal repair of such a DSB, a frequent outcome is deletion of the intervening sequence as a result of recombination promoted by the DSB and associated cellular repair processes.

[0250] However, in some situations, selection of a target sequence within the region of homology may also result in much larger deletions, including gene fusions (if the deletion is in a coding region), which may or may not be desirable given the particular situation.

[0251] The examples provided herein further illustrate the selection of various target regions for the creation of DSBs designed to induce insertions, deletions, or mutations that result in the reduction or elimination of SCN9A protein activity, as well as the selection of specific target sequences within such regions that are designed to minimize off-target events relative to on-target events.

[0252] human cells As described and exemplified herein, the primary target of gene editing to improve pain or any disorder associated with SCN9A is human cells. For example, in ex vivo methods, the human cells can be somatic cells, which can be modified using the described techniques to give rise to differentiated cells, such as neurons or progenitor cells of the peripheral nervous system. For example, in in vivo methods, the human cells can be neurons of the peripheral nervous system or cells derived from other affected organs.

[0253] By performing gene editing on autologous cells that are derived from, and therefore already perfectly matched to, a patient in need, it is possible to generate cells that can be safely reintroduced into the patient, effectively generating a cell population that will be effective in ameliorating one or more clinical conditions associated with the patient's disease.

[0254] Stem cells can proliferate and give rise to more progenitor cells, which in turn have the potential to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can proliferate and subsequently produce progeny that differentiate into one or more mature cell types, while retaining one or more cells of the parent's developmental potential. The term "stem cell" then refers to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retains the ability to proliferate without substantial differentiation. In one embodiment, the term progenitor cell or stem cell refers to a generalized mother cell whose descendants (progeny) often specialize in different directions, e.g., by differentiation by acquiring entirely individual characteristics, as occurs, for example, in the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, such as those that are themselves derived from pluripotent cells. While each of these multipotent cells can be considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells of greater developmental potential. Such ability may be natural or may be artificially induced during treatment with various factors. In many biological cases, stem cells may also be "pluripotent" because they can produce descendants of one or more different cell types, but this is not required for "stemness."

[0255] Self-renewal may be another important aspect of stem cells. Theoretically, self-renewal can occur through either of two major mechanisms. Stem cells can divide asymmetrically, with one daughter retaining a stem state and the other daughter expressing some other specific function and phenotype. Alternatively, some stem cells within a population can divide symmetrically into two stems, thus maintaining some stem cells within the population as a whole, while other cells within the population give rise to only differentiated progeny. Generally, "progenitor cells" have a more primitive cell phenotype (i.e., at an earlier stage along a developmental pathway or progression than fully differentiated cells). In many cases, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which they develop and differentiate.

[0256] In the context of cellular ontogeny, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is one that has progressed further along a developmental pathway than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-restricted progenitor cells (such as muscle progenitor cells), which can differentiate into other types of progenitor cells further down the pathway (such as muscle progenitor cells), and then into end-stage differentiated cells such as muscle cells that play characteristic roles in specific tissue types and may or may not retain the ability to proliferate further.

[0257] induced pluripotent stem cells The genetically engineered human cells described herein may be induced pluripotent stem cells (iPSCs). An advantage of using iPSCs is that the cells can be derived from the same subject to which progenitor cells are administered. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells (e.g., autologous cells) to be administered to the subject. Because the precursors are essentially derived from an autologous source, the risk of engraftment rejection or allergic reaction can be reduced compared to the use of cells from another subject or group of subjects. Furthermore, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one embodiment, the stem cells used in the disclosed methods are not embryonic stem cells.

[0258] Although differentiation is generally irreversible under physiological conditions, several methods have recently been developed for reprogramming somatic cells into iPSCs. Exemplary methods are known to those skilled in the art and are briefly described below.

[0259] The term "reprogramming" refers to the process of changing or reversing the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming refers to the process of reversing cell differentiation toward a more undifferentiated or primitive type of cell. It should be noted that many primary cell cultures may lose some of their fully differentiated characteristics when cultured. Therefore, simply culturing such cells within the term differentiated cells does not render them undifferentiated (e.g., undifferentiated) or pluripotent. The transition of differentiated cells to pluripotency requires a reprogramming stimulus that exceeds the stimulus that results in partial loss of differentiated characteristics in culture. Reprogrammed cells also possess the characteristic of being able to be passaged for extended periods without losing their proliferative potential, compared to their primary cell parents, which generally only have the capacity for a limited number of divisions in culture.

[0260] Reprogrammed cells may be partially or terminally differentiated prior to reprogramming. Reprogramming may involve the complete reversion of a differentiated cell (e.g., a somatic cell) from its differentiated state to a pluripotent or multipotent state. Reprogramming may involve the complete or partial reversion of a differentiated cell (e.g., a somatic cell) from its differentiated state to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming may result in the expression of specific genes by the cell, which expression further contributes to reprogramming. In certain examples described herein, reprogramming a differentiated cell (e.g., a somatic cell) can cause the differentiated cell to be in an undifferentiated state (e.g., an undifferentiated cell). The resulting cell is referred to as a "reprogrammed cell" or "induced pluripotent stem cell (iPSC or iPS cell)."

[0261] Reprogramming can involve altering, e.g., reversing, at least some of the genetic patterns that occur during cell differentiation, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting. Reprogramming is distinct from simply maintaining the existing undifferentiated state of an already pluripotent cell or from maintaining the existing, fully undifferentiated state of an already multipotent cell (e.g., a myogenic stem cell). Reprogramming is also distinct from promoting self-renewal or proliferation of an already pluripotent or multipotent cell, although in some instances, the compositions and methods described herein may also be useful for such purposes.

[0262] Many methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Any such method that reprograms somatic cells to a pluripotent phenotype is suitable for use in the methods described herein.

[0263] A reprogramming methodology for generating pluripotent cells using defined combinations of transcription factors has been described. Mouse somatic cells can be converted into ES cell-like cells with expanded developmental potential by direct transduction of Oct4, Sox2, Klf4, and c-Myc. See, e.g., Takahashi and Yamanaka, Cell 126(4):663-76 (2006). iPSCs resemble ES cells because they restore pluripotency-associated transcriptional circuitry and much of the epigenetic landscape. Furthermore, mouse iPSCs fulfill all standard assays for pluripotency, specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, chimera participation, germline transmission [see, e.g., Maherali and Hochedlinger, Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

[0264] Human iPSCs can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, have been established as a core set of transcription factors governing pluripotency. See, e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57 (2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121 (2014); Focosi et al., Blood Cancer Journal 4:e211 (2014); and references cited therein. Historically, iPSC production could be achieved by using viral vectors to introduce nucleic acid sequences encoding stem cell-associated genes into adult somatic cells.

[0265] iPSCs can be generated or derived from terminally differentiated somatic cells, as well as adult or somatic stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming. In such cases, it may not be necessary to include the number of reprogramming factors required to reprogram terminally differentiated cells. Furthermore, reprogramming can be induced by non-viral introduction of reprogramming factors, for example, by introducing the protein itself, or by introducing a nucleic acid encoding the reprogramming factor, or by introducing a messenger RNA that produces the reprogramming factor upon translation (see, e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010)). Reprogramming can be achieved, for example, by introducing a combination of nucleic acids encoding stem cell-associated genes, including Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming using the methods and compositions described herein can further include introducing one or more of Oct-3 / 4, a member of the Sox family, a member of the Klf family, and a member of the Myc family into somatic cells. The methods and compositions described herein can further include introducing one or more of each of Oct-4, Sox2, NANOG, c-Myc, and Klf4 for reprogramming. As noted above, the precise method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, when cells differentiated from reprogrammed cells are to be used, for example, in human therapy, in one embodiment, reprogramming is not affected by methods that alter the genome. Thus, in such instances, reprogramming can be achieved without the use of, for example, viral or plasmid vectors.

[0266] The efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a population of starting cells can be enhanced by the addition of various agents (e.g., small molecules), as shown by Shi et al., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., Nature Biotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, agents or combinations of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used to generate patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA).

[0267] Other non-limiting examples of reprogramming promoters include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VPA) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate ( Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic acid hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming promoters include, for example, dominant-negative forms (e.g., catalytically inactive forms) of HDACs, siRNA inhibitors of HDACs, and antibodies that specifically bind to HDACs. Such inhibitors are available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.

[0268] To confirm the induction of pluripotent stem cells for use in the methods described herein, isolated clones can be tested for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecat1, ESgl, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utfl, and Natl. In one example, cells expressing, for example, Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods, such as Western blotting or flow cytometry, to detect the presence of the encoded polypeptide. Detection can include not only RT-PCR but also detection of protein markers. Intracellular markers can best be identified by protein detection methods such as RT-PCR or immunocytochemistry, while cell surface markers are readily identified by, for example, immunocytochemistry.

[0269] The pluripotent stem cell characteristics of isolated cells can be confirmed by testing the iPSCs to differentiate into cells of each of the three germ layers. As an example, teratoma formation in nude mice can be used to assess the pluripotency of isolated clones. The cells can be introduced into nude mice, and histology and / or immunohistochemistry can be performed on tumors arising from the cells. For example, the growth of tumors containing cells from all three germ layers further indicates that the cells are pluripotent stem cells.

[0270] Cells of the peripheral nervous system In some embodiments, the genetically engineered human cells described herein are neurons and nerves outside the brain and spinal cord. Neurons, which process information, and glial cells, which provide mechanical and metabolic support to the nervous system, are the two major classes of cells in the peripheral nervous system. Non-limiting examples of neurons include sensory neurons (which collect impulses from sensory receptors in areas such as skin, muscles, and organs and transmit those impulses through nerves to the CNS) and motor neurons (which collect outgoing messages from the CNS and deliver them to the appropriate body organs, instructing them on what action needs to be taken). Non-limiting examples of glial cells include Schwann cells in nerves or satellite cells in ganglia.

[0271] Generation of patient-specific iPSCs One step of the ex vivo method of the present disclosure may include generating patient-specific iPS cells, patient-specific iPS cell(s), or patient-specific iPS cell lines. As described in Takahashi and Yamanaka (2006) and Takahashi, Tanabe et al. (2007), there are many established methods for generating patient-specific iPS cells. For example, the generating step may include: a) isolating somatic cells, such as skin cells or fibroblasts, from a patient; and b) introducing a set of pluripotency-associated genes into the somatic cells to induce them to become pluripotent stem cells. The set of pluripotency-associated genes may be one or more genes selected from the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT, and LIN28.

[0272] Performing a biopsy or aspiration of the patient's tissue A biopsy or aspiration is a tissue or fluid sample taken from the body. There are various types of biopsies and aspirations. Almost all of them involve using a sharp instrument to remove a small amount of tissue. If the biopsy is on the skin or other sensitive areas, a numbing agent may be applied first. Biopsies or aspirations can be performed according to any method known in the art. For example, in bone marrow aspiration, a large needle is inserted into the pelvic bone to collect bone marrow. In the case of a nerve biopsy, for example, from the skin or leg to isolate neurons of the peripheral nervous system, the nerve portion is excised, causing minimal mechanical damage. Compression or stretching of the nerve is avoided, and no attempt is made to remove excess fat or connective tissue.

[0273] Isolation of peripheral nervous system neurons Neurons of the peripheral nervous system can be isolated according to any known method in the art. For example, nerve segments are excised under sterile conditions with minimal mechanical damage. Compression or stretching of the nerve is strictly avoided, and excessive removal of fat or connective tissue is not attempted. Because nerve fibers are highly sensitive to mechanical damage, proximal nerve sectioning is performed first. After isolation, the outermost connective tissue layer, the epiallantois, is removed and collected for enzymatic digestion. The fibers are torn with the aid of fine forceps until all bundles are separated into individual fibers. The epiallantois and torn fibers are then subjected to overnight enzymatic digestion using dispase II and type I collagenase. The digested product is filtered and collected by centrifugation. The resulting cell suspension is plated on an adhesive PLL / laminin substrate. Adherent cells are cultured for analysis (Andersen et al., Scientific Reports-Nature, 2016, 6:31781).

[0274] Isolation of mesenchymal stem cells Mesenchymal stem cells can be isolated according to any method known in the art, such as from patient's bone marrow or peripheral blood.For example, bone marrow aspirate can be collected in a syringe with heparin.Cells can be washed and centrifuged with Percoll.Cells can be cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger MF, Mackay AM, Beck SC et al., Science 1999;284:143-147).

[0275] Genetically modified cells The term "genetically modified cell" refers to a cell containing at least one genetic modification introduced by genome editing (e.g., using the CRISPR / Cas9 or CRISPR / Cpf1 system). In some ex vivo examples herein, the genetically modified cell may be a genetically modified progenitor cell. In some in vivo examples herein, the genetically modified cell may be a genetically modified neuron of the peripheral nervous system. Genetically modified cells containing an exogenous genome-targeting nucleic acid and / or an exogenous nucleic acid encoding a genome-targeting nucleic acid are contemplated herein.

[0276] The term "control-treated population" refers to a cell population that has been treated with the same medium, viral induction, nucleic acid sequence, temperature, confluency, flask size, pH, etc., except for the addition of the genome-edited component. Any method known in the art can be used to measure SCN9A gene transcription or protein expression or activity, for example, Western blot analysis of SCN9A protein or real-time PCR to quantify SCN9A mRNA.

[0277] The term "isolated cell" refers to a cell that has been removed from an organism in which it was originally found, or the progeny of such a cell. Optionally, the cell may be cultured in vitro, e.g., under defined conditions or in the presence of other cells. Optionally, the cell may be subsequently introduced into a second organism or reintroduced into the organism from which it was isolated (or the cell from which it was derived).

[0278] With respect to isolated cell populations, the term "isolated population" refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some cases, an isolated population can be a substantially pure population of cells compared to the heterogeneous population from which the cells are isolated or enriched. In some cases, an isolated population can be an isolated population of human progenitor cells (e.g., a substantially pure population of human progenitor cells) compared to a heterogeneous population of cells that includes human progenitor cells and the cells from which the human progenitor cells are derived.

[0279] With respect to a particular cell population, the term "substantially enhanced" refers to a population of cells in which the occurrence of a particular type of cell is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, at least 5000-fold, at least 20000-fold, at least 100000-fold, or more, compared to existing or reference levels, depending on the desired level of such cells, e.g., for ameliorating pain.

[0280] With respect to a particular cell population, the term "substantially enriched" refers to a population of cells that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more of the cells that make up the total cell population.

[0281] With respect to a particular cell population, the term "substantially pure" refers to a population of cells that is at least about 75%, at least about 85%, at least about 90%, or at least about 95% pure with respect to the cells that make up the total cell population. That is, with respect to a population of progenitor cells, the term "substantially pure" or "essentially purified" refers to a population of cells that contains less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1% of the cells that are not progenitor cells as defined by that term herein.

[0282] Differentiation of genome-edited iPSCs into peripheral nervous system cells Another step of the ex vivo method of the present disclosure may include differentiating the genome-edited iPSCs into cells of the peripheral nervous system (e.g., neurons or glial cells, such as Schwann cells in nerves or satellite glial cells in ganglia). The differentiation step may be performed according to any method known in the art. For example, neural differentiation of iPSCs is induced using a combination of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and dibutyryl cyclic AMP (dbcAMP). The iPSC-derived neural cells are then further differentiated into Schwann cells using ciliary neurotrophic factor (CNTF), neuregulin 1β, and dbcAMP (Wang et al., Biomaterials. 2011;32(22):5023-5032).

[0283] Differentiation of genome-edited mesenchymal stem cells into peripheral nervous system cells Another step of the ex vivo method of the present disclosure may include differentiating the genome-edited mesenchymal stem cells into cells of the peripheral nervous system (e.g., neurons or glial cells, such as ganglionic Schwann cells or ganglionic satellite glial cells). The differentiation step may be performed according to any method known in the art. For example, MSCs are treated with various factors and hormones, including basic fibroblast growth factor, human recombinant platelet-derived growth factor, forskolin, and glial growth factor-2 (Ladak et al., Experimental Neurology 228 (2011) 242-252).

[0284] Cell transplantation into patients Another step of the ex vivo method of the present disclosure can include transplanting genome-edited neurons of the peripheral nervous system into a patient. This transplantation step can be achieved using any transplantation method known in the art. For example, the genetically modified cells can be directly injected into the patient's blood or administered to the patient by other means.

[0285] III. Formulation and Delivery Pharmaceutically acceptable carrier The ex vivo methods of administering progenitor cells to a subject contemplated herein involve the use of a therapeutic composition comprising the progenitor cells.

[0286] Therapeutic compositions may contain a physiologically acceptable carrier together with the cell composition dissolved or dispersed therein as an active ingredient, and optionally at least one additional bioactive agent as described herein. In some cases, the therapeutic composition will not be substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired.

[0287] Generally, the progenitor cells described herein can be administered as a suspension with a pharmaceutically acceptable carrier. Those skilled in the art will understand that the pharmaceutically acceptable carrier used in the cell composition does not contain buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the survival of the cells delivered to a subject. Cell-containing formulations may include, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and optionally, nutrients that maintain cell survival or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the progenitor cells described herein using routine experimentation.

[0288] The cell composition can be emulsified or provided as a liposomal composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients can be mixed with additives that are pharmaceutically acceptable, compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0289] The additional agent contained in the cell composition may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0290] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing only the active ingredient and water, or containing a buffer such as sodium phosphate at a physiological pH value, physiological saline, or both, e.g., phosphate-buffered saline. Furthermore, aqueous carriers may contain more than one buffer salt, including salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to and in addition to water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in a cell composition effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0291] Guide RNA formulations Guide RNAs of the present disclosure can be formulated with pharmaceutically acceptable additives, such as carriers, solvents, stabilizers, adjuvants, diluents, and the like, depending on the particular mode of administration and dosage form. Guide RNA compositions can be formulated to achieve a physiologically compatible pH, ranging from about pH 3 to about pH 11, depending on the formulation and route of administration, with a range of about pH 3 to about pH 7. In some cases, the pH can be adjusted to a range of about pH 5.0 to about pH 8. In some cases, the composition can comprise a therapeutically effective amount of at least one compound described herein, along with one or more pharmaceutically acceptable additives. Optionally, the composition can include a combination of compounds described herein, or can include a second active ingredient useful for treating or preventing bacterial growth (e.g., but not limited to, an antibacterial or antimicrobial agent), or can include a combination of reagents of the present disclosure.

[0292] Suitable additives include, for example, carrier molecules including large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary additives can include antioxidants (for example, but not limited to, ascorbic acid), chelating agents (for example, but not limited to, EDTA), carbohydrates (for example, but not limited to, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example, and without limitation, oil, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

[0293] delivery The guide RNA polynucleotide (RNA or DNA) and / or endonuclease polynucleotide(s) (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, the endonuclease polypeptide(s) can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In further alternative embodiments, the DNA endonuclease can be delivered as one or more polypeptides, alone or pre-complexed with one or more guide RNAs or one or more crRNAs together with tracrRNA.

[0294] Polynucleotide can be delivered by non-viral delivery vehicle, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18:1127-1133 (2011) (which focuses on the non-viral delivery vehicle for siRNA, which is also useful for the delivery of other polynucleotides).

[0295] For polynucleotides of the present disclosure, formulations may be selected from, for example, any of those taught in International Application PCT / US2012 / 069610.

[0296] Polynucleotides, such as guide RNAs, sgRNAs, and mRNAs encoding endonucleases, may be delivered to cells or patients by lipid nanoparticles (LNPs).

[0297] LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, nanoparticles may range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm.

[0298] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the membrane-fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as "helper" lipids to enhance transfection activity and nanoparticle stability. The limitations of cationic lipids include poor stability and low efficacy due to rapid clearance, as well as the occurrence of inflammatory or anti-inflammatory responses.

[0299] LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0300] Any lipid or lipid combination known in the art can be used to generate LNPs. Examples of lipids used to generate LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of natural lipids are DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0301] Lipids can be combined in any molar ratio to produce LNPs. In addition, polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce LNPs.

[0302] As mentioned above, the site-specific polypeptide and the genome-targeting nucleic acid can be administered separately to cells or patients. Alternatively, the site-specific polypeptide can be pre-complexed with one or more guide RNAs or one or more crRNAs together with the tracrRNA. The pre-complexed material can then be administered to cells or patients. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

[0303] RNA can interact specifically with either RNA or DNA. This property is utilized in many biological processes, but there is also a risk of indiscriminate interactions in the nucleic acid-rich cellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which RNA is pre-complexed with endonucleases. Another advantage of RNPs is that they protect the RNA from degradation.

[0304] The endonuclease in RNP can be modified or unmodified.Similarly, gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.Many modifications are known in the art and can be used.

[0305] The endonuclease and sgRNA can be combined in a molar ratio of typically 1:1. Alternatively, the endonuclease, crRNA, and tracrRNA can be combined in a molar ratio of typically 1:1:1. However, a wide range of molar ratios can be used to generate RNPs.

[0306] AAV (adeno-associated virus) Recombinant adeno-associated virus (AAV) vectors can be used for delivery. Techniques for generating rAAV particles, which provide cells with a packaged AAV genome containing the polynucleotide to be delivered, the rep and cap genes, and helper virus functions, are standard in the art. rAAV production typically requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separate from (i.e., not within) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype capable of deriving recombinant virus, and may be from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, the AAV serotypes described herein. The production of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication WO 01 / 83692.

[0307] AAV serotype AAV particles of the present disclosure packaging compositions of the present disclosure, e.g., polynucleotides encoding endonucleases, donor sequences, or RNA guide molecules, can comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, AAV particles can include, but are not limited to, AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.40, AAV12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.11, AAV16.12 / hu.11, AAV16.13 / hu.12, AAV16.14 / hu.13, AAV16.15 / hu.15, AAV16.16 / hu.16, AAV16.17 / hu.17, AAV16.18 / hu.18, AAV16.19 / hu.19, AAV16.20 / hu.20, AAV16.21 / hu.21, AAV16.22 / hu.22, AAV16.23 / hu.23, AAV16.24 / hu.24, AAV16.25 / hu.25, AAV16.26 / hu.26, AAV16.27 / hu.27, AAV16.28 / hu.28, AAV16.29 / hu.29, AAV16.30 / hu.30, AAV16.31 / hu.31, AAV16.32 / hu.32, AAV1 .3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2, AAV2.5T, AAV2-15 / rh.62, AAV223.1 , AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV27.3, AAV29.3 / b b.1, AAV29.5 / bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-11 / rh.53, AAV3-3, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AA V42-13, AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1 , AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29、AAV4-8 / r11.64、AAV4-8 / rh.64、AAV4-9 / rh.54、AAV5、AAV52.1 / hu.20、AAV52 / hu.19、AAV5-22 / rh.58、AAV5-3 / rh.57、AAV54.1 / hu.21、AAV54.2 / hu.22、AAV54.4R / hu.27、AAV54.5 / hu.23、AAV54.7 / hu.24、AAV58.2 / hu.25、AAV6、AAV6.1、AAV6.1.2、AAV6.2、AAV7、AAV7.2、AAV7.3 / hu.7、AAV8、AAV-8 b、AAV-8h、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV3.3、AAV3.4、AAV3.5、AAV3.7 、AAV-b、AAVC1、AAVC2、AAVC5、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVcy. V-DJ、AAV-DJ8、AAVF3、AAVF5、AAV-h、AAVH-1 / hu.1、AAVH2、AAVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhER1.14、AAVhEr1.16、AAVhEr1.18、AAVhER1.23、AAVh Er1.35、AAVhEr1.36、AAVhEr1.5、AAVhEr1.7、AAVhEr1.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr2.4、AAVhEr3.1、AA Vhu.1、AAVhu.10、AAVhu.11、AAVhu.11、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.19、AAVhu.2、AAVhu.20、 AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.3、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.4、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46 、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.5、AAVhu.51、AAVhu.52、AAVhu.53、AAVhu.54、AAVhu.55、AAVhu.56、AA Vhu.57、AAVhu.58、AAVhu.6、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.7、AAVhu.8、AAVhu.9、AAVhu.t19、AAVLG-10 / rh. 40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVLG-9 / hu.39、AAV-LK01、AAV-LK02、AAVLK03、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、 AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK17、AAV-LK18、AAV-LK19、AAVN721-8 / rh.43、AAV-PAEC、AAV-PAEC1 1、AAV-PAEC12、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAVpi.1、AAVpi.2、AAVpi.3、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVpi.1 AVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.2、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.2R、AAVrh.31、AAVrh.32、 AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.43、AAVrh.44、AAVrh.45、AAVrh.46、AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh .51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAV rh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh. 68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R The vector may utilize or be based on a serotype selected from any of the serotypes including the A586R mutant, AAVrh8R R533A mutant, BAAV, BNP61AAV, BNP62AAV, BNP63AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV10, AAV-LK16, AAAV, AAV Shuffle100-1, AAV Shuffle100-2, AAV Shuffle100-3, AAV Shuffle100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAV SM100-10, AAV SM100-3, AAV SM10-1, AAV SM10-2, and / or AAV SM10-8, and variants thereof.

[0308] In some embodiments, as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011)), the AAV serotype may be or may have a variant of the AAV9 sequence, such as, but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

[0309] In some aspects, the AAV serotype may be or have a sequence as described in U.S. Patent No. 6,156,303, such as, but not limited to, AAV3B (SEQ ID NOs: 1 and 10 of U.S. Patent No. 6,156,303), AAV6 (SEQ ID NOs: 2, 7, and 11 of U.S. Patent No. 6,156,303), AAV2 (SEQ ID NOs: 3 and 8 of U.S. Patent No. 6,156,303), AAV3A (SEQ ID NOs: 4 and 9 of U.S. Patent No. 6,156,303), or a derivative thereof.

[0310] In some embodiments, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12):5887-5911 (2008)). The amino acid sequence of AAVDJ8 may contain two or more mutations to remove the heparin-binding domain (HBD). As a non-limiting example, the AAV-DJ sequence set forth as SEQ ID NO: 1 in U.S. Patent No. 7,588,772 may contain two mutations: (1) R587Q, in which arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (2) R590T, in which arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). Another non-limiting example may include three mutations: (1) K406R, in which lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q, in which arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, in which arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0311] In some embodiments, the AAV serotype may be or have a sequence as described in International Publication WO2015121501, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 in WO2015121501), "UPenn AAV10" (SEQ ID NO: 8 in WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 in WO2015121501), or a variant thereof.

[0312] According to the present disclosure, the selection or use of AAV capsid serotypes can be from various species. In one example, the AAV can be avian AAV (AAAV). The AAAV serotype can be or have a sequence as described in U.S. Patent No. 9,238,800, such as, but not limited to, AAAV (SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Patent No. 9,238,800), or a variant thereof.

[0313] In one example, the AAV may be bovine AAV (BAAV). The BAAV serotype may be or have a sequence as described in U.S. Patent No. 9,193,769, such as, but not limited to, BAAV (SEQ ID NOS: 1 and 6 in U.S. Patent No. 9,193,769), or a variant thereof. The BAAV serotype may be or have a sequence as described in U.S. Patent No. 7,427,396, such as, but not limited to, BAAV (SEQ ID NOS: 5 and 6 in U.S. Patent No. 7,427,396), or a variant thereof.

[0314] In one example, the AAV may be a caprine AAV. The caprine AAV serotype may be or have a sequence as described in U.S. Patent No. 7,427,396, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Patent No. 7,427,396), or a variant thereof.

[0315] In other examples, the AAV may be engineered as a hybrid AAV derived from two or more parent serotypes. In one example, the AAV may be AAV2G9, which contains sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be or have sequences as described in U.S. Patent Application Publication No. 20160017005.

[0316] In one example, the AAV may be a serotype generated by an AAV9 capsid library with mutations at amino acids 390-627 (VP1 numbering) as described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011). Serotypes and corresponding nucleotide and amino acid substitutions include, but are not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F41 7S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1 500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C; 667C;D556A), AAV9.34(A1534G, C1794T;N512D), AAV9.35(A1289T, T1450A, C1494T, A1515T, C1794A, G1816A;Q430L, Y484N, N98K, V606I), AAV 9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G13 01A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54(C1531A, T1609A; L511I, L537M ), AAV9.55(T1605A;F535L), AAV9.58(C1475T, C1579A;T492I, H527N), AAV.59(T1336C;Y446H), AAV 9.61(A1493T;N498I), AAV9.64(C1531A, A1617T;L511I), AAV9.65(C1335T, T1530C, C1568A;A523D) , AAV9.68(C1510A;P504T), AAV9.80(G1441A,;G481R), AAV9.83(C1402A, A1500T;P468T, E500D), AA V9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806 C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

[0317] In one example, the AAV may be a serotype that includes at least one AAV capsid CD8+ T-cell epitope. By way of non-limiting example, the serotype may be AAV1, AAV2, or AAV8.

[0318] In one example, the AAV may be a variant, such as PHP.A or PHP.B, as described in Deverman. 2016. Nature Biotechnology. 34(2):204-209.

[0319] In one example, the AAV may be a serotype selected from any of those found in SEQ ID NOs: 4734-5302 and Table 2.

[0320] In one example, the AAV may be encoded by a sequence, fragment or variant as disclosed in SEQ ID NOs: 4734-5302 and Table 2.

[0321] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches have been proposed such as Ratschin et al.,Mol.Cell.Biol.4:2072(1984);Hermonat et al.,Proc.Natl.Acad.Sci.USA,81:6466(1984);Tratschin et al.,Mo1.Cell.Biol.5:3251(1985);McLaughlin et al. al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988). Samulski et al. al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

[0322] AAV vector serotypes can be matched to the target cell type. For example, the following exemplary cell types can be transduced, among others, by the AAV serotypes indicated:

[0323] [Table 2]

[0324] In addition to adeno-associated viral vectors, other viral vectors can be used, including, but not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpesviruses, Epstein-Barr virus, papovaviruses, poxviruses, vaccinia viruses, and herpes simplex viruses.

[0325] In some embodiments, the Cas9 mRNA, the sgRNA targeting one or two sites in the SCN9A gene, and the donor DNA can each be formulated separately into lipid nanoparticles or co-formulated into a single lipid nanoparticle.

[0326] In some embodiments, the Cas9 mRNA can be formulated in lipid nanoparticles, while the sgRNA and donor DNA can be delivered in an AAV vector.

[0327] Options are available for delivering Cas9 nuclease as a DNA plasmid, mRNA, or protein. Guide RNA can be expressed from the same DNA or delivered as RNA. RNA can be chemically modified to alter or improve half-life or reduce the likelihood or severity of an immune response. The endonuclease protein can be complexed with the gRNA before delivery. Viral vectors allow for efficient delivery; split versions of Cas9 and smaller orthologs of Cas9 can be packaged into AAV, as can donors for HDR. Various non-viral delivery methods exist that can deliver each of these components, or non-viral and viral methods can be used together. For example, nanoparticles can be used to deliver proteins and guide RNAs, while AAV can be used to deliver donor DNA.

[0328] In some embodiments of the in vivo-based therapies described herein, viral vector(s) encoding the endonuclease, guide RNA and / or donor DNA may be delivered to neurons of the peripheral nervous system, e.g., primary sensory and motor neurons, via direct intraganglionic or intrathecal injection or intrathecal delivery (Hoyng et al., Front Mol Neurosci. 2015 Jul 15;8:32).

[0329] IV. Medication and Administration The terms "administering," "introducing," and "transplanting" are used interchangeably in the context of placing cells, e.g., progenitor cells, in a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, e.g., a site of injury or repair, so that a desired effect(s) occurs. Cells, e.g., progenitor cells, or their differentiated progeny, can be administered by any suitable route that results in delivery to a desired location within a subject, while keeping at least some of the transplanted cells or components of the cells alive. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to a few days, to as long as several years, or even the lifespan of the patient, i.e., long-term engraftment. For example, in some embodiments described herein, an effective amount of progenitor cells is administered via a systemic route of administration, e.g., intraperitoneal or intravenous.

[0330] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0331] When provided prophylactically, the progenitor cells described herein can be administered to a subject prior to any symptoms of pain. Thus, prophylactic administration of the progenitor cell population serves to prevent pain.

[0332] The progenitor cell population administered by the methods described herein can include allogeneic progenitor cells obtained from one or more donors. Such progenitor cells can be of any cell or tissue origin, such as liver, muscle, or heart. "Allogeneic" refers to a single progenitor cell or a biological sample containing progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical. For example, a liver progenitor cell population administered to a subject can be derived from one or more unrelated donor subjects or from one or more non-identical siblings. In some cases, a syngeneic progenitor cell population can be used, such as one obtained from genetically identical animals or identical twins. The progenitor cells can be autologous; that is, the progenitor cells are harvested or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same.

[0333] The term "effective amount" refers to the amount of a population of progenitor cells or their progeny necessary to prevent or alleviate at least one or more signs or symptoms of pain, and relates to the amount of a composition sufficient to achieve a desired effect, for example, to treat a subject with pain. Thus, the term "therapeutically effective amount" refers to the amount of progenitor cells or a composition containing progenitor cells sufficient to promote a particular effect when administered to a typical subject, for example, a subject with or at risk of pain. An effective amount would also include an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (for example, but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It is understood that in any given case, one skilled in the art can determine the appropriate "effective amount" using routine experimentation.

[0334] For use in the various embodiments described herein, an effective amount of progenitor cells is at least 10 2 progenitor cells, at least 5 x 10 2 progenitor cells, at least 10 3 progenitor cells, at least 5 x 10 3progenitor cells, at least 10 4 progenitor cells, at least 5 x 10 4 progenitor cells, at least 10 5 progenitor cells, at least 2 x 10 5 progenitor cells, at least 3 x 10 5 progenitor cells, at least 4 x 10 5 progenitor cells, at least 5 x 10 5 progenitor cells, at least 6 x 10 5 progenitor cells, at least 7 x 10 5 progenitor cells, at least 8 x 10 5 progenitor cells, at least 9 x 10 5 progenitor cells, at least 1 x 10 6 progenitor cells, at least 2 x 10 6 progenitor cells, at least 3 x 10 6 progenitor cells, at least 4 x 10 6 progenitor cells, at least 5 x 10 6 progenitor cells, at least 6 x 10 6 progenitor cells, at least 7 x 10 6 progenitor cells, at least 8 x 10 6 progenitor cells, at least 9 x 10 6 The progenitor cells may be derived from one or more donors or from an autologous source. In some examples described herein, the progenitor cells may be expanded in culture before being administered to a subject in need thereof.

[0335] A small, incremental reduction in the level of SCN9A expressed in cells of a patient suffering from pain may be advantageous for ameliorating one or more symptoms of the disease, increasing long-term survival, and / or reducing side effects associated with other treatments. Upon administration of such cells to a human patient, the presence of progenitor cells with reduced levels of SCN9A is advantageous. In some cases, effective treatment of a subject results in a reduction of SCN9A by at least about 3%, 5%, or 7% relative to total SCN9A in the treated subject. In some cases, the reduction in SCN9A is at least about 10% of total SCN9A. In some cases, the reduction in SCN9A is at least about 20%-30% of total SCN9A. Similarly, in some situations, the introduction of a more limited subpopulation of cells with significantly reduced levels of SCN9A may be advantageous in various patients, as normalized cells may have a selective advantage over diseased cells. However, progenitor cells with moderately reduced levels of SCN9A may also be advantageous for ameliorating one or more aspects of a patient's pain. In some examples, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the progenitor cells in a patient receiving such cells have reduced levels of SCN9A.

[0336] "Administered" refers to the delivery of a progenitor cell composition to a subject by a method or route that results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in the subject, i.e., administration results in delivery to a desired location in the subject, where at least a portion of the delivered composition, i.e., at least 1 x 10 4 The cells are delivered to the desired site over a period of time.

[0337] In one aspect of the method, the pharmaceutical composition is administered by any of a variety of routes, including but not limited to, enteral (into the small intestine), gastrointestinal, epidural (into the dura mater), oral (by mouth), transdermal, epidural, intracerebral (into the cerebrum), intraventricular (into the ventricles of the brain), epicutaneous (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal (through the nose), intravenous (into a vein), intravenous bolus, intravenous infusion, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous injection (into bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavity injection (into a diseased cavity), intracavitary (into the base of the penis), intravenous (into the rectum ... penis), intravaginal administration, intrauterine administration, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (snorting), sublingual, sublabial, enema, ophthalmic (on the conjunctiva), eardrop, auricular (into or by the ear canal), buccal (against the cheek), conjunctival, dermal, dental (into the teeth), electroosmotic, intracervical, intravenous sinus, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrasynovial, intrachondral (inside the cartilage), intracauda equina (inside the cauda equina), intracisternal (inside the cisterna magna cerebellomedularis), intracorneal (inside the cornea), intracoronaryintracornal), intracoronary (inside the coronary arteries), intracavernosal (inside the distensible spaces of the corpora cavernosa of the penis), intradiscal (inside the intervertebral disc), intraductal (inside the glandular ducts), intraduodenal (inside the duodenum), intradural (inside or beneath the dura), intraepidermal (against the epidermis), intraesophageal (against the esophagus), intragastric (inside the stomach), intragingival (inside the gums), intraileal (inside the distal small intestine), intralesional (introduced directly into or into a localized lesion), intraluminal (inside the lumen), intralymphatic (inside the lymph), intramedullary (inside the bone marrow cavity) , intrameningeal (inside the meninges), intraocular (inside the eye), intraovarian (inside the ovaries), intrapericardial (inside the pericardium), intrapleural (inside the pleura), intraprostatic (inside the prostate), intrapulmonary (inside the lungs or their bronchi), intrasinus (inside the nasal or periorbital sinuses), intrathecal (inside the spinal column), intrasynovial (inside the synovial cavities of a joint), intratendon (inside tendons), intratesticular (inside the testicles), intrathecal (inside the cerebrospinal fluid at any level of the neuraxis), intrathoracic (inside the chest), intraductal (inside organ tubules), intratumoral (inside a tumor), intratympanic (inside the middle ear media), intravascular (inside one or more blood vessels), intraventricular (inside the ventricles of the brain), iontophoresis (using an electric current, in which ions of soluble salts are moved into body tissues), irrigation (to bathe or flush an open wound or body cavity), laryngeal (directly onto the larynx), nasogastric (through the nose into the stomach), occlusive dressing therapy (topical administration, then covering the area with a bandage to occlude), ocular (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, transdermal, periarticular, peridural, perineural, periodontal, rectal, respiratory (local Administration may be via routes such as intrapulmonary (inside the respiratory tract), retrobulbar (behind the pons or behind the eyeball), intramyocardial (inside the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transcutaneous intratracheal (through the tracheal wall), transtympanic (through or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), intravaginal, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and spinal.

[0338] Modes of administration include injection, infusion, infusion, and / or oral ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, percutaneous intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, intracerebrospinal, and intrasternal injection and infusion. In some cases, the route is intravenous. Cell delivery can be administered by injection or infusion.

[0339] The cells can be administered systemically. The phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to administration of a population of progenitor cells other than directly to a target site, tissue, or organ, but instead such that they enter the subject's circulatory system and are therefore subject to metabolic and other similar processes.

[0340] The effectiveness of a treatment, including a composition for treating pain, can be determined by a skilled clinician. However, a treatment is considered "effective" if any or all of the signs or symptoms of SCN9A, but by way of example only, their levels are favorably altered (e.g., reduced by at least 10%) or other clinically recognized symptoms or markers of the disease are improved or ameliorated. Efficacy can also be measured by the absence of deterioration of the individual (e.g., the halting or at least slowing of disease progression) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease, e.g., halting or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptoms occurring.

[0341] Treatment according to the present disclosure can ameliorate one or more symptoms associated with pain by reducing or altering the amount of SCN9A protein in an individual.

[0342] V. Characteristics and properties of the voltage-gated sodium channel type IX alpha subunit (SCN9A) gene SCN9A is a neurotransmitter that is used to treat diseases and disorders such as, but not limited to, congenital analgesia, anosmia, schizophrenic personality, borderline personality disorder, malignant breast neoplasm, non-small cell lung cancer, cold intolerance, heat convulsions, diabetes, diabetes mellitus, dissociative disorders, epilepsy, erythromyelitis, primary erythromyelitis, facial pain, herpes virus infection, hereditary sensory neuropathy type 5, hyperplasia, neuralgia, hereditary sensory and autonomic neuropathy, degenerative polyarthritis, pain, limb pain, post-surgical pain, Parkinson's disease, post-herpetic neuralgia, prostate neoplasm, pruritus, seizures, somatoform disorders, smoking disorders, trigeminal neuralgia, synovial cysts, chronic pain, acute onset pain, congenital paramyotonia, discomfort, sensory discomfort SCN9A has been associated with: burning pain, insensitivity to pain, inflammatory pain, mechanical pain, scalp pain, hereditary motor and sensory neuropathy type II, common migraine, lack of pain sensation, malignant prostate neoplasm, pain disorders, knee osteoarthritis, neuropathy, complex regional pain syndrome, tonic-clonic seizures, hereditary neuropathy, prostate cancer, breast cancer, severe myoclonic epilepsy of infancy, myxoid cysts, channelopathy, paroxysmal severe pain disorder, painful neuropathy, compression neuropathy, autosomal recessive congenital insensitivity to pain, generalized epilepsy with febrile seizures plus type 2, generalized epilepsy with febrile seizures plus 7, febrile seizures familial 3B, and small fiber neuropathy (the adult-onset form is referred to as small fiber neuropathy). Editing the SCN9A gene using any of the methods described herein may be used to treat, prevent, and / or alleviate the symptoms of the diseases and disorders described herein.

[0343] The SCN9A gene encodes the alpha subunit of a sodium channel designated NaV1.7. NaV1.7 is primarily expressed in sensory neurons and plays an important role in nociceptive signal transduction. Mutations in the SCN9A gene are known to result in pain perception disorders, including primary erythromelalgia, paroxysmal severe pain disorder, congenital insensitivity to pain, and small fiber neuropathy. Gain-of-function mutations in the SCN9A gene result in spontaneous pain, as observed in primary erythromelalgia and paroxysmal severe pain disorder. Therefore, knockout or knockdown of the SCN9A gene in patients with primary erythromelalgia or paroxysmal severe pain disorder can be used to treat, prevent, and / or alleviate associated symptoms.

[0344] Primary erythromelalgia is a rare autosomal dominant disorder characterized by episodes of burning pain in the hands and feet in response to heat and movement. Affected individuals typically develop signs and symptoms in early childhood, although in milder cases, symptoms may appear later in life. Management of the condition is primarily symptomatic. Besides avoidance of pain triggers (such as heat, movement, and alcohol), treatment options include cooling and elevating the extremities, the use of anesthetics such as lidocaine and mexilitine, and, in extreme cases, the use of opioid medications.

[0345] Paroxysmal acute pain disorder is another rare disorder characterized by severe episodic pain in the rectum, eyes, and mandible area, as well as reddening of the skin. Symptoms of this condition often begin in the neonatal period or early childhood and can persist throughout life. Medications for treating chronic neuropathic pain disorders are often used to alleviate pain episodes caused by the disease. Carbamazepine, a sodium channel blocker, has proven to be the most effective for these treatments.

[0346] In one example, the gene is voltage-gated sodium channel type IX alpha subunit (SCN9A), which is also referred to as sodium voltage-gated channel alpha subunit 9, sodium channel voltage-gated type IX alpha polypeptide, voltage-gated sodium channel subunit alpha Nav1.7, sodium channel protein type IX subunit alpha, neuroendocrine sodium channel, peripheral sodium channel 1, HNE-Na, NENA, PN1, GEFSP7, HSAN2D, Nav1.7, FEB3B, ETHA, and SFNP. SCN9A is located at cytogenetic location 2q24.3, with genomic coordinates from 166,195,185 to 166,375,993 on chromosome 2 on the forward strand. The nucleotide sequence of SCN9A is set forth as SEQ ID NO: 5303. SCN7A is a gene upstream of SCN9A on the reverse strand, and RN7SKP152 is a gene downstream of SCN9A on the reverse strand. AC010127.3 is a gene located on the opposite forward strand of SCN9A. SCN9A has an NCBI gene ID of 6335, a Uniprot ID of Q15858, and an Ensembl gene ID of ENSG00000169432. SCN9A has 3906 SNPs, 39 introns, and 38 exons. The Ensembl exon identifiers and the start / stop sites of the introns and exons are shown in Table 3. [Table 3-1] [Table 3-2]

[0347] Table 4 provides information on all transcripts of the SCN9A gene based on the Ensembl database. Table 4 provides the Ensembl transcript ID and the corresponding NCBI RefSeq ID for the transcript, the Ensembl translation ID and the corresponding NCBI RefSeq ID for the protein, the biotype of the transcript sequence as classified by Ensembl, and the exons and introns of the transcript based on the information in Table 3. [Table 4]

[0348] SCN9A has 3906 SNPs, and the NCBI rs numbers and / or Uniprot VAR numbers for this SCN9A gene are VAR_019947, VAR_019948, VAR_019949, VAR_019950, VAR_030444, VAR_032014, VAR_032015, VAR_032016, VAR_032017, VAR_032018, VAR_032019, VAR_032020, VAR_032021, VAR_032022, VAR_032023, VAR_064595, and VAR_064596. , VAR_064597, VAR_064598, rs71428908, VAR_064600, VAR_064601, VAR_064602, VAR_064603, VAR_064604, VAR_064605, VAR_064606, VAR_064607, VAR _064608, VAR_064609, VAR_064610, VAR_064611, VAR_064612, VAR_064613, VAR_072279, VAR_072280, VAR_072280, VAR_072281, rs951510, rs952462, rs952463, rs1011778, rs1358532, rs1406272, rs1540870, rs1540871, rs1540875, rs1919177, rs1881436, rs1881437, rs1881438, rs1881439, rs199 7291, rs1528481, rs1528483, rs1528484, rs1528487, rs2893013, rs4131159, rs4131160, rs4131632, rs4488677, rs4408747, rs4331519, rs4429487, rs4001001, rs4273234, rs4583483, rs4438497, rs4286289, rs4546021, rs4455168, rs4455169, rs4605385, rs4465779, rs4384809, rs4386335, rs466 7512, rs5836099, rs6432898, rs6432899, rs6432900, rs6432901, rs6432902, rs6432903, rs6432904, rs6432905, rs6432906, rs6432907, rs6432910,<h2 style=";text-align:left;direction:ltr">rs6723900、rs6720769、rs6715214、rs6712015、rs6712019、rs6708450、rs6708715、rs6725355、rs6725732、rs6722807、rs6716736、rs6723160、rs67 26575、rs6760472、rs6723789、rs6728885、rs6738419、rs6729030、rs6732627、rs6719276、rs6729980、rs6738102、rs6750593、rs6756635、rs671892 2、rs6718791、rs7576631、rs7569509、rs7563366、rs6708467、rs6715470、rs6757555、rs6731773、rs6715367、rs7577446、rs6732135、rs6714902、rs 7570862、rs6746587、rs6721003、rs7558866、rs6432909、rs7584766、rs7589477、rs7584892、rs7559171、rs6432908、rs7590179、rs7572553、rs7566 965, rs7602898, rs6432897, rs6432896, rs7580299, rs7603335, rs7597876, rs4667883, rs4667882, rs7582061, rs9287866, rs5836096, rs10171225 、rs4564789、rs7594979、rs4459751、rs7606521、rs4599137、rs9646771、r s4076255、rs4447616、rs4443015、rs10188002、rs3956542、rs4132348、rs 4132347、rs4131162、rs4131161、rs1528486、rs1528485、rs1881440、rs16 09311、rs1540876、rs1540874、rs10930216、rs10930217、rs10930218、rs1 0930219、rs1540873、rs1540872、rs6728894、rs11280107、rs11686478、rs 7424841、rs12105034、rs13000874、rs6734919、rs11674528、rs12692794、rs12692795、rs6753017、rs6744871、rs6736291、rs11340941、rs12478446、rs6757314、rs13004059、rs11693091、rs6757502、rs6737120、rs6732242、rs12463992、rs12612992、rs12464575、rs13417859、rs13395456、rs12615462、rs6746615、rs7420705、rs13027418、rs12616699、rs12467272、rs11898284、rs7589743、rs16851931、rs7597083、rs7597178、rs16851958、rs16851960、rs7598695、rs7592978、rs16851968、rs9759544、rs7606196、rs16852023、rs16852031、rs16852043、rs16852048、rs16852054、rs16852069、rs13030011、rs11899120、rs13013237、rs12469343、rs7606274、rs13401294、rs12992260、rs10190952、rs9646772、rs7608288、rs10193767、rs10538478、rs12994338、rs13016545、rs12994880、rs13405544、rs13034090、rs13017469、rs10556841、rs13017637、rs11888456、rs10600584、rs13035164、rs10611611、rs10634344、rs12473416、rs13431341、rs13020056、rs10638743、rs13021236、rs12999243、rs13000233、rs13000683、rs10655845、rs13387119、rs11435802、rs35244507、rs10688081、rs10696005、rs10710576、rs11463044、rs35280187、rs10497283、rs34327610、rs13390803、rs34920657、rs10497284、rs34355606、rs35730267、rs34926643、rs35333199、rs34936668、rs36101458、rs12476306、rs12105169、rs58078583、rs12692796、rs58159440、rs41268677、rs11890824、rs58184464、rs35789805、rs56058874、rs35353363、rs34490209、rs35821032、rs35360495、rs34449821、rs35425598、rs56999748、rs11681893、rs35473199、rs11682860、rs11676630、rs11693152、rs13004690、rs12615972、rs34617014、rs13026637、rs56149857、rs16851928、rs35911982、rs16851935、rs16851943、rs55681559、rs16851964、rs16851966、rs35542360、rs16851974、rs16852010、rs11885693、rs17766038、rs17766243、rs17766561、rs17766807、rs11901118、rs13426370、rs57275972、rs12993435、rs13427940、rs12472033、rs12622435、rs12622743、rs35187227、rs13035599、rs36035794、rs12996068、rs13021074、rs34784693、rs60461278、rs59265133、rs56282252、rs36096260、rs59348143、rs56292816、rs16826112、rs35257630、rs34813283、rs55945526、rs13411967、rs60930703、rs67175205、rs73025509、rs73025522、rs73025523、rs73025528、rs28532836、rs73025590、rs35683510、rs13391197、rs62176964、rs62176965、rs55989063、rs62178525、rs35433129、rs33999505、rs34533574、rs71428911、rs71428912、rs71428915、rs71428916、rs71428917、<h2 style=";text-align:left;direction:ltr">rs71428918、rs71428919、rs60050530、rs71428910、rs34038642、rs72149794、rs35077026、rs35080467、rs34582751、rs57073619、rs57087440、rs3 4082367、rs73972306、rs73972316、rs73972318、rs74336612、rs75477543、rs56152529、rs76673713、rs71031239、rs34099849、rs34101964、rs3512 9718, rs76710665, rs76711511, rs79030107, rs74445522, rs79031716, rs74449889, rs75597232, rs76740831, rs75608447, rs34132334, rs3464919 5、rs35552412、rs77937563、rs72882854、rs72882859、rs72882882、rs728 82899、rs72882901、rs72884703、rs17766014、rs17766982、rs72884732、r s72884741、rs72884745、rs72884753、rs72884762、rs72884775、rs72884780、rs72884785、rs72884786、rs35962214、rs72884797、rs72884798、rs17 817307、rs72884802、rs75675344、rs72886606、rs72886608、rs17817547、 rs79140856、rs79147031、rs17817679、rs80356466、rs35182375、rs80356 469, rs35595054, rs55754250, rs75773428, rs60310134, rs60392208, rs36073771, rs71395218, rs115848509, rs66472972, rs79202357, rs7579416 8、rs57861261、rs71405933、rs73025538、rs112373468、rs62176960、rs75818857、rs62176961、rs67339035、rs62178526、rs115911128、rs75848558、rs60056351、rs71428914、rs115946138、rs74706664、rs113603623、rs111285706、rs111302354、rs77046324、rs60145586、rs113610968、rs72330491、rs112490413、rs739、 69684、rs112504933、rs111337704、rs74757084、rs116013127、rs73969685、rs74768783、rs73969700、rs116037918、rs73969701、rs72520704、rs74385849、rs71031240、rs79417925、rs74791755、rs71031242、rs74423851、rs75620229、rs79061212、rs75645300、rs113739900、rs72884720、rs113758466、rs72884726、rs72884789、rs72884800、rs78215125、rs75709114、rs77151034、rs77154470、rs111426485、rs80356465、rs80356468、rs114760918、rs80356470、rs114985967、rs79187718、rs74613934、rs112644902、rs76904702、rs111528010、rs78278362、rs115847294、rs112347596、rs115028579、rs113518536、rs78304745、rs79221441、rs79222523、rs112718465、rs115074698、rs113962992、rs111624869、rs75818591、rs115099720、rs74658674、rs75006765、rs115881388、rs113988866、rs114811473、rs111281728、rs111679051、rs75029843、rs112761603、rs78428931、rs112768548、rs113629537、rs111705022、rs111705421、rs75053746、rs78118515、rs79645790、rs115979359、rs113660887、rs114893976、rs78162977、rs114048544、rs115193623、rs75093813、rs78510947、rs77373314、rs76244722、rs112835924、rs76247525、rs113698317、rs78552930、rs74778123、rs116048770、rs116373723、rs75949647、rs114920745、rs116069340、rs112869318、rs114923155、rs114926041、rs115322950、rs113737673、rs112561342、rs77143068、rs79446427、rs111404258、rs112919255、rs111415017、rs75225428、rs111458233、rs112945476、rs111470787、rs79493055、rs116451742、rs111899000、rs77487847、rs113850984、rs77192374、rs76448913、rs78250271、rs116188445、rs112675405、rs111948101、rs111953170、rs77231154、rs114267271、rs115435826、rs79853207、rs77579497、rs113911089、rs78334485、rs79876905、rs76510000、rs76102017、rs116547783、rs115473061、rs111643968、rs115489522、rs77639503、rs116268225、rs111654843、rs113989468、rs114344033、rs77682648、rs115119378、rs77328118、rs112779076、rs78452939、rs113173376、rs76571042、rs115522370、rs114032727、rs78863850、rs113195803、rs78454756、rs116653005、rs77784194、rs77797040、rs78906046、rs75468572、rs80095598、rs138982989、rs138989210、rs114358903、rs113283978、rs113182934、rs114525222、rs115555277、rs75069209、rs79684957、rs115244856、rs113309940、rs113311364、rs80160529、rs115251943、rs115274753、rs112867239、rs114118902、rs139080215、rs114143095、rs116773598、rs148713442、rs142480223、rs77432282、rs140701967、rs148765803、rs145478117、rs79741845、rs142518463、rs139170417、rs77441037、rs75200610、rs148846057、rs145575900、rs116422705、rs111858722、rs116435828、rs116962683、rs75230883、rs148931877、rs112947029、rs121908920、rs121908921、rs140792426、rs115783892、rs113436493、rs111884740、rs139281380、rs139288692、rs112970706、rs115393048、rs112995109、rs75273355、rs77539813、rs115812636、rs142707672、rs78731163、rs184266839、rs140830160、rs111972606、rs149102734、rs78763917、rs116547537、rs114319629、rs145811912、rs184317901、rs77642761、rs149157378、rs115495114、rs79918973、rs142779717、rs149194222、rs112047115、rs181405366、rs76564908、rs78843438、rs181423521、rs184404793、rs80126387、rs149285180、rs137950241、rs184447464、rs149297295、rs78947176、rs117581394、rs187270227、rs139017042、rs116710246、rs149301325、rs142909834、rs140574148、rs137962506、rs190380742、rs137966449、rs116713443、rs149350025、rs112222195、rs184515932、rs116748713、rs142952951、rs139064023、rs181563231、rs181572301、rs181577442、rs78998616、rs145366755、rs113361139、rs115726491、rs184560681、rs184561606、rs148819190、rs190457189、rs142559100、rs149429956、rs143007711、rs142585699、rs114681048、rs140761356、rs145608272、rs121908916、rs142666506、rs187410330、rs115802383、rs187428818、rs149490896、rs149507189、rs113450348、rs149034908、rs146213117、rs145737444、rs184675901、rs146234445、rs181717210、rs184685668、rs181276399、rs187500785、rs145769973、rs190597640、rs181734340、rs184275120、rs143161505、rs146267489、rs149592596、rs190625704、rs115823295、rs141042794、rs138081618、rs190666356、rs190668591、rs184760546、rs149648597、rs145798468、rs184289118、rs142753783、rs187145433、rs181814822、rs146333890、rs187164514、rs187187436、rs181818571、rs181414054、rs184784994、rs181414423、rs181840211、rs187627632、rs145969336、rs187630476、rs187638933、rs181864169、rs117600380、rs143281561、rs149739106、rs187660096、rs184471275、rs139433712、rs143307775、rs187686954、rs143323637、rs146453073、rs184491945、rs184503606、rs149812467、rs187735078、rs181543382、rs187739615、rs149846122、rs181976860、rs181544216、rs184525265、rs187760599、rs190446880、rs137993592、rs184557454、rs182005122、rs184956974、rs184558307、rs184973913、rs184975345、rs190455017、rs146114242、rs182041228、rs149440243、rs190971448、rs139677117、rs138238059、rs143020225、rs187396655、rs149989224、rs191004585、rs187404611、rs150021077、rs146148893、rs182164896、rs182169962、rs149458240、rs187420437、rs187457184、rs141218614、rs146210131、rs146758972、rs185134458、rs187483230、rs187499307、rs138051487、rs139537793、rs190630868、rs150115235、rs149656478、rs191129855、rs187982120、rs182221863、rs184769972、rs181809487、rs141258921、rs146330125、rs184777485、rs139562000、rs188023656、rs150184169、rs139564447、rs191220059、rs146882468、rs184798240、rs187628994、rs143699917、rs143713899、rs184829398、rs143292594、rs188117229、rs143733438、rs141345692、rs188123311、rs188145250、rs146953750、rs141083667、rs143769263、rs181942663、rs146963548、rs149811195、rs190856490、rs191334405、rs190873215、rs191343221、rs139815430、rs181981693、rs191359321、rs149865252、rs150371036、rs143829355、<h2 style=";text-align:left;direction:ltr">rs191372613、rs138452077、rs138452908、rs185386849、rs143842609、rs188237643、rs150393847、rs187773100、rs187775085、rs184966574、rs184977393、rs1、<h2 style=";text-align:left;direction:ltr"> 91427726、rs188275827、rs182035257、rs184996404、rs141453198、rs188284141、rs190995816、rs138488726、rs139848912、rs182512669、rs191445085、rs146642805、rs191006637、rs182521362、rs188309132、rs150023650、rs191085791、rs185460522、rs185466137、rs182175100、rs191091185、rs191109954、rs185493398、rs146761679、rs182203285、rs185502977、rs150107947、rs191122461、rs139722857、rs141521157、rs185512688、rs188369589、rs191126523、rs139884364、rs139732025、rs143623645、rs138334413、rs185538321、rs182251259、rs138341036、rs182626281、rs191206839、rs182313512、rs141315817、rs147236226、rs144030757、rs141338230、rs182655507、rs191614297、rs150639101、rs143731425、rs188153399、rs144066296、rs141603539、rs150324876、rs141375532、rs185622259、rs191640401、rs146970406、rs188513575、rs141629021、rs150341781、rs191351429、rs141401072、rs188532495、rs188248808、rs147057677、rs188544520、rs191692191、rs144131152、rs147074129、rs182496397、rs191429109、rs182500442、rs141673661、rs139846473、rs150446261、rs150785198、rs182820808、rs182520122、rs182835951、rs182529061、rs188657481、rs182852687、rs138621998、rs182529589、rs185798678、rs150473901、rs141757967、rs182553426、rs185815125、rs143943780、rs185496131、rs150902480、rs185498433、rs191884045、rs185502994、rs185503388、rs188365371、rs188776686、rs185517165、rs141530060、rs188383084、rs191914888、rs191918328、rs191918870、rs150560390、rs191920350、rs144365987、rs144372216、rs191935835、rs188403481、rs188811649、rs185898649、rs147221067、rs182626458、rs150991753、rs144016113、rs191958942、rs150590478、rs183013605、rs182641899、rs183026897、rs139927718、rs144435180、rs144435267、rs140119151、rs188473580、rs147697530、rs139930314、rs151064878、rs150652684、rs185979948、rs185640009、rs140151023、rs188519035、rs183095706、rs188522328、rs150690124、rs191679645、rs183115017、rs183125824、rs183133746、rs186044551、rs141940569、rs151168288、rs138746989、rs191681855、rs144136503、rs150725477、rs191708188、rs191711647、rs182806094、rs144573884、rs147831105、rs147833108、rs147430160、rs185756419、rs188657198、rs141742493、rs144270467、rs138636241、rs186110761、rs186125436、rs151258913、rs150885548、rs192222550、rs192233893、rs147939855、rs183284329、rs147539362、rs185839773、rs188756672、rs138823585、rs188769302、rs140057840、rs183353183、rs188780390、rs183367774、rs188784746、rs185874593、rs188790411、rs192366053、rs188810319、rs188814235、rs189287946、rs182991290、rs144392389、rs147640034、rs151008517、rs192402484、rs183036253、rs138700080、rs138700579、rs183064984、rs185993526、rs189320567、rs183090065、rs192417863、rs142157838、rs180836507、rs186006161、rs148149379、rs144500679、rs151120735、rs180884797、rs180886733、rs186060853、rs144552092、rs189017951、rs189022051、rs186460536、rs140492487、rs186461495、rs189023255、rs186466472、rs141954437、rs189522976、rs189523541、rs189049999、rs148313223、rs151210240、rs189054543、rs147849719、rs189539871、rs189064184、rs189068219、rs192210846、rs147956569、rs186209759、rs189569164、rs192674981、rs144747253、rs186238424、rs183351491、rs183367424、rs192699758、rs183370069、rs189262728、rs192363212、rs183403867、rs192726587、rs142134626、rs183711149、rs142138406、rs181081805、rs192738555、rs142140153、rs200131264、rs140380560、rs181094108、rs200134765、rs144841509、rs183732115、rs192406551、rs142150794、rs148096531、rs200162971、rs186321392、rs370110583、rs148456006、rs192433693、rs186364672、rs186633344、rs180802944、rs200178927、rs200194515、rs375200578、rs200182914、rs145193907、rs183502133、rs140445008、rs180893354、rs138915544、rs145244774、rs148241180、rs186442256、rs200240989、rs186465363、rs148278257、rs145257168、rs189524843、rs148315085、rs183628282、rs189533865、rs183640056、rs148556202、rs200292819、rs192655074、rs192658579、rs189564784、rs138962140、rs148362057、rs142347131、rs189586446、rs200341902、rs200063079、rs370018897、rs200099565、rs192906198、rs183704773、rs192723631、rs181075520、rs186767700、rs181079869、rs370059723、rs186771296、rs529190860、rs370067909、rs529209086、rs186784826、rs200394559、rs200399998、rs183730817、rs538838362、rs370082676、rs200408344、rs142400656、rs200411606、rs145182185、rs145187060、rs186806270、rs200172456、rs192975309、rs375204316、rs370455867、rs200449501、rs186648437、rs534019932、rs529286738、rs375210239、rs370472054、rs192801395、rs370474533、rs186654635、rs200213811、rs186674692、rs538940239、rs375250531、rs200482081、rs375566600、rs181175263、rs200486515、rs370211696、rs181183562、rs181186118、rs193052322、rs200518320、rs200258203、rs186875141、rs181200412、rs375314088、rs184015802、rs375635737、rs186886737、rs529393841、rs375684756、rs539081952、rs200296022、rs189773617、rs375344768、rs375717078、rs189783847、rs193147774、rs189793333、rs189802138、rs375383262、rs200341995、rs148603098、rs370331208、rs183883370、rs190038995、rs190041054、rs200356611、rs192916635、rs148622242、rs539165024、rs534237861、rs189833651、rs200381002、rs539201442、rs529545926、rs190079309、rs529224329、rs538840251、rs200677133、rs538845547、rs370750101、rs534286923、rs375828071、rs200417967、rs539238807、rs184127871、rs375484262、rs190100237、rs200702592、rs538865759、rs200441726、rs370458490、rs375873835、rs370471482、rs370474188、rs534038802、rs534350762、rs534357040、rs200464271、rs184169321、rs193007084、rs529641451、rs529320212、rs375570049、rs529339341、rs538978492、rs193043836、rs200774110、rs189961043、rs200780217、rs534113220、rs370879581、rs534409097、rs534414126、rs534415144、rs370562444、rs200579922、rs199528742、rs200815709、rs534171106、 <h2 style=";text-align:left;direction:ltr">rs539098927、rs539117115、rs200830395、rs186937907、rs370939400、rs 539140140、rs200617539、rs200619065、rs184228905、rs200835066、rs37 6036522、rs539143083、rs200849406、rs539479843、rs376051261、rs529755984、rs534486080、rs534488673、rs193152357、rs375749001、rs3757630 51、rs193166864、rs539520802、rs529783271、rs200878465、rs534232221 、rs375776919、rs200884318、rs190061595、rs534278901、rs190082049、r s545837483、rs375825189、rs187135957、rs375828938、rs193224590、rs376111502、rs553880123、rs193232467、rs375856563、rs553890257、rs5345 45311, rs200709311, rs539583140, rs200720228, rs199625995, rs539285858, rs375886661, rs539314440, rs529638862, rs200757990, rs19329082 1、rs200764978、rs370859545、rs193298128、rs190162179、rs529872397、rs554016514、rs370874132、rs539661794、rs375980172、rs200809157、rs5 45964402、rs539677853、rs200826539、rs200972952、rs554071139、rs534456457、rs529727269、rs184226889、rs554100480、rs376022705、rs20083 1251、rs200999865、rs201000497、rs529730407、rs200843960、rs1842465 46、rs190358218、rs554163756、rs554165274、rs199576878、rs539500156、<h2 style=";text-align:left;direction:ltr">rs376288233、rs199589622、rs184260785、rs371207844、rs200882280、rs199599557、rs199756028、rs529983093、rs200885240、rs187133567、rs56 3464573、rs201061055、rs539547053、rs554273987、rs529802379、rs199609865、rs553889410、rs546154866、rs553889944、rs534551405、rs5539117 96、rs371042731、rs554309833、rs554312712、rs199784484、rs201087484 rs371264016、rs199791955、rs563543796、rs554341821、rs546194264、r s371271639、rs545893207、rs539907191、rs546211175、rs539907650、rs554376047、rs534785673、rs200925482、rs201111027、rs546225573、rs1902 83527, rs553954986, rs199824489, rs200939963, rs199828294, rs539633485, rs534590465, rs530084774, rs199836776, rs553992440, rs20094656 5、rs376437229、rs534834256、rs545955340、rs529878459、rs534839750、rs539669267、rs563692075、rs546307681、rs199679924、rs554081720、rs5 34850887、rs554088921、rs540043962、rs554092522、rs190336684、rs199 879584、rs530137975、rs200995410、rs530143022、rs371164212、rs19034 9718, rs546362979, rs199717009, rs371397880, rs540082625, rs201198963, rs201021546, rs563799556, rs199737519, rs371414170, rs554199061<h2 style=";text-align:left;direction:ltr">rs199746311、rs546391192、rs190379146、rs199753578、rs201051190、rs199757256、rs199915769、rs546428044、rs554663323、rs199924841、rs54 0148616、rs563477777、rs199933920、rs546146748、rs563898086、rs201243874、rs546456054、rs199940586、rs530215503、rs563913729、rs5302186 81、rs376579866、rs199948709、rs540192719、rs540198060、rs554750370 rs563492226rs554287627rs546498422rs546158464rs540216332r s554299241、rs199968973、rs546516362、rs563983733、rs199780289、rs199780984、rs371286659、rs371523166、rs530267604、rs199987389、rs5402 58346、rs534786295、rs546227682、rs535009588、rs371537833、rs546570808、rs546572548、rs534792758、rs554861011、rs530289862、rs53995506 7、rs199832245、rs199832681、rs199841742、rs554463637、rs535055981、 rs564125461、rs200028908、rs546625033、rs199848155、rs376692400、rs5 64139470、rs199850424、rs201155458、rs554499889、rs535082824、rs554501599、rs371367087、rs563737735、rs200056934、rs564203583、rs54634 0639、rs371630270、rs540400302、rs376746720、rs201185327、rs546352773、rs575423992、rs376493135、rs546717270、rs371397187、rs540431428、<h2 style=";text-align:left;direction:ltr">rs534892966、rs201205411、rs199903431、rs376515297、rs563817371、rs563824187、rs546746556、rs555098627、rs575492002、rs201217172、rs37 6529993, rs554686400, rs546760948, rs534939379, rs563953797, rs540206408, rs546500427, rs540485824, rs376608116, rs554799633, rs5405056 12、rs201445594、rs546799753、rs540240449、rs534999520、rs540519101 rs564030716、rs376835362、rs530454237、rs564030746、rs199998975、r s200004737、rs564394299、rs540551673、rs575674513、rs564399141、rs564446433、rs200011236、rs575695879、rs530483113、rs530485751、rs5757 07171、rs564466723、rs376885423、rs535039496、rs546880794、rs371565974、rs546882633、rs371773562、rs535234661、rs540598706、rs56410784 3、rs201343843、rs201354058、rs201503351、rs530342843、rs546907614、 rs376908183、rs201510411、rs554978785、rs201511761、rs200048101、rs5 46921591、rs530350892、rs376922975、rs564200680、rs200060978、rs201 533181、rs575403459、rs564241971、rs564580989、rs546961299、rs20154 4671、rs201545926、rs371836327、rs540688834、rs201398365、rs575434258、rs564263518、rs564274418、rs376775745、rs575466805、rs555444508、<h2 style=";text-align:left;direction:ltr">rs201414746、rs546745027、rs564310081、rs376792809、rs564315036、rs 547017479、rs530416775、rs564331757、rs547021107、rs564332268、rs56 4662103、rs540733381、rs555135086、rs555471824、rs576009530、rs530597787、rs576019908、rs555139503、rs201589695、rs546790645、rs5760551 60、rs564376709、rs564714000、rs201599943、rs530619739、rs547065965 、rs575600523、rs540522467、rs540547244、rs547082685、rs564445595、r s540567098、rs540796514、rs371731981、rs564769026、rs546879622、rs377073478、rs540589456、rs555621992、rs371916205、rs371958712、rs5306 34413, rs575756239, rs376897349, rs371780831, rs376904842, rs535249716, rs575804340, rs555336411, rs540652005, rs530685640, rs37693773 8、rs555659985、rs201659566、rs530547787、rs575882807、rs201664439、rs564870320、rs376965890、rs564871823、rs546993077、rs575943952、rs2 01560701、rs540907392、rs564896638、rs564896690、rs547000677、rs564899870、rs201563763、rs576373898、rs564640008、rs555445641、rs57597 3973、rs372028184、rs371863491、rs555773796、rs564960625、rs547253736、rs576455029、rs540974086、rs564986496、rs564993510、rs201726729、<h2 style=";text-align:left;direction:ltr">rs371864190、rs565010528、rs540730019、rs201574893、rs376994924、rs530783315、rs201577842、rs201741634、rs201743233、rs547309884、rs371884028、rs3720883、<h2 style=";text-align:left;direction:ltr"> 34、rs377222716、rs576572380、rs576041557、rs547054615、rs201761066、rs377231210、rs576600072、rs576089157、rs530626933、rs541061398、rs530815395、rs576098220、rs565110303、rs540810897、rs555612139、rs541084553、rs576662260、rs547128261、rs540853945、rs555633602、rs377085474、rs547403854、rs565167501、rs530849811、rs576712624、rs547415198、rs201649913、rs201651716、rs377093160、rs535596388、rs576773731、rs576676610、rs535558092、rs565234556、rs377328308、rs555955176、rs565256471、rs556091995、rs372131207、rs201845924、rs555645605、rs541215463、rs564833136、rs565311283、rs565318120、rs540868515、rs576946641、rs556156424、rs547539536、rs201883775、rs535414368、rs372262107、rs530959005、rs540871938、rs577021824、rs535419397、rs201895529、rs540879571、rs372269123、rs377116511、rs547186293、rs565405762、rs547588654、rs201905108、rs556236282、rs201905758、rs547191791、rs540903705、rs547201834、rs530979178、rs372002155、rs576377214、rs555741423、rs577086434、rs201915876、rs530989045、rs540941092、rs556278477、rs535735811、rs201707595、rs576479846、rs541356065、rs541358959、<h2 style=";text-align:left;direction:ltr">rs535499788、rs541362812、rs565031158、rs535511711、rs577214491、rs372339994、rs377206205、rs576544612、rs377226193、rs576585621、rs54 1054278、rs577266682、rs565097315、rs565571242、rs577289107、rs556405965、rs576629767、rs535795174、rs530825627、rs565155125、rs5651864 43、rs201992546、rs541131401、rs377540035、rs377540898、rs541131406 rs565224250、rs201821518、rs541464325、rs577369845、rs565254553、r s577404457、rs372203675、rs565602281、rs202014643、rs576881042、rs577440102、rs547742253、rs577443766、rs531116715、rs377543079、rs5657 09853, rs201999985, rs377592540, rs541216093, rs201867764, rs565371438, rs372263773, rs547573283, rs201896555, rs535696383, rs20189962 7、rs530977363、rs577068583、rs201908866、rs547600407、rs547600820、rs535712483、rs556270749、rs541350117、rs577567218、rs577154533、rs5 65779439、rs541577543、rs577175836、rs531022872、rs565800474、rs565 519424、rs541591158、rs547675018、rs541594618、rs565809092、rs37249 5273、rs541395767、rs202083986、rs556650900、rs565547663、rs577251799、rs556665730、rs556373614、rs531050542、rs565585913、rs531203453、<h2 style=";text-align:left;direction:ltr">rs565871418、rs535797353、rs202109432、rs577720248、rs201987450、rs531083937、rs577393911、rs531100303、rs556716582、rs577747466、rs55 6487424、rs372559804、rs556713829、rs531103846、rs565682322、rs372563835、rs577815172、rs377588911、rs565947211、rs547813798、rs5312507 91、rs556789899、rs531125112、rs541713728、rs556819410、rs377754745、rs556713324、rs547819277、rs536000213、rs377597385、rs202165845、r s556543920、rs535860380、rs556549617、rs577508369、rs556552102、rs548057554、rs565736915、rs202180059、rs556878947、rs547836679、rs2020 51580、rs577538470、rs577539276、rs531148427、rs556584085、rs531302 114、rs202061011、rs535883810、rs556612694、rs531317317、rs20207101 6、rs372490157、rs372654175、rs556940143、rs372655989、rs541594525、rs578059299、rs541831570、rs577625085、rs547898574、rs372511122、rs2 02219028、rs531188532、rs547913013、rs541630786、rs547938227、rs578134604、rs541870309、rs556706372、rs202110802、rs372557663、rs54797 5937、rs566204756、rs566207903、rs577796497、rs556764057、rs557048933、rs202241586、rs531379120、rs557050535、rs541923828、rs556774205、<h2 style=";text-align:left;direction:ltr">rs578243189、rs541702079、rs535980549、rs531270141、rs535997775、rs566269762、rs556841303、rs557109946、rs745311902、rs548256739、rs55 6848020、rs548043362、rs536155895、rs566022284、rs577936356、rs541985091、rs548053319、rs556871781、rs536168223、rs566049151、rs5779674 98、rs566340038、rs386391721、rs386391722、rs557188530、rs367584747 、rs386391723、rs566366442、rs536028182、rs542030965、rs578005069、r s566085715、rs566086387、rs578024455、rs367621683、rs548346140、rs3 72833423、rs548105826、rs202211195、rs745531940、rs202215137、rs5780 72210, rs372665054, rs531338785, rs536243045, rs566133697, rs578090160, rs367666265, rs578114636, rs548158077, rs566180671, rs39776519 7、rs536089884、rs557356456、rs397767714、rs367683945、rs566528668、rs745659906、rs267598976、rs557071588、rs267598977、rs367701477、rs5 66564807、rs536281976、rs578242489、rs566583346、rs548214486、rs548476797、rs536129992、rs548482202、rs566262563、rs566594213、rs26760 7030、rs372744611、rs557461504、rs745754029、rs566617243、rs566617297、rs548258391、rs566310144、rs548519839、rs745338187、rs536158076、<h2 style=";text-align:left;direction:ltr">rs548274093、rs745823712、rs536163412、rs397868358、rs548542674、rs536168659、rs745839147、rs566338405、rs566348964、rs557535791、rs54 8558816, rs548565032, rs745383244, rs397870091, rs548565940, rs542287393, rs566709971, rs372990045, rs557204387, rs745418676, rs5664090 45、rs566730224、rs566752170、rs566753733、rs372818817、rs397722222、rs745983399、rs367830408、rs745480989、rs566793485、rs397734324、r s548645786、rs557669888、rs745516750、rs548657650、rs548381702、rs745552432、rs536425753、rs397973279、rs746070730、rs548387911、rs5577 08733、rs372854250、rs745569838、rs548698515、rs566482323、rs557734 401、rs746118213、rs542420738、rs367667669、rs397986566、rs56690215 7、rs531528009、rs548736753、rs566500747、rs542456172、rs746187360、rs367671512、rs566932278、rs745660727、rs367697326、rs548464529、rs5 31756169、rs542471936、rs566573122、rs566962886、rs542498214、rs566 982218、rs542504451、rs557806739、rs398090827、rs746306232、rs53648 9917、rs531803128、rs745714010、rs367720972、rs557918693、rs746350953、rs745725339、rs367991453、rs527241020、rs531579666、rs746381295、<h2 style=";text-align:left;direction:ltr">rs542208653、rs397818828、rs746391927、rs557954773、rs531600512、rs566644132、rs548866946、rs536555587、rs566656331、rs372959324、rs548538509、rs5671、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">02431、rs745838757、rs397868838、rs746437505、rs397868962、rs548885146、rs531845059、rs536570765、rs372983721、rs557571631、rs5667195 71、rs536584686、rs566723075、rs542325703、rs542643487、rs368055368 rs373019820、rs567192923、rs548936551、rs542349880、rs558065104、r s542355012、rs368062692、rs536415135、rs397963856、rs746552388、rs557690548、rs542394071、rs746083843、rs548693261、rs557732835、rs746 143575、rs542699414、rs558122779、rs567298923、rs536465674、rs55813 4318、rs531746406、rs527346644、rs548743673、rs368107300、rs3679175 48、rs746198188、rs557871766、rs567022998、rs567363028、rs373168195、rs567043047、rs567054046、rs558207456、rs542763237、rs746378441、 rs536545503、rs542568822、rs548865256、rs368147111、rs542580556、rs 558228752、rs536555646、rs746772302、rs536557590、rs531975462、rs74 6796860、rs527408032、rs746420496、rs536564089、rs368176642、rs558272974、rs746431232、rs531844054、rs527275962、rs746843910、rs567526 414、rs542614723、rs549120414、rs549124141、rs536578689、rs54914020 6、rs567586614、rs536744845、rs558044120、rs746516190、rs558055629、<h2 style=";text-align:left;direction:ltr">rs746920567、rs558060550、rs368061706、rs527461217、rs567208316、rs531886773、rs746554596、rs567220976、rs542895321、rs746974389、rs56 7222661、rs549185419、rs567237436、rs527336026、rs527483460、rs558121403、rs548993925、rs567691347、rs747002615、rs548995543、rs7466504 12、rs368271911、rs567723777、rs747040934、rs747050192、rs746670993、rs567347819、rs527512680、rs531938194、rs549242146、rs549032856、r s567373871、rs567394422、rs368307024、rs542980503、rs558508221、rs5 36676061、rs536681562、rs558516052、rs549052565、rs368145556、rs5320 90630、rs527554992、rs532120905、rs558490145、rs747176695、rs542991566、rs542774236、rs368150079、rs373340022、rs558242385、rs54935778 9、rs531980410、rs368389084、rs746808580、rs549368350、rs567506427、 rs531991551、rs536910845、rs549105394、rs746848184、rs373384143、rs5 49407905、rs558344252、rs527453833、rs549417854、rs746919102、rs746928276、rs527655882、rs368229380、rs558364672、rs549168229、rs36846 3219、rs568161057、rs558762757、rs532232329、rs549168547、rs7474705 20、rs549464271、rs543190810、rs543192154、rs368241422、rs536968067、<h2 style=";text-align:left;direction:ltr">rs747507965、rs558804203、rs542903509、rs747524681、rs567666371、rs558400652、rs549194158、rs527487277、rs368267915、rs536991033、rs56 8290255、rs558428181、rs567744716、rs527721202、rs568321346、rs568324171、rs568330046、rs567746937、rs568347691、rs747644321、rs7476460 48、rs567759326、rs747078793、rs536831479、rs568368464、rs558924666、rs543302057、rs543307320、rs747708818、rs558953446、rs568439076、r s532106809、rs558517679、rs558548847、rs747224561、rs568476272、rs567841142、rs549626904、rs373748468、rs747772882、rs747777569、rs5590 08851、rs549332923、rs373735201、rs747760347、rs568467648、rs568540915、rs568478645、rs568552353、rs368374445、rs747265095、rs56797831 6、rs559065366、rs527824027、rs537109464、rs747287657、rs747918167、rs536895056、rs373815330、rs536905377、rs537128261、rs558672062、rs5 68679804、rs537140055、rs747362082、rs543122161、rs558701088、rs747 382624、rs373841126、rs532429751、rs568724063、rs543132278、rs74742 6187、rs543154656、rs373616597、rs747448727、rs558755199、rs543176875、rs558793028、rs549483878、rs559205579、rs549801876、rs748064481、<h2 style=";text-align:left;direction:ltr">rs549805500、rs568786830、rs368724653、rs368741155、rs558812967、rs373650798、rs558827971、rs527935102、rs549505744、rs532266308、rs56 8910791、rs543588977、rs568921105、rs543252094、rs568938831、rs568308434、rs748230779、rs558889724、rs543276363、rs532518466、rs5495538 42、rs558900091、rs568478922、rs373953968、rs549933491、rs559388880、rs569021337、rs748301976、rs527792590、rs748304870、rs537274423、r s527797934、rs549957716、rs559020013、rs747831682、rs747839515、rs537094901、rs568565984、rs549993366、rs373786499、rs568616456、rs5280 24377、rs569138531、rs373808223、rs748419478、rs568648416、rs747947163、rs543458934、rs527866121、rs748000776、rs748452908、rs54347453 4、rs559511141、rs532596683、rs528051938、rs748032198、rs559524225、rs559189600、rs748504526、rs537164960、rs569247301、rs748039029、rs5 49789421, rs559564579, rs543501994, rs748048145, rs748564719, rs368926174, rs532635443, rs373862049, rs559205490, rs532651681, rs36894 9323、rs374099497、rs543549351、rs537201581、rs550141100、rs537202219、rs532674344、rs543574164、rs748190208、rs559321177、rs559677369、<h2 style=";text-align:left;direction:ltr">rs569424535、rs748690640、rs748695381、rs527959809、rs748236906、rs537243887、rs568970879、rs559373028、rs373953011、rs569027161、rs54 3882002、rs559723934、rs549952543、rs528001989、rs748754609、rs569502538、rs532712684、rs374154008、rs369022060、rs569525422、rs5436688 08、rs550218083、rs369031999、rs532562751、rs537295453、rs543930117、rs537468875、rs550244319、rs569098216、rs550247150、rs569123332、r s748398622、rs569610503、rs569138600、rs559826745、rs559831361、rs5 37315654、rs374011315、rs569152152、rs559871284、rs550298627、rs5500 18415、rs374021078、rs748455811、rs369107141、rs569197685、rs569789 352、rs544062895、rs550041205、rs749055063、rs544064839、rs56981468 5、rs532613900、rs537352949、rs550065962、rs569844606、rs559995442、 rs569277903、rs550412301、rs569291008、rs368934346、rs569896484、rs5 50421090、rs748602707、rs374102129、rs560036890、rs528121884、rs537 409772、rs569406301、rs749187435、rs560050695、rs374116302、rs74921 5366、rs560073328、rs749217434、rs569413716、rs569435862、rs537425379、rs559701154、rs550504055、rs537429508、rs537648288、rs528365663、<h2 style=";text-align:left;direction:ltr">rs748720721、rs559707544、rs748725716、rs570053547、rs748745862、rs528373215、rs537657709、rs749311576、rs569486858、rs544215781、rs374387803、rs748789887、rs3、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">69261656、rs544222244、rs559774900、rs369037062、rs528392678、rs569589072、rs569594721、rs537484448、rs560194858、rs532943697、rs52820 7220, rs550610763, rs570175462, rs543970760, rs532756083, rs749422762, rs537706167, rs550630579, rs528430659, rs569673848, rs570223084 rs544008440, rs748956951, rs532972999, rs560270291, rs749479220, rs560277925, rs559882854, rs749000031, rs544323417, rs570287651, rs7 49525560、rs528461391、rs537558317、rs749079120、rs374481593、rs550724860、rs560344389、rs559975872、rs374497861、rs569835399、rs74960 2473, rs749608721, rs570379112, rs749616223, rs570383918, rs560388069, rs550404028, rs550788771, rs532851603, rs532852070, rs528314905 、rs570419018、rs528315588、rs374522881、rs569919976、rs550435904、r s570456628、rs569926252、rs550450442、rs749695781、rs749701713、rs3 74544338、rs528533074、rs537818590、rs570492575、rs570495057、rs544454714、rs374350913、rs570000771、rs570005998、rs570551342、rs36922 9821、rs749284610、rs570558380、rs528366547、rs550901951、rs550527209、rs570048995、rs550533340、rs570063206、rs369464511、rs528383682、rs374397762、rs550927757、rs550923680、rs560539816、rs533107673、rs374594002、rs369476119、rs749864558、rs570646108、rs550966261、rs749881351、rs749897918、rs550925375、rs544544030、rs544231457、rs560608042、rs560181527、rs544551194、rs369520775、rs533153225、rs528632986、rs749364100、rs544577577、rs369534920、rs570119696、rs570782066、rs528411030、rs550621249、rs551063575、rs750002442、rs750005785、rs528650251、rs369296369、rs560255202、rs374670592、rs551081825、rs570227654、rs570232101、rs550676640、rs560705198、rs551103406、rs750060579、rs570253110、rs570861231、rs551126930、rs560742915、rs532994198、rs537750141、rs528481236、rs374500828、rs528693904、rs544387925、rs528695618、rs544671979、rs749640667、rs528701463、rs570957540、rs528504065、rs528504422、rs560405294、rs570985462、rs560819326、rs560408978、rs533248873、rs544410928、rs570467358、rs571031386、rs544422821、rs571046788、rs550875084、rs544456517、rs533083149、rs528558257、rs570555980、rs749782980、rs571112937、rs533289922、rs560511642、rs533297953、rs528567800、rs544487770、rs750377110、rs369709511、rs750395605、rs560523856、rs570685646、rs560602815、rs533104605、rs571201597、rs369722062、rs538095397、rs537898453、rs560644731、rs533167279、rs560669576、rs551401679、rs369545306、rs561042263、rs551429062、rs750017693、rs369556343、rs551090369、rs538124443、rs750042378、rs537945859、rs374872804、rs571350458、rs561086455、rs571361273、rs570859811、rs570896555、rs570898206、rs544911099、rs561103484、rs750110593、rs544921736、rs528872305、rs571410824、rs571416933、rs369592808、rs528882230、rs551531848、rs551164285、rs528891024、rs533405417、rs750148529、rs571464240、rs750175883、rs551569090、rs560811710、rs544697883、rs571001720、rs544980017、rs571007860、rs551236739、rs551249244、rs528924602、rs571065554、rs528745212、rs750724319、rs538210953、rs528933846、rs571071311、rs750755032、rs528942614、rs538036504、rs538040234、rs544760124、rs560927301、rs544802212、rs538235148、rs528956488、rs551342807、rs533481144、rs369899370、rs551359194、rs369900308、rs750399158、rs538259887、rs528795367、rs750864433、rs750411662、rs533498494、rs560998810、rs571707506、rs561331700、rs375028919、rs545103329、rs571703952、rs544837618、rs528807734、rs551400406、rs571269005、rs544875089、rs529020508、rs533357560、rs750951154、rs551440261、rs750514774、rs544886311、rs551792787、rs561404804、rs571366310、rs528855590、rs751001567、rs375069289、rs533379792、rs571842712、rs533382711、rs561137186、rs571879284、rs750631806、rs529077450、rs561470191、rs750638359、rs529093387、rs551558117、rs551573774、rs551579214、rs571500446、rs561525428、rs545229175、rs751136819、rs551581886、rs750707641、rs571528077、rs755813688、rs561214522、rs572015044、rs571536730、rs369860008、rs528947505、rs551963416、rs538225517、rs545277826、rs533634090、rs561260903、rs571600152、rs369874176、rs571638665、rs552017588、rs571666279、rs571668997、rs369907063、rs769057914、rs552049341、rs769065818、rs751325083、rs561669214、rs552061128、rs572187861、rs756005334、rs538475444、rs756013985、rs545099670、rs552106230、rs545375585、rs572237867、rs561726025、rs769150127、rs529011948、rs538288286、rs571755014、rs571757393、rs538515826、rs545115612、rs533712836、rs545115642、rs750944314、<h2 style=";text-align:left;direction:ltr">rs529029702、rs571778387、rs529035618、rs552177570、rs571826748、rs751000526、rs369968332、rs571851662、rs545431594、rs561799159、rs56 1460464、rs571880265、rs769270578、rs533571445、rs571940318、rs756189754、rs545217019、rs571959447、rs751128080、rs538381958、rs7558049 03、rs751159961、rs751175785、rs572018867、rs756228072、rs751582483 、rs561574977、rs561576248、rs572437382、rs755851030、rs561611663、r s552286628、rs545506688、rs768991354、rs769402083、rs769406063、rs572087546、rs538428981、rs769026589、rs755939083、rs572579811、rs7694 91361、rs561982435、rs769046166、rs751371429、rs572259896、rs545578378、rs756446498、rs751798524、rs572656276、rs756457749、rs76955409 0、rs538674345、rs751813574、rs552419780、rs756076130、rs552432031、rs769194199、rs533853260、rs572717577、rs769195919、rs572295505、rs5 33714676, rs538700995, rs545631150, rs538526606, rs572321690, rs572759437, rs538715990, rs562093903, rs552491193, rs552493086, rs75192 0813、rs572323525、rs552522135、rs769705492、rs751482341、rs572334430、rs751492391、rs545682563、rs751495665、rs538752299、rs545696456、<h2 style=";text-align:left;direction:ltr">rs538535248、rs572354221、rs751523153、rs545446264、rs572377516、rs533919054、rs572892333、rs769771450、rs769774967、rs572382379、rs572388571、rs756700849、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">rs756704425、rs769807240、rs769295468、rs572950216、rs562222303、rs751550702、rs752097894、rs769296900、rs572978709、rs572982359、rs56 1835001、rs552229556、rs552242552、rs533760063、rs552650520、rs752133481、rs561858372、rs756247571、rs769912734、rs756277853、rs5455086 86、rs561928325、rs572544925、rs561951163、rs756369671、rs752191138、rs769511874、rs751770785、rs769520412、rs756476767、rs545617350、r s573120275、rs552452362、rs552743787、rs552722759、rs573125010、rs572720574、rs752267943、rs572723257、rs562364859、rs769633498、rs7522 89745, rs770063371, rs756983343, rs552799307, rs756541837, rs756588238, rs752355486, rs770120624, rs572827353, rs751960880, rs55284713 7、rs573278742、rs769713298、rs752418228、rs573311661、rs533903238、rs552555197、rs562163326、rs573336871、rs770205263、rs562492571、rs5 52901035, rs573360915, rs770226861, rs552927404, rs752501530, rs752008244, rs756662298, rs752010591, rs752027589, rs562558634, rs57345 6683、rs769784952、rs752598240、rs572946713、rs756749599、rs756758706、rs752107556、rs769861427、rs562628042、rs573532135、rs752123654、<h2 style=";text-align:left;direction:ltr">rs573539819、rs752651234、rs757322465、rs545769534、rs757324658、rs573014318、rs752159218、rs752669367、rs545789688、rs562288190、rs57 3583431、rs752694131、rs553077185、rs752705175、rs562290070、rs573057895、rs756846139、rs752187308、rs757406912、rs573077799、rs5527108 47、rs757431594、rs573668899、rs553131814、rs562733566、rs562322012、rs573698013、rs770570377、rs769977143、rs752262387、rs752842135、r s553198489、rs562795927、rs573769337、rs752265531、rs770014639、rs757542280、rs573778414、rs573160530、rs573173020、rs756996519、rs7575 74398、rs573226807、rs573830638、rs573832648、rs770130183、rs770143495、rs770714259、rs752395219、rs757620158、rs562472371、rs75709188 7、rs757651862、rs573329574、rs552932842、rs757678036、rs562906731、rs573398771、rs573931855、rs757698701、rs573423419、rs757196334、rs5 73956248、rs562590653、rs770844073、rs753073195、rs757264923、rs770368521、rs562966673、rs553375250、rs757286231、rs562624182、rs55302 8786、rs562631637、rs553396451、rs757816346、rs752659859、rs770424435、rs753158893、rs753161012、rs553429432、rs757326234、rs562658586、<h2 style=";text-align:left;direction:ltr">rs757848676、rs563030100、rs757851276、rs770440360、rs553444080、rs562685659、rs770998439、rs757903328、rs752715463、rs753243305、rs57 4158047, rs753248999, rs573615359, rs771049218, rs553520048, rs574218458, rs574218497, rs770484072, rs770507859, rs757415695, rs5736929 93、rs771127117、rs753348681、rs770572267、rs574279980、rs573729847、rs553217886、rs752874223、rs574313437、rs770646119、rs753375127、r s553594673、rs753413529、rs563204921、rs563204956、rs573793230、rs553615720、rs770669377、rs752915464、rs752935458、rs771254688、rs5632 50976、rs752936829、rs573845641、rs758171359、rs553671701、rs757627939、rs770755089、rs770761836、rs573905888、rs758213818、rs57445887 9、rs758231448、rs770785487、rs563313026、rs758251421、rs758252419、rs573944942、rs753048798、rs771377765、rs563340232、rs553350853、rs7 58286245、rs562957866、rs553362365、rs757786023、rs574020661、rs753630953、rs562989702、rs574575080、rs753648561、rs753119257、rs75366 8162、rs753149666、rs563015753、rs753169837、rs553433381、rs770963601、rs574621082、rs753732662、rs758421005、rs574670231、rs757870156、<h2 style=";text-align:left;direction:ltr">rs771562041、rs574145206、rs574724729、rs757927255、rs553537463、rs771111770、rs574251637、rs553550103、rs563161459、rs758527549、rs57 4771040、rs753843081、rs553570026、rs771649415、rs574292008、rs771662229、rs574805977、rs574854330、rs771738593、rs563201986、rs7586513 82、rs574315059、rs758656020、rs574340577、rs758660161、rs771774637、rs758691949、rs758107372、rs754017346、rs771227297、rs563235099、r s553661695、rs754045274、rs563260674、rs754055297、rs563269397、rs553682936、rs574438316、rs771304275、rs758800428、rs753559306、rs7719 19739, rs753582578, rs754141945, rs575081551, rs563336388, rs575092590, rs753600502, rs575125159, rs771392468, rs563351273, rs75888819 0、rs563353671、rs758905037、rs771413333、rs553772622、rs574587211、rs753682189、rs758373659、rs772039702、rs772039784、rs772048626、rs7 54275081、rs771477905、rs753690596、rs553815137、rs575278958、rs574681748、rs575303100、rs771582810、rs758485743、rs759087854、rs57534 8590、rs753812341、rs759111100、rs772225059、rs758499442、rs754462186、rs754476252、rs574742878、rs754490813、rs772285262、rs753828829、<h2 style=";text-align:left;direction:ltr">rs574761563、rs772336893、rs754543076、rs759290654、rs772395366、rs753849639、rs754638250、rs758555473、rs759384662、rs574887579、rs75 4694440、rs772505511、rs754710057、rs753978827、rs771768782、rs772542224、rs772565872、rs574947376、rs759493094、rs771816020、rs5749621 88、rs758720517、rs754046765、rs772538411、rs754056327、rs759582553、rs754072901、rs759604608、rs759618021、rs759618971、rs772713563、r s754073647、rs754934055、rs754977237、rs772800001、rs758784984、rs754113212、rs575064964、rs575092333、rs771950039、rs575127361、rs7719 84656, rs771998562, rs772005602, rs759809047, rs772905789, rs575158631, rs755129606, rs754241027, rs755139637, rs754241912, rs75986898 8、rs575188566、rs755204073、rs773012423、rs773015695、rs773030286、rs575222561、rs759000378、rs575270437、rs772133229、rs759054169、rs7 60019571、rs575323599、rs772196281、rs755344662、rs755345735、rs759150088、rs773161532、rs755360952、rs755361509、rs772283807、rs76008 8897、rs773197006、rs773259205、rs760189302、rs755471441、rs755501706、rs754512913、rs760244093、rs755553046、rs772335325、rs759300259、<h2 style=";text-align:left;direction:ltr">rs772449743、rs773425925、rs772489586、rs754719129、rs755653914、rs755666694、rs760410743、rs759482720、rs759520309、rs754870625、rs760471093、rs7595230、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">63、rs754822550、rs773686703、rs754840578、rs760600919、rs760603637、rs772672495、rs754892941、rs760523838、rs760451171、rs755736948、r s760557658、rs760816228、rs772714587、rs755000613、rs772804611、rs755017523、rs759736603、rs759736945、rs760912489、rs774006498、rs7728 43217、rs759766086、rs755067851、rs759784688、rs761013330、rs772909429、rs772930184、rs761116601、rs761152276、rs774249327、rs774251259 rs774274995, rs772944744, rs761216742, rs774309167, rs772964132, rs761267824, rs761373885, rs759949565, rs755275181, rs774519927, rs77 4522732、rs761441210、rs761463732、rs773084045、rs773099131、rs773104432、rs760030368、rs774666634、rs761564783、rs761565126、rs7553244 98、rs773159916、rs761601211、rs755370020、rs761671328、rs774795813 rs774799502 rs760228901 rs774812992 rs774858304 rs761769631 rs 755586452、rs761781232、rs774899950、rs773396301、rs760333909、rs755619723、rs774976082、rs761920258、rs761927300、rs761950663、rs77344 6299、rs761963623、rs775079499、rs755705775、rs773689919、rs760624162、rs760699862、rs762086202、rs775205757、rs762111294、rs762125145、<h2 style=";text-align:left;direction:ltr">rs760733738、rs773860024、rs775323392、rs762225419、rs775369857、rs775376322、rs773919023、rs773782386、rs760694869、rs775387692、rs77 3967655、rs760882331、rs762340832、rs762148321、rs762202141、rs775464792、rs775131844、rs762435356、rs775544399、rs762034007、rs7625102 04、rs775614853、rs775627209、rs773976748、rs760891383、rs760906241、rs774029965、rs775822699、rs762720619、rs774047193、rs774053322、r s762804252、rs774058465、rs774129861、rs761104208、rs762966087、rs762968069、rs776089462、rs761185751、rs774332033、rs774469859、rs7761 58237, rs774482073, rs774589810, rs761511663, rs763179120, rs761511750, rs774646303, rs761588559, rs774694132, rs774728176, rs76169728 8、rs774886139、rs774922349、rs761826946、rs761845760、rs775063793、rs762003012、rs776637872、rs776654464、rs763546628、rs775110547、rs7 62276860、rs775383881、rs775461240、rs762422181、rs776740578、rs763649134、rs775386066、rs762309744、rs762328054、rs762349561、rs76382 6888、rs776958522、rs763851219、rs763608389、rs776992703、rs763887905、rs776685090、rs763905132、rs763962939、rs763974094、rs777096875、<h2 style=";text-align:left;direction:ltr">rs764029915、rs763580952、rs775580427、rs762627922、rs777288188、rs762666941、rs762675794、rs775794317、rs762726532、rs762730738、rs76 2889511, rs762892329, rs762959657, rs763002281, rs763002290, rs763012243, rs776218442, rs777563631, rs776244370, rs777622383, rs7762513 66、rs776332292、rs763230781、rs776360258、rs776427552、rs764653324、rs776476300、rs763360058、rs763363216、rs776553236、rs763457677、r s777864926、rs763480814、rs763480911、rs776628475、rs763552752、rs763566743、rs778010014、rs778022974、rs778036245、rs763698303、rs7768 74241、rs777012344、rs763688323、rs778143440、rs777215702、rs778289549、rs778300575、rs764096123、rs776939122、rs776981873、rs77811978 2、rs778380666、rs778267601、rs765112370、rs778321318、rs778494297、rs765311513、rs764145516、rs777290815、rs765318979、rs765320933、rs7 64873930、rs764888631、rs778333649、rs778374010、rs764194425、rs778674007、rs777339467、rs777382966、rs777418612、rs777478358、rs76554 0232、rs764346157、rs764347894、rs778800909、rs764349631、rs764377819、rs764399440、rs778929063、rs779079123、rs777546243、rs764445619、<h2 style=";text-align:left;direction:ltr">rs765838143、rs779164966、rs777646023、rs779192695、rs764496311、rs777703989、rs777740982、rs777812604、rs777829093、rs764661916、rs77 7840632、rs779461736、rs777844017、rs777855721、rs766045094、rs766058889、rs764699511、rs764719051、rs764746314、rs764794225、rs7796810 69、rs777969265、rs779840641、rs779874157、rs778055850、rs778396766、rs779957394、rs766239824、rs780023870、rs780043145、rs766263796、r s766274346、rs778441261、rs765258572、rs780109322、rs778501127、rs765316534、rs766324084、rs778543495、rs765369653、rs778602129、rs7663 93434、rs780316047、rs765423637、rs778611965、rs780347157、rs765492974、rs765497371、rs778714853、rs780447105、rs765533323、rs76648184 3、rs780503041、rs780639638、rs765541870、rs780693341、rs778736307、rs765643844、rs765653200、rs765673912、rs765800389、rs766622658、rs7 80798844、rs779132370、rs766649583、rs779175706、rs779230028、rs779282960、rs779344629、rs779375751、rs765948436、rs779425422、rs78112 4123、rs779450397、rs765993652、rs766010851、rs766066655、rs766071038、rs779649545、rs779679057、rs779738791、rs766206300、rs766879944、<h2 style=";text-align:left;direction:ltr">rs779928230、rs766901141、rs766933679、rs780083168、rs766940667、rs766287305、rs766299085、rs766300702、rs781535786、rs781539021、rs78 1551678, rs780193488, rs780222163, rs780296350, rs766400518, rs796125137, rs767194024, rs780344643, rs767261742, rs780357335, rs7664536 51、rs766456874、rs780494441、rs766560176、rs796454833、rs767330330、rs766575026、rs780717433、rs796834572、rs780751522、rs796879015、r s796895878、rs796908878、rs796943897、rs780779741、rs767568953、rs767585211、rs767665163、rs780791041、rs780805821、rs767821897、rs7678 74262、rs780879054、rs767995767、rs768064437、rs766687711、rs766688014、rs766704428、rs768290765、rs781060177、rs781072045、rs76846622 3、rs781072572、rs781158522、rs781159679、rs768587771、rs768603693、rs768623038、rs768653386、rs768702122、rs768711176、rs768744959、rs7 68781393、rs781188766、rs768844255、rs781231699、rs768884828、rs768 886558、rs781253062、rs781284676、rs766863424、rs781317195、rs78132 7688、rs781361862、rs781448754、rs781467412、rs766956430、rs781531817、rs767039336、rs767055311、rs781777533、rs767184929、rs796249942、rs796309254, rs767265990, rs796349008, rs796352290, rs796380704, rs7967300 02, rs796797940, rs796870168, rs796981058, rs767788312, rs767804500, rs7679, 05631, rs768152740, rs768239772, rs768260693, rs768385834, rs768416620, rs768531332, rs768574136, rs768585281, rs768804885, and rs768853312.

[0349] In one example, a guide RNA used in the present disclosure may include at least one 20-nucleotide (nt) target nucleic acid sequence listed in Table 5. Table 5 provides the gene symbol and sequence identifier for the gene (Gene SEQ ID NO:), the gene sequence including 1-5 kilobase pairs upstream and / or downstream of the target gene (Extended Gene SEQ ID NO:), and the 20-nt target nucleic acid sequence (20-nt Target Sequence SEQ ID NO:). The Sequence Listing lists each target gene, the strand for targeting that gene (designated as (+) strand or (-) strand in the Sequence Listing), the associated PAM type, and its PAM sequence for each 20-nt target nucleic acid sequence (SEQ ID NOs: 5305-125469). It is understood in the art that the spacer sequence when "T" is "U" can be an RNA sequence corresponding to the 20-nt sequence listed in Table 5. [Table 5]

[0350] In one example, a guide RNA used in the present disclosure may include at least one spacer sequence, which, when "T" is "U," may be an RNA sequence corresponding to a 20 nucleotide (nt) target sequence, such as, but not limited to, any of SEQ ID NOs: 5305-125469.

[0351] In one example, the guide RNA used in the present disclosure may include at least one spacer sequence, which, when "T" is "U", is a 20 nt sequence, for example, but not limited to, an RNA sequence corresponding to any of SEQ ID NOs: 5305-125469.

[0352] In one example, a guide RNA may include a 20 nucleotide (nt) target nucleic acid sequence associated with a PAM type such as, but not limited to, NAAAAC, NNAGAAW, NNGRRT, NNNNGHTT, NRG, or YTN. As a non-limiting example, the 20 nt target nucleic acid sequence for a particular target gene and a particular PAM type may be an RNA sequence corresponding to any one of the 20 nt nucleic acid sequences in Table 6, where "T" is "U". [Table 6]

[0353] In one example, a guide RNA may include a 20-nucleotide (nt) target nucleic acid sequence associated with a PAM type of YTN. As a non-limiting example, the 20-nt target nucleic acid sequence of a particular target gene may include a 20-nt core sequence, where the 20-nt core sequence may be an RNA sequence corresponding to SEQ ID NOs: 56864-125469 when "T" is "U." As another non-limiting example, the 20-nt target nucleic acid sequence of a particular target gene may include a core sequence, where the core sequence may be a fragment, portion, or region of an RNA sequence corresponding to any of SEQ ID NOs: 56864-125469 when "T" is "U."

[0354] VI. Other Treatment Approaches Gene editing can be performed using nucleases engineered to target specific sequences. To date, there are four major types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 nuclease system. These nuclease platforms differ in design difficulty, target density, and mechanism of action. In particular, the specificity of ZFNs and TALENs is due to protein-DNA interactions, while RNA-DNA interactions are primarily responsible for Cas9.

[0355] CRISPR endonucleases such as Cas9 can be used in the methods of the present disclosure.However, the teachings described herein, such as therapeutic targeting sites, can be applied to other forms of endonucleases, such as ZFN, TALEN, HE or MegaTAL, or using a combination of nucleases.However, in order to apply the teachings of the present disclosure to such endonucleases, it will be necessary to engineer proteins that are specifically targeted to specific targeting sites.

[0356] Additional binding domains can be fused to the Cas9 protein to enhance specificity. The target site of these constructs will be located at the identified gRNA-specific site, but additional binding motifs, for example, for zinc finger domains, will be required. In the case of Mega-TALs, a meganuclease can be fused to the DNA-binding domain of the TALE. The meganuclease domain can enhance specificity and provide cleavage. Similarly, inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain and requires adjacent binding sites for the sgRNA / Cas9 target site and the fused DNA-binding domain. This will likely require some protein engineering of dCas9 to reduce binding without additional binding sites, in addition to catalytic inactivation.

[0357] Zinc finger nuclease Zinc finger nucleases (ZFNs) are modular proteins consisting of an engineered zinc finger DNA-binding domain linked to the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target "half-site" sequences on opposite DNA strands with precise spacing between them to form catalytically active FokI dimers. Dimerization of the FokI domain, which itself has no sequence specificity, generates a DNA double-strand break between the ZFN half-sites, the initiating step of genome editing.

[0358] The DNA-binding domain of each ZFN typically consists of three to six zinc fingers with abundant Cys2-His2 structures, each of which primarily recognizes a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interactions with the fourth nucleotide may also be important. Amino acid modifications of a finger at a position that makes critical contact with DNA alter the sequence specificity of a given finger. Thus, a four-finger zinc finger protein selectively recognizes a 12-bp target sequence, which is the composite of the triplet preferences contributed by each finger, although triplet preferences can be influenced to varying degrees by adjacent fingers. A key aspect of ZFNs is that they can be easily retargeted to almost any genomic address simply by modifying individual fingers, although this requires considerable expertise to achieve. Most applications of ZFNs use proteins with four to six fingers, each recognizing 12 to 18 bp. Thus, a pair of ZFNs typically recognizes a 24-36 bp binding target sequence, excluding a typical 5-7 bp spacer between the half sites. The binding sites can be further separated by a larger spacer, such as 15-17 bp. Assuming that repetitive sequences or gene homologs are eliminated during the design process, target sequences of this length are likely unique in the human genome. Nevertheless, ZFN protein-DNA interactions are not absolute in their specificity, and off-target binding and cleavage events can occur either as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" mutants, also known as obligate heterodimer mutants, which can dimerize only with each other and not with themselves. Forcing obligate heterodimerization prevents homodimer formation. This greatly enhances the specificity of ZFNs and any other nucleases that employ these FokI mutants.

[0359] Various ZFN-based systems have been described in the art, and their modifications are regularly reported, and numerous references describe the rules and parameters used to guide the design of ZFNs. See, for example, Segal et al., Proc Natl Acad Sci USA 96(6):2758-63(1999), Dreier B et al., J Mol Biol.303(4):489-502(2000), Liu Q et al., J Biol Chem.277(6):3850-6(2002), Dreier et al., J Biol Chem 280(42):35588-97(2005), and Dreier et al., J Biol Chem.276(31):29466-78(2001).

[0360] Transcription activator-like effector nucleases (TALENs) TALENs, like ZFNs, represent another form of modular nuclease in which an engineered DNA-binding domain is linked to a FokI nuclease domain, and a pair of TALENs act in tandem to achieve target DNA cleavage. The primary difference between TALENs and ZFNs is the nature of the DNA-binding domain and its associated target DNA sequence recognition properties. The DNA-binding domain of TALENs is derived from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs consist of tandem arrays of 33–35 amino acid repeats, each of which recognizes a single base pair in a target DNA sequence typically up to 20 bp long, giving a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable dinucleotide (RVD), which contains only two amino acids at positions 12 and 13. Guanine, adenine, cytosine, and thymine bases are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This constitutes a much simpler recognition code than zinc fingers, and thus represents a significant advantage over the latter in terms of nuclease design. Nevertheless, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs also benefit from the use of obligate heterodimer mutants of the FokI domain to reduce off-target activity.

[0361] Further mutants of the FokI domain have been created that inactivate its catalytic function. When either half of a TALEN or ZFN pair contains an inactive FokI domain, only single-strand DNA nicking occurs at the target site, rather than a DSB. The results are comparable to those achieved using CRISPR / Cas9 or CRISPR / Cpfl "nickase" mutants in which one of the Cas9 cleavage domains is inactivated. DNA nicks can be used to facilitate genome editing via HDR, although with lower efficiency than DSBs. A key advantage is that off-target nicks are repaired quickly and accurately, unlike DSBs, which are prone to NHEJ repair errors.

[0362] A variety of TALEN-based systems have been described in the art, and their modifications are regularly reported.For example, see Boch, Science 326(5959):1509-12(2009); Mak et al., Science 335(6069):716-9(2012); and Moscou et al., Science 326(5959):1501(2009).The use of TALEN based on "Golden Gate" platform or cloning scheme has been described by several groups. See, for example, Cermak et al., Nucleic Acids Res. 39(12):e82(2011), Li et al., Nucleic Acids Res. 39(14):6315-25(2011), Weber et al., PLoS One. 6(2):e16765(2011), Wang et al., J Genet Genomics 41(6):339-47, Epub 2014 May 17(2014), and Cermak T et al., Methods Mol Biol. 1239:133-59(2015).

[0363] Homing endonucleases Homing endonucleases (HEs) are sequence-specific endonucleases with long recognition sequences (14–44 base pairs) that cleave DNA with high specificity, i.e., often at specific sites in the genome. There are at least six known HE families, classified by structure, including GIY-YIG, His-Cis box, HNH, PD-(D / E)xK, and VSR-like, which originate from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages. Similar to ZFNs and TALENs, HEs can be used to create DSBs at target loci as the first step in genome editing. Furthermore, some natural and engineered HEs cleave only a single strand of DNA and function as site-specific nickases. The large target sequences of HEs and the specificity they offer make them attractive candidates for creating site-specific DSBs.

[0364] Various HE-based systems have been described in the art, and modifications thereof are regularly reported. See, for example, the reviews by Steentoft et al., Glycobiology 24(8):663-80 (2014), Belfort and Bonocora, Methods Mol Biol. 1123:1-26 (2014), Hafez and Hausner, Genome 55(8):553-69 (2012), and references therein.

[0365] MegaTAL / Tev-mTALEN / MegaTev As further examples of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms utilize a fusion of the DNA-binding domain of a TALE with a catalytically active HE, taking advantage of both the tunable DNA binding and specificity of the TALE and the cleavage sequence specificity of the HE. See, e.g., Boissel et al., NAR 42:2591-2601 (2014), Kleinstiver et al., G3 4:1155-65 (2014), and Boissel and Scharenberg, Methods Mol. Biol. 1239:171-96 (2015).

[0366] In a further variant, the MegaTev construct is a fusion of a meganuclease (Mega) and a nuclease domain (Tev) derived from the GIY-YIG homing endonuclease I-TevI. These two active sites are located approximately 30 bp apart on the DNA substrate, resulting in two DSBs with incompatible overhanging ends. See, for example, Wolfs et al., NAR 42, 8816-29 (2014). It is anticipated that other combinations of existing nuclease-based approaches will be developed and useful for achieving the targeted genome modifications described herein.

[0367] dCas9-FokI or dCpf1-Fok1 and other nucleases Combining the structural and functional properties of the above nuclease platforms offers an additional approach to genome editing that can potentially overcome some of the inherent deletions. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to create DSBs. Targeting specificity is promoted by a 20- or 24-nucleotide sequence in the guide RNA that undergoes Watson-Crick base pairing with the target DNA (plus, in the case of S. pyogenes-derived Cas9, an additional two bases in the adjacent NAG or NGG PAM sequence). While such sequences are long enough to be unique in the human genome, the specificity of the RNA / DNA interaction is not absolute, and considerable promiscuity can be tolerated, particularly in the 5' half of the target sequence, effectively reducing the number of bases required to promote specificity. One solution to this problem has been to completely inactivate the catalytic function of Cas9 or Cpf1, i.e., retain only the RNA-guided DNA-binding function, and instead fuse the FokI domain to the inactivated Cas9. See, e.g., Tsai et al., Nature Biotech 32:569-76 (2014) and Guilinger et al., Nature Biotech. 32:577-82 (2014). Because FokI must dimerize to be catalytically active, two guide RNAs are required to bring two FokI fusions into close proximity to form dimers and cleave DNA. This essentially doubles the number of bases in the binding target site, thereby increasing the stringency of targeting by CRISPR-based systems.

[0368] As a further example, fusing the DNA binding domain of a TALE to a catalytically active HE, such as I-TevI, takes advantage of both the tunable DNA binding and specificity of the TALE and the cleavage sequence specificity of I-TevI, with the expectation that off-target cleavage may be further reduced.

[0369] VII. Kit The present disclosure provides kits for carrying out the methods described herein. A kit may include one or more of a genome-targeting nucleic acid, a polynucleotide encoding a genome-targeting nucleic acid, a site-directed polypeptide, a polynucleotide encoding a site-directed polypeptide, and / or any nucleic acid or proteinaceous molecule necessary to carry out aspects of the methods described herein, or any combination thereof.

[0370] A kit may include (1) a vector containing a nucleotide sequence encoding a genome-targeting nucleic acid, (2) a site-directed polypeptide or a vector containing a nucleotide sequence encoding the site-directed polypeptide, and (3) reagents for reconstituting and / or diluting the vector(s) and / or polypeptide.

[0371] A kit may include (1) a vector comprising (i) a nucleotide sequence encoding a genome-targeting nucleic acid and (ii) a nucleotide sequence encoding a site-specific polypeptide, and (2) reagents for reconstituting and / or diluting the vector.

[0372] In any of the above kits, the kit can include a single molecular guide genome-targeting nucleic acid. In any of the above kits, the kit can include a dual molecular genome-targeting nucleic acid. In any of the above kits, the kit can include two or more dual molecular guides or single molecular guides. The kit can include a vector encoding the nucleic acid target nucleic acid.

[0373] In any of the above kits, the kit may further comprise a polynucleotide to be inserted to achieve the desired genetic modification.

[0374] The components of the kit may be in separate containers or may be combined in a single container.

[0375] Any of the above kits may further include one or more additional reagents. Such additional reagents may be selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, washing buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, adapters for sequencing, etc. The buffers may be stabilization buffers, reconstitution buffers, dilution buffers, etc. The kits may also include one or more components that can be used to promote or enhance on-target binding or cleavage of DNA by the endonuclease, or to increase targeting specificity.

[0376] In addition to the components described above, the kit may further include instructions for using the components of the kit to practice the method. The instructions for practicing the method may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be included as a package insert, on a label on the container of the kit or its components (i.e., associated with its packaging or subpackaging), or the like. The instructions may be included as an electronic storage data file contained on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, or the like. In some cases, the actual instructions may not be included in the kit, but rather a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0377] VIII. CERTAIN METHODS AND COMPOSITIONS OF THE INVENTION Accordingly, the present disclosure particularly relates to the following non-limiting methods in accordance with the present disclosure: In a first method, i.e., Method 1, the present disclosure provides a method for editing a voltage-gated sodium channel alpha subunit 9 (SCN9A) gene of a cell by genome editing, comprising: introducing one or more deoxyribonucleic acid (DNA) endonucleases into the cell to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the SCN9A gene or an SCN9A regulatory element, which result in one or more permanent insertions, deletions, or mutations of at least one nucleotide in or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

[0378] In another method, i.e., Method 2, the present disclosure provides an ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising editing patient-specific induced pluripotent stem cells (iPSCs) in or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene, differentiating the edited iPSCs into neurons of the peripheral nervous system, and administering the peripheral nervous system neurons to the patient.

[0379] In another method, Method 3, the present disclosure provides an ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising obtaining patient-specific induced pluripotent stem cells (iPSCs), editing the iPSCs in or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene, differentiating the edited iPSCs into neurons of the peripheral nervous system, and administering the peripheral nervous system neurons to the patient.

[0380] In another method, i.e., Method 4, the disclosure provides the method of Method 2 or 3, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSCs to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the SCN9A gene or SCN9A regulatory elements that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide in or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

[0381] In another method, i.e., Method 5, the present disclosure provides an ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising editing mesenchymal stem cells in or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene, differentiating the edited mesenchymal stem cells into neurons of the peripheral nervous system, and administering the neurons of the peripheral nervous system to the patient.

[0382] In another method, i.e., Method 6, the present disclosure provides an ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising obtaining mesenchymal stem cells from the patient, editing the mesenchymal stem cells in or near the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene, differentiating the edited mesenchymal stem cells into neurons of the peripheral nervous system, and administering the neurons of the peripheral nervous system to the patient.

[0383] In another method, i.e., Method 7, the present disclosure provides the method of Method 5 or 6, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the mesenchymal stem cells to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the SCN9A gene or SCN9A regulatory elements that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide in or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0384] In another method, i.e., Method 8, the present disclosure provides an in vivo method for treating a patient having an SCN9A-associated disorder, comprising: editing the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene in cells of the patient.

[0385] In another method, i.e., Method 9, the disclosure provides the method of Method 8, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the cell to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the SCN9A gene or an SCN9A regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide in or near the SCN9A gene, thereby reducing or eliminating expression or function of the SCN9A gene product.

[0386] In another method, method 10, the disclosure provides any one of methods 8-9, wherein the cell is a neuron of the peripheral nervous system.

[0387] In another method, i.e., Method 11, the present disclosure provides the method of Method 10, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are delivered to neurons of the peripheral nervous system via direct intraganglionic or intraspinal injection, or by intrathecal delivery.

[0388] In another method, i.e., Method 12, the disclosure provides a method for modifying a contiguous genomic sequence of the SCN9A gene in a cell, comprising contacting the cell with one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs).

[0389] In another method, i.e., method 13, the disclosure provides the method of method 12, wherein the modification of the contiguous genomic sequence occurs in one or more exons of the SCN9A gene.

[0390] In another method, i.e., Method 14, the present disclosure provides any one of Methods 1-13, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are selected from any of SEQ ID NOs: 1-620 and variants having at least 90% homology to any of SEQ ID NOs: 1-620.

[0391] In another method, method 15, the disclosure provides the method of method 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more proteins or polypeptides.

[0392] In another method, i.e., method 16, the present disclosure provides the method of method 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more polynucleotides encoding the one or more DNA endonucleases.

[0393] In another method, i.e., method 17, the present disclosure provides the method of method 16, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

[0394] In another method, method 18, the present disclosure provides the method of method 17, wherein the one or more ribonucleic acids (RNAs) are one or more chemically modified RNAs.

[0395] In another method, method 19, the present disclosure provides the method of method 18, wherein the one or more ribonucleic acids (RNAs) are chemically modified in a coding region.

[0396] In another method, i.e., method 20, the disclosure provides any one of methods 16-19, wherein the one or more polynucleotides or one or more ribonucleic acids (RNAs) are codon-optimized.

[0397] In another method, i.e., Method 21, the present disclosure provides any one of Methods 1 to 20, wherein the method further comprises introducing one or more gRNAs or one or more sgRNAs into the cell.

[0398] In another method, i.e., method 22, the present disclosure provides the method of method 21, wherein the one or more gRNAs or one or more sgRNAs comprise a spacer sequence complementary to a DNA sequence in or near the SCN9A gene.

[0399] In another method, i.e., Method 23, the present disclosure provides any one of Methods 21-22, wherein the one or more gRNAs or one or more sgRNAs are chemically modified.

[0400] In another method, i.e., Method 24, the present disclosure provides any one of Methods 21-23, wherein the one or more gRNAs or one or more sgRNAs are pre-complexed with the one or more deoxyribonucleic acid (DNA) endonucleases.

[0401] In another method, i.e., Method 25, the present disclosure provides the method of Method 24, wherein the pre-complexing involves covalently binding the one or more gRNAs or one or more sgRNAs to the one or more deoxyribonucleic acid (DNA) endonucleases.

[0402] In another method, i.e., method 26, the disclosure provides any one of methods 14-25, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle.

[0403] In another method, i.e., Method 27, the present disclosure provides any one of Methods 21-25, wherein the one or more deoxyribon...

Claims

1. 1. A method for editing a voltage-gated sodium channel alpha subunit 9 (SCN9A) gene in a cell by genome editing, comprising introducing into the cell one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or an SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

2. 1. An ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising: (a) editing patient-specific induced pluripotent stem cells (iPSCs) within or near the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of the SCN9A gene; (b) differentiating the edited iPSCs into neurons of the peripheral nervous system; and (c) administering said peripheral nervous system neurons to said patient. The method comprising:

3. 3. The method of claim 2, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSCs to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

4. 1. An ex vivo method for treating a patient having an SCN9A-associated condition or disorder, comprising: (a) editing mesenchymal stem cells within or near the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene or other DNA sequences encoding regulatory elements of said SCN9A gene; (b) differentiating the edited mesenchymal stem cells into neurons of the peripheral nervous system; and (c) administering said peripheral nervous system neurons to said patient. The method comprising:

5. 5. The method of claim 4, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the mesenchymal stem cells to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

6. 1. An in vivo method for treating a patient having an SCN9A-associated disorder, the method comprising editing the voltage-gated sodium channel alpha subunit 9 (SCN9A) gene in the cells of the patient.

7. 7. The method of claim 6, wherein the editing step comprises introducing into the cell one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the SCN9A gene or SCN9A regulatory element, resulting in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or near the SCN9A gene, thereby reducing or eliminating expression or function of an SCN9A gene product.

8. The method according to any one of claims 6 to 7, wherein the cells are neurons of the peripheral nervous system.

9. 9. The method of claim 8, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are delivered to neurons of the peripheral nervous system via direct intraganglionic or intrathecal injection, or intrathecal delivery.

10. A method for altering the contiguous genomic sequence of the SCN9A gene in a cell, the method comprising contacting the cell with one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs).

11. 11. The method of claim 10, wherein the alterations in the contiguous genomic sequence occur in one or more exons of the SCN9A gene.

12. 12. The method of any one of claims 1 to 11, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are selected from any of the sequences of SEQ ID NOs: 1 to 620 and variants having at least 90% homology to any of the sequences disclosed in SEQ ID NOs: 1 to 620.

13. 13. The method of claim 12, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more proteins or polypeptides.

14. 13. The method of claim 12, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more polynucleotides encoding the one or more DNA endonucleases.

15. 15. The method of claim 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

16. 16. The method of claim 15, wherein the one or more ribonucleic acids (RNAs) are one or more chemically modified RNAs.

17. 17. The method of claim 16, wherein the one or more ribonucleic acids (RNAs) are chemically modified in the coding region.

18. 18. The method of any one of claims 14 to 17, wherein the one or more polynucleotides or one or more ribonucleic acids (RNAs) are codon-optimized.

19. The method of any one of claims 1 to 18, further comprising introducing one or more gRNAs or one or more sgRNAs into the cell.

20. 20. The method of Claim 19, wherein the one or more gRNAs or one or more sgRNAs comprise a spacer sequence that is complementary to a DNA sequence within or near the SCN9A gene.

21. 21. The method of any one of claims 19 to 20, wherein the one or more gRNAs or one or more sgRNAs are chemically modified.

22. 22. The method of any one of claims 19-21, wherein the one or more gRNAs or one or more sgRNAs are pre-complexed with the one or more deoxyribonucleic acid (DNA) endonucleases.

23. 23. The method of Claim 22, wherein the pre-complexing involves covalent attachment of the one or more gRNAs or one or more sgRNAs to the one or more deoxyribonucleic acid (DNA) endonucleases.

24. 24. The method of any one of claims 12 to 23, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle.

25. 24. The method of any one of claims 19 to 23, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are formulated in liposomes or lipid nanoparticles that also comprise the one or more gRNAs or one or more sgRNAs.

26. 21. The method of any one of claims 10 or 19-20, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are encoded within an AAV vector particle.

27. 21. The method of any one of claims 19 to 20, wherein the one or more gRNAs or one or more sgRNAs are encoded within an AAV vector particle.

28. 21. The method of any one of claims 19-20, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are encoded within an AAV vector particle that also encodes the one or more gRNAs or one or more sgRNAs.

29. 29. The method of any one of claims 26-28, wherein the AAV vector particle is selected from the group consisting of any of those disclosed in SEQ ID NOs: 4734-5302 and Table 2.

30. A single-molecule guide RNA comprising at least one spacer sequence that is an RNA sequence corresponding to any of SEQ ID NOs: 5305 to 125469.

31. 31. The single-molecule guide RNA of Claim 30, wherein the single-molecule guide RNA further comprises a spacer extension region.

32. The single guide RNA of Claim 30, wherein the single guide RNA further comprises a tracrRNA extension region.

33. The single-molecule guide RNA according to claims 30 to 32, wherein the single-molecule guide RNA is chemically modified.

34. The single-molecule guide RNA of any one of claims 30 to 33, which is pre-complexed with a DNA endonuclease.

35. 35. The single-molecule guide RNA of Claim 34, wherein the DNA endonuclease is Cas9 or Cpf1 endonuclease.

36. 36. The single-molecule guide RNA of Claim 35, wherein the Cas9 or Cpf1 endonuclease is selected from the group consisting of S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpf1, and Acidaminococcus sp. BV3L6 Cpf1, and variants having at least 90% homology to the endonuclease.

37. 37. The single-molecule guide RNA of Claim 36, wherein the Cas9 or Cpf1 endonuclease comprises one or more nuclear localization signals (NLS).

38. 38. The single-molecule guide RNA of Claim 37, wherein at least one NLS is at or within 50 amino acids of the amino terminus of the Cas9 or Cpfl endonuclease and / or at least one NLS is at or within 50 amino acids of the carboxy terminus of the Cas9 or Cpfl endonuclease.

39. A DNA encoding the single-molecule guide RNA according to any one of claims 30 to 33.

Images