Compositions and methods for excising transcription factor 4 (TCF4) repeat expansions

Abstract

An RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence set forth in any one of SEQ ID NOs: 1 to 2325, as well as compositions, methods and uses thereof.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 482,870 (filed February 2, 2023) and 63 / 382,108 (filed November 2, 2022), the contents of which are incorporated herein by reference.

[0002] Throughout this application, various publications are referenced, including those within parentheses. The entire disclosures of all publications mentioned in this application are incorporated by reference into this application in their entireties to inform the reader of the technology that may be used in or to which the present invention pertains.

[0003] Sequence Listing Reference This application was created on November 2, 2023 on an IBM PC using an operating system compatible with MS-Windows®, and incorporates by reference the nucleotide sequence contained in an XML file having a size of 2,044 kilobytes and filed as part of this application on November 2, 2023, entitled "231102_92056-A-PCT_Sequence_Listing_AWG.xml." [Background technology]

[0004] The transcription factor 4 (TCF4) gene (also known as ITF2 or SEF-2) is located on chromosome 18 and consists of 20 exons. Exons 1 and 20 are non-coding exons, while the remaining exons encode basic helix-loop-helix (bHLH) proteins that function as homodimers or heterodimers with other bHLH proteins. These dimers bind to DNA at Ephrussi (E) box sequences.

[0005] TCF4 has been reported to be expressed in developing corneal endothelial cells. It functions as a crucial effector in the Wnt signaling pathway. TCF4 expression is a key factor in controlling the balance between proliferation and differentiation. Wnt / TCF4 signaling has been reported to regulate human corneal endothelial cells (hCECs).

[0006] Intron trinucleotide CTG repeat expansions in TCF4 may account for 50-70% of cases of Fuchs endothelial corneal dystrophy (FECD), a degenerative disease affecting the corneal endothelial cell monolayer. Expression of the repeat expansion leads to abnormal expression of TCF4 due to RNA toxicity and missplicing. Most subjects without FECD have 12-40 repeats of the CTG sequence in intron 3 of TCF4. CTG repeat sequences measured in peripheral blood leukocytes contain 50-3000 repeats in 77.7% of FECD cases in the United States.

[0007] FECD is characterized by localized proliferation and loss of endothelial cells and edema. Because human corneal endothelial cells (hCECs) have minimal regenerative capacity in vivo, the decline in hCEC density as FECD progresses leads to severe corneal damage and vision loss. Currently, the only treatment is corneal transplantation. Summary of the Invention

[0008] In an embodiment of the invention, there is provided a method for excising an intronic trinucleotide CTG repeat expansion from an allele of transcription factor 4 (TCF4) in a cell, said method comprising: at least one CRISPR nuclease or a nucleotide molecule encoding a CRISPR nuclease; a first RNA molecule comprising a first guide sequence portion, or a nucleotide molecule encoding said first RNA molecule; and a second RNA molecule comprising a second guide sequence portion, or a nucleotide molecule encoding said second RNA molecule; introducing into said cells a composition comprising a complex of the CRISPR nuclease and the first RNA molecule that makes a double-stranded break upstream of the intronic trinucleotide CTG repeat expansion, and a complex of the CRISPR nuclease and the second RNA molecule that makes a double-stranded break downstream of the intronic trinucleotide CTG repeat expansion; The first guide sequence portion comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1216 to 2325, and the second guide sequence portion comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 1215.

[0009] In an embodiment of the present invention, a composition containing an RNA molecule comprising a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 2325 is also provided.

[0010] In some embodiments of this invention, there is also provided a method of treating Fuchs' endothelial corneal dystrophy (FECD) in a human subject, the method comprising delivering to the subject any of the compositions described herein or transplanting into the cornea of ​​the human subject any of the modified cells described herein. [Brief explanation of the drawings]

[0011] [Figure 1] A schematic diagram of intron 3 of the TCF4 gene. An outline of the TCF4 guide is also shown below.

[0012] [Table A] [Figure 2] Activity of sgRNA targeting intron 3 of TCF4. RNP containing SpCas9 protein and synthetic RNA guide was electroporated into U2OS cells. 72 hours after DNA transfection, cells were harvested, genomic DNA was extracted, and the TCF4 region targeted by the guide was amplified and analyzed by next-generation sequencing (NGS). The graph shows the editing rate ± STDV after two electroporations of RNP. [Figure 3] TCF4 mRNA levels measured by qRT-PCR. U2OS cells were electroporated with the indicated RNP combinations, and 7 days later, RNA was extracted and cDNA was prepared. qRT-PCR was performed using fast SYBR and primers specific for the exon 11-exon 12 junction of the TCF4 gene. Results are shown as mean ± standard deviation (STDV) (n = 3). [Figure 4] TCF4 protein levels were measured by Western blot (WB). U2OS cells were electroporated with the indicated RNP combinations, and 7 days later, proteins were extracted and lysed with RIPA. Anti-TCF4 and anti-GAPDH antibodies were used for WB. [Figure 5] Excision percentage (%) measured by ddPCR. U2OS cells were electroporated with the indicated RNP combinations, and genomic DNA was extracted 7 days later. ddPCR was performed on a QX200 BioRad system using primers specific for the g4 + g6 (FAM) excision pattern and RPP30 as a probe or housekeeping gene (HEX). Results of the excision percentage calculated from the FAM / HEX ratio are shown (mean ± standard deviation, n = 3). [Figure 6A-B] Editing and excision in HeLa cells by DNA transfection of OMNI-103 and OMNI-110. HeLa cells were transfected with plasmids encoding the indicated nucleases and sgRNAs. Figure 6A: Cells were harvested 72 hours after DNA transfection, genomic DNA was extracted, and the target guide region was amplified and analyzed by next-generation sequencing. The graph shows the editing rate ± STDV in triplicate transfection wells. Figure 6B: Genomic DNA was extracted 7 days after electroporation, and ddPCR was performed using EvaGreen on a QX200 BioRad system with excision-specific primers or RPP30 as a housekeeping gene. The results show the excision rate calculated from the excision / HKG ratio (mean ± STDV, n = 2). [Figure 7]TCF4 protein levels measured by Western blot. HeLa cells were electroporated with the indicated combinations, and 14 days after transfection, proteins were extracted and dissolved in RIPA. Anti-TCF4 and anti-GAPDH antibodies were used for WB. [Figure 8A-B] Editing and excision by OMNI-50 in U2OS cells using LVLP. Figure 8A: U2OS cells were infected with downstream and upstream repeat constructs. Three days post-infection, cells were harvested, genomic DNA was extracted, and the targeting guide region was amplified and analyzed by NGS. Figure 8B: U2OS cells were infected with a mixture of upstream and downstream constructs packaged in LVLP, or with LVLP containing both constructs in the same particle (all-in-one). Eighteen days post-infection, cells were harvested, genomic DNA was extracted, and excision was measured by ddPCR. [Figure 9] TCF4 mRNA levels were measured by qRT-PCR after excision using LVLP. U2OS cells were infected with downstream and upstream repeat constructs, and 21 days after electroporation, RNA was extracted and cDNA was prepared. qRT-PCR was performed using fast SYBR and primers specific for the exon 11-exon 12 junction of the TCF4 gene. Results are shown as mean ± standard deviation (STDV) (n=2). DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description In healthy individuals, TCF4 is a transcription factor expressed in corneal endothelial cells and regulates the switch between proliferation and differentiation. However, in many patients with Fuchs endothelial corneal dystrophy (FECD), repeat expansions in TCF4 lead to the formation of toxic RNA, dysregulation of TCF4 protein, or toxic protein aggregates (through repeat-associated non-AUG-dependent (RAN) translation), ultimately leading to transcriptional dysfunction and cell death.

[0014] Fuchs endothelial corneal dystrophy (FECD) is characterized by the formation of guttae—microscopic collagenous aggregates—in corneal endothelial cells. These aggregates initially cause blurred vision, worsen over time, and can ultimately lead to corneal blindness. 70% of FECD patients in the United States have a TCF4 repeat expansion. The disease is inherited in an autosomal dominant manner.

[0015] This disclosure provides methods for excising a repeat expansion from at least one TCF4 allele in a cell.

[0016] This disclosure also provides methods of treating, preventing, or alleviating Fuchs' endothelial corneal dystrophy (FECD) by excising the repeat expansion from at least one TCF4 allele in a subject.

[0017] In some aspects, this disclosure provides methods that utilize at least one naturally occurring nucleotide difference or polymorphism (e.g., a single nucleotide polymorphism (SNP)) to distinguish / distinguish between two alleles of a gene, i.e., an allele having a mutation (e.g., a repeat expansion in TCF4) that encodes a variant product that causes a disease phenotype and a specific sequence at the SNP location (REF / SNP) (a "mutant allele"), and an allele that encodes a functional product (a "functional allele"). In some aspects, the SNP location is used to distinguish / distinguish between two alleles of a gene that have one or more disease-associated mutations, e.g., targeting one of the alleles that has both the specific sequence at the SNP location (SNP / REF) and the disease-associated mutation. In some aspects, the disease-associated mutation is targeted. In some aspects, the method further comprises knocking out expression of the mutant protein, allowing expression of a functional protein.

[0018] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, representative methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0019] The term "a" or "an" is understood to refer to "one or more" of the listed components. It will be apparent to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Thus, the terms "a" and "at least one" have the same meaning in this application.

[0020] For the purpose of better understanding the present teachings, and without in any way limiting the scope of the teachings, unless otherwise indicated, all numbers and other numerical values ​​expressing quantities, percentages or proportions used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should be construed in light of the number of recited significant digits and by applying ordinary rounding techniques.

[0021] Unless otherwise stated, adjectives such as "substantially" and "about," modifying a condition or relationship characterizing an embodiment of this invention, are understood to mean that the condition or characteristic is defined to the extent acceptable in the operation of the embodiment for its intended application. Unless otherwise indicated, the term "or" in this specification and claims is considered to be an inclusive "or," indicating at least one or any combination of the items it connects, rather than an exclusive "or."

[0022] In this specification and claims, the verbs "comprise," "contain," and "have," and their conjugations, are used to indicate that the object of the verb is not necessarily an exhaustive list of components, elements, or moieties of the subject of the verb. Other terms in this specification are intended to be defined by their well-known meanings in the art.

[0023] In some embodiments of the present invention, DNA nucleases are used to cleave DNA at target sites and trigger cellular repair mechanisms, such as, but not limited to, non-homologous end joining (NHEJ). In classical NHEJ, the two ends of a double-strand break (DSB) are ligated in a rapid but imprecise manner (i.e., frequently resulting in DNA mutations at the break site in the form of small insertions or deletions).

[0024] As used herein, the term "modified cell" refers to a cell in which a complex of an RNA molecule and a CRISPR nuclease makes a double-stranded break as a result of hybridization with a target sequence, i.e., on-target hybridization.

[0025] The invention provides modified cells obtained by use of the methods described herein. In some embodiments, these modified cells are capable of giving rise to progeny cells. In some embodiments, these modified cells are capable of giving rise to progeny cells after engraftment. By way of non-limiting example, the modified cells may be hematopoietic stem cells (HSCs) or cells suitable for allogeneic or autologous cell transplantation.

[0026] As used herein, the term "targeting sequence" or "targeting molecule" refers to a nucleotide sequence or molecule comprising a nucleotide sequence capable of hybridizing to a specific target sequence; for example, a targeting sequence has a nucleotide sequence along its length that is at least partially complementary to the sequence being targeted. A targeting sequence or targeting molecule may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, either alone or in combination with other RNA molecules, with the targeting sequence serving as the targeting portion of the CRISPR complex. When a molecule having a targeting sequence is present simultaneously with a CRISPR nuclease, the RNA molecule, alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), can target the CRISPR nuclease to a specific target sequence. As a non-limiting example, a CRISPR RNA molecule or the guide sequence portion of a single-guide RNA molecule may serve as a targeting molecule. Each possibility is a separate embodiment. A targeting sequence can be custom designed to target a desired sequence.

[0027] In this specification, the term "targeting" refers to the targeting sequence of targeting molecule being preferentially hybridized with the nucleic acid having the target nucleotide sequence.It is understood that the term "targeting" includes various hybridization capabilities, such as the nucleic acid having the target nucleotide sequence being preferentially targeted, but in addition to on-target hybridization, unintended off-target hybridization may also occur.It is understood that when an RNA molecule targets a sequence, the complex of the RNA molecule and the CRISPR nuclease molecule will target this sequence for nuclease activity.

[0028] The "guide sequence portion" of an RNA molecule refers to a nucleotide sequence that can hybridize to a specific target DNA sequence; for example, the guide sequence portion has a nucleotide sequence along its length that is partially or fully complementary to the targeted DNA sequence. In some embodiments, the length of the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, or about 17-50, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17-42, 17-41, 17-40, 17-41, 17-42, 17-43, 17-44, 17-45, 17-46, 17-47, 17-48, 17-49, 17-49, 17-50, 17-51, 17-52, 17-53, 17-54, 17-55, 17-56, 17-57, 17-58, 17-5 ...60, 17-61, 7 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 17 to 24, 17 to 22, 17 to 21, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 22, 18 to 20, 20 to 21, 21 to 22, or 17 to 20. Preferably, the entire length of the guide sequence portion is completely complementary to the target DNA sequence along its length. The guide sequence portion may be a portion of an RNA molecule that can form a complex with a CRISPR nuclease, and the guide sequence portion serves as the DNA-targeting portion of the CRISPR complex. When an RNA molecule having a guide sequence portion is present simultaneously with a CRISPR molecule, alone or in combination with one or more other RNA molecules (e.g., a tracrRNA molecule), the RNA molecule can target the CRISPR nuclease to a specific target DNA sequence. Thus, a CRISPR complex can be formed by directly binding a CRISPR nuclease to an RNA molecule having a guide sequence portion, or by binding a CRISPR nuclease to an RNA molecule having a guide sequence portion and one or more other RNA molecules. Each possibility is a separate embodiment. The guide sequence portion can be custom designed and directed to a desired sequence. Thus, a molecule containing a "guide sequence portion" is a type of targeting molecule.In some embodiments, the guide sequence portion has the same sequence as a guide sequence portion described herein (e.g., a guide sequence set forth in any of SEQ ID NOS: 1-2325), or a sequence that differs by no more than 1, 2, 3, 4, or 5 nucleotides. Each possibility is a separate embodiment. In some of these embodiments, the guide sequence portion has the same sequence as a sequence set forth in any of SEQ ID NOS: 1-2325. Throughout this application, the terms "guide molecule," "RNA guide molecule," "guide RNA molecule," and "gRNA molecule" are synonymous with a molecule that includes a guide sequence portion. In some embodiments, the molecule that includes a guide sequence portion is a crRNA molecule. In some embodiments, the molecule that includes a guide sequence portion is a crRNA molecule, preferably along with a compatible tracrRNA molecule that can form a crRNA:tracrRNA complex with the crRNA molecule. In some embodiments, the molecule that includes a guide sequence portion is an sgRNA molecule.

[0029] As used herein, the term "non-discriminatory" refers to the guide sequence portion of an RNA molecule that targets a specific DNA sequence that is common to all alleles of a gene.

[0030] The term "single nucleotide polymorphism (SNP) location," as used herein, refers to a location where a single base DNA sequence variation occurs between species or between pairs of chromosomes in an individual. When an SNP location exists on a pair of chromosomes in an individual, the SNP on one chromosome is a "heterozygous SNP." The term SNP location refers to a specific nucleic acid location where a specific variation occurs, and encompasses both sequences containing a variation from the most frequently occurring base at a particular nucleic acid location (also referred to as an "SNP" or alternative "ALT"), and sequences containing the most frequently occurring base at a particular nucleic acid location (also referred to as a reference or "REF"). Thus, the sequence at an SNP location may reflect either an SNP (i.e., an alternative sequence variation relative to a consensus reference sequence within a population) or the reference sequence itself.

[0031] In some aspects, this disclosure provides methods that utilize at least one naturally occurring nucleotide difference or polymorphism (e.g., a single nucleotide polymorphism (SNP)) to distinguish / distinguish between two alleles of a gene, i.e., an allele with a mutation (e.g., an expanded trinucleotide repeat in TCF4) that encodes a variant product that causes a disease phenotype and a specific sequence at the SNP location (REF / SNP) (the "variant allele"), and an allele that encodes a functional product (the "functional allele"). The method further comprises knocking out expression of the variant protein to allow expression of the functional protein. In some aspects, the method is for treating, ameliorating, or preventing a dominant-negative genetic disease.

[0032] In some embodiments, RNA molecules are provided that bind / associate with and / or target an RNA-guided DNA nuclease to a sequence containing at least one nucleotide (e.g., a SNP) that differs between a mutant allele and a functional allele of a gene of interest (i.e., a sequence in the mutant allele that is not present in the functional allele). The sequence may be located within the disease-associated mutation. The sequence may be located upstream or downstream of the disease-associated mutation. The RNA molecules of the invention may target the sequence difference between the mutant allele and the functional allele.

[0033] In an embodiment of the present invention, the RNA molecule comprises a guide sequence portion having 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 2325.

[0034] The RNA molecule and / or the guide sequence portion of the RNA molecule may contain modified nucleotides. Exemplary modifications to nucleotides / polynucleotides may be synthetic and may include polynucleotides with nucleotides containing bases other than the naturally occurring adenine, cytosine, thymine, uracil, or guanine. Modifications to polynucleotides include polynucleotides with synthetic, non-naturally occurring nucleosides, e.g., locked nucleic acids. Modifications to polynucleotides may be used to increase or decrease RNA stability. An example of a modified polynucleotide is mRNA with 1-methylpseudouridine. For examples of modified polynucleotides and their uses, see U.S. Pat. No. 8,278,036, WO 2015 / 006747, and Weissman and Kariko (2015), incorporated herein by reference.

[0035] As used herein, "consecutive nucleotides" as set forth in a SEQ ID NO: refers to the nucleotides of the sequence in the order set forth in the SEQ ID NO:, without any intervening nucleotides.

[0036] In embodiments of the present invention, the guide sequence portion may be 17 to 50 nucleotides in length, or may contain 20 to 22 consecutive nucleotides within the sequence set forth in any of SEQ ID NOS: 1 to 2325. In embodiments of the present invention, the guide sequence portion may be less than 22 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion may be 17, 18, 19, 20, or 21 nucleotides in length. In such embodiments, the guide sequence portion may consist of 17, 18, 19, 20, or 21 nucleotides, respectively, within the 17 to 22 consecutive nucleotide sequence set forth in any of SEQ ID NOS: 1 to 2325. For example, the guide sequence portion of the 17 consecutive nucleotide sequence set forth in SEQ ID NOS: 2324 may be any of the following nucleotide sequences (nucleotides removed from the consecutive sequence are strikethrough):

[0037] [ka]

[0038] In embodiments of the present invention, the guide sequence portion may be greater than 20 nucleotides in length. For example, in embodiments of the present invention, the guide sequence portion may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In such embodiments, the guide sequence portion has 17 to 50 nucleotides comprising a sequence of 20, 21, or 22 consecutive nucleotides set forth in any of SEQ ID NOs: 1 to 2325, and nucleotides that are completely complementary to the nucleotides (sequences) adjacent to the 3' end, 5' end, or both, of the target sequence.

[0039] In an embodiment of the present invention, a CRISPR nuclease and an RNA molecule comprising a guide sequence portion bind to a target DNA sequence to form a CRISPR complex that cleaves the target DNA sequence. The CRISPR nuclease (e.g., Cpf1) may form a CRISPR complex comprising the CRISPR nuclease and the RNA molecule without an additional tracrRNA molecule. Alternatively, the CRISPR nuclease (e.g., Cas9) may form a CRISPR complex between the CRISPR nuclease, the RNA molecule, and the tracrRNA molecule. The guide sequence portion, which has a nucleotide sequence capable of hybridizing to a specific target DNA sequence, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA sequence portion) can be present in the same RNA molecule. Alternatively, the guide sequence portion can be present in one RNA molecule, and the sequence portion involved in CRISPR nuclease binding (e.g., the tracrRNA portion) can be present in a separate RNA molecule. A single RNA molecule comprising a guide sequence portion (e.g., a DNA-targeting RNA sequence) and at least one CRISPR protein-binding RNA sequence portion (e.g., a tracrRNA sequence portion) can form a complex with a CRISPR nuclease and serve as a DNA-targeting molecule. In some embodiments, a first RNA molecule comprising a DNA-targeting RNA portion that includes a guide sequence portion and a second RNA molecule comprising a CRISPR protein-binding RNA sequence interact by base pairing to form an RNA complex that targets the CRISPR nuclease to a DNA target site, or they are fused to each other to form an RNA molecule that complexes with a CRISPR nuclease and targets the CRISPR nuclease to a DNA target site.

[0040] In embodiments of the invention, the RNA molecule containing the guide sequence portion may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of the guide portion of the RNA molecule and a transactivating crRNA (tracrRNA) (see Jinek et al., 2012). In such embodiments, the RNA molecule is a single-guide RNA (sgRNA) molecule. Some embodiments of the invention may also form CRISPR complexes utilizing individual tracrRNA molecules and individual RNA molecules containing the guide sequence portion. In such embodiments, the tracrRNA may hybridize with the RNA molecule via base pairing, which may be advantageous in certain applications of the invention described herein.

[0041] The term "tracr mate sequence" refers to a sequence sufficiently complementary to the tracrRNA molecule to hybridize with the tracrRNA through base pairing and promote the formation of a CRISPR complex (see U.S. Patent No. 8,906,616). In embodiments of this invention, the RNA molecule may further comprise a tracr mate sequence portion.

[0042] As used herein, a "gene" includes a DNA region that encodes a gene product and all DNA regions that control the production of that gene product, whether or not the regulatory sequence is contiguous with the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation control sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0043] "Eukaryotic" cells include, but are not limited to, fungal cells (eg, yeast), plant cells, animal cells, mammalian cells, and human cells.

[0044] As used herein, the term "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases may be isolated from or derived from natural sources. Natural sources may be living organisms. Alternatively, nucleases may be engineered or synthetic proteins that retain phosphodiester bond cleavage activity. Genetic modification can be achieved using nucleases (e.g., CRISPR nucleases).

[0045] In an embodiment of the invention, there is provided a method for excising an intronic trinucleotide CTG repeat expansion from an allele of transcription factor 4 (TCF4) in a cell, comprising: The method comprises: at least one CRISPR nuclease or a nucleotide molecule encoding a CRISPR nuclease; a first RNA molecule comprising a first guide sequence portion, or a nucleotide molecule encoding said first RNA molecule; and a second RNA molecule comprising a second guide sequence portion, or a nucleotide molecule encoding said second RNA molecule; introducing into said cells a composition comprising a complex of the CRISPR nuclease and the first RNA molecule that makes a double-stranded break upstream of the intronic trinucleotide CTG repeat expansion, and a complex of the CRISPR nuclease and the second RNA molecule that makes a double-stranded break downstream of the intronic trinucleotide CTG repeat expansion; The first guide sequence portion comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1216 to 2325, and the second guide sequence portion comprises 17 to 50 consecutive nucleotides within the sequence shown in any one of SEQ ID NOs: 1 to 1215.

[0046] In some embodiments, the RNA molecule is a crRNA molecule and the composition further contains a tracrRNA molecule that forms a crRNA:tracrRNA molecule with the crRNA molecule. In some embodiments, the RNA molecule is an sgRNA molecule.

[0047] In some embodiments, the first guide sequence portion comprises 17-50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1216-2325, the second guide sequence portion comprises 17-50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1-1215, and at least one of the TCF4 alleles in the cell has a heterozygous SNP at position rs34071688.

[0048] In some embodiments, the first guide sequence portion comprises 17-50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1216-2325, the second guide sequence portion comprises 17-50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1-1215, and at least one of the TCF4 alleles in the cell has a heterozygous SNP at one of positions selected from the group consisting of rs746872826, 18:5558615, rs879522127, and rs1268568114.

[0049] In some embodiments, the first guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 1215, the second guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 1215, and at least one of the TCF4 alleles in the cell is selected from the group consisting of rs71670792, rs1261085, rs1261084, rs11431395, rs373174214, rs35555522, rs66807288, rs141970461, rs71674214, rs8766, rs10221362 , rs2276195, rs569385112, rs398041379, rs72925008, rs8090085, rs5825130, rs56887277, rs397836157, rs55755941, rs781691419, rs11409023, r s10221357, rs11661961, rs11660565, rs61656112, rs60565673, rs72925018, rs1261073, rs1261076, rs748718974, rs13381800, rs34773632, rs1942 265, rs7241077, rs62092440, rs1893431, rs3838898, rs1893430, 18:55251930_G_GTTTT, rs771385941, rs4800988, rs1942264, rs1261093, rs15399 50, rs1539951, rs62092442, rs62092444, rs11662842, rs11664992, rs1261134, rs1261114, rs1788027, rs397858367, rs113662542, rs1261118, rs10 701336, rs397809469, rs150323043, rs1038226655, rs149454001, rs796169498, rs9955026, rs1046741326, rs754435392, rs867471715, rs77760825 6, rs762153709, rs1153636, rs1153637, rs5825134, rs35480166, rs893946, rs374155330, rs781053385, rs899293868, rs996440450, rs1349129287,rs780424692、rs34702622、rs11428164、rs1660235、rs1440473、rs1788026、rs11332509、rs1660237、rs1631486、rs1025804、rs753933037、rs1660233、rs200650987、rs71951255、rs368762262、rs751007744、rs780342991、rs1660241、rs796749696、rs61468075、rs777518462、rs1660242、rs1440476、rs1011392、18:55374871_T_TA、rs1788030、rs1623427、rs1621581、rs1788025、rs1788023、rs1348047、rs1788019、rs9950000、rs9958125、rs9320010、rs3794891、rs757629087、rs772409228、rs12607679、rs3794889、rs4801149、rs12605773、rs2872041、rs4801150、rs1020169、rs7238888、rs7235757、rs2958178、rs2958165、rs2958171、rs1328839434、rs796792902、rs2958175、rs11309751、rs2958186、rs2919446、rs2958161、rs2860511、rs2958162、rs2919451、rs2919450、rs201657057、rs2958163、rs1440477、rs2958166、rs377458803、rs2958169、rs8098843、rs11412305、rs386387765、18:55423173_T_TA、rs781071274、rs4374254、rs370693034、rs761981780、rs8084308、rs77540208、rs9320016、rs4524013、rs1025639279、rs4500831、rs7229456、rs12967143、rs12963334、rs12963463、rs398100891、rs12958048、18:55434419_C_CTTT、rs375388593、rs140134419、rs4801153、rs4801154, rs745460290, rs527450659, rs4341827, rs4468713, rs7228159, rs145330990, rs7231748, rs34577882, rs34578042, rs1452789, rs14527 88, rs12606995, rs188225813, rs732779, rs11385247, rs9966430, rs2924321, rs151196106, rs3760600, rs2924328, rs1377243, rs2924329, rs58251 42、rs199707137、18:55468891_C_CCCA、rs11338618、rs149728054、rs112 98284、rs7233312、rs2924331、18:55480276_C_CAA、rs2958182、rs2958183 rs2958184、rs2924332、rs2924333、rs2060889、rs2958187、rs2924335、r s138885827、rs2924336、rs59413482、rs796565215、18:55496896_C_CAA、r s4801157、rs2958188、rs2958189、rs2060886、rs3017183、rs2958158、rs2 924338、rs12956276、rs55812411、rs776881842、rs1452791、rs9957668、rs 9954890, rs9964328, rs67387556, rs1491335073, 18:55511330_T_TAAA, rs751932079, rs2957261, 18:55511331_T_TAAAA, rs8090106, rs140221855 rs17089851, rs398032944, rs1341922999, rs624244, rs627685, rs9948513, rs9965067, rs9965195, rs35371867, rs11441646, rs9949107, rs7240986 rs4801158, rs72627231, rs11412432, rs33938531, rs4800990, rs4458089, rs4572488, rs12968271, rs9636107, rs2123389, rs9947814, rs71352207The patient has a heterozygous SNP at any of the SNP positions selected from the group consisting of rs1452787, rs2123392, rs2123393, 18:55559041_T_TA, rs34935191, rs74182105, rs139870092, rs76053687, rs150848781, rs564960433, rs41396445, and rs34232463.

[0050] In some aspects, the composition is introduced into cells of a subject or into cells in culture.

[0051] In some aspects, the cells are corneal cells or corneal endothelial cells.

[0052] In some embodiments, the composition is introduced into a cell in vivo.

[0053] In some embodiments, the composition is introduced into the cell by a lentivirus-like particle (LVLP).

[0054] In some aspects, the cells are stem cells, fibroblasts, blood cells, hepatocytes, keratinocytes, other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPS cells), iPS-derived cells, or iPS-derived corneal endothelial cells.

[0055] In some embodiments, the composition is introduced into the cell ex vivo.

[0056] In some embodiments, the CRISPR nuclease, the first RNA molecule, and the second RNA molecule are introduced into the cell substantially simultaneously or at different times.

[0057] In some embodiments, the first or second RNA molecule is a crRNA molecule or an sgRNA molecule.

[0058] In an embodiment of the present invention, a composition is provided that contains an RNA molecule having a guide sequence portion comprising 17 to 50 consecutive nucleotides within a sequence shown in any one of SEQ ID NOs: 1 to 2325.

[0059] In some embodiments, the composition further comprises a second RNA molecule, wherein the first RNA molecule has a guide sequence portion comprising 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1216 to 2325, and the second RNA molecule has a guide sequence portion comprising 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1 to 1215.

[0060] In some embodiments, the composition further comprises a CRISPR nuclease.

[0061] In some embodiments, the composition further comprises a tracrRNA molecule.

[0062] In embodiments of the invention, cells modified by any of the disclosed methods or using any of the disclosed compositions are provided.

[0063] In some aspects, the cells are corneal cells or corneal endothelial cells.

[0064] In some aspects, the cells are stem cells or other cells that can be reprogrammed into fibroblasts, blood cells, hepatocytes, keratinocytes, or other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), iPS-derived cells, or iPS-derived corneal endothelial cells.

[0065] In some aspects, the stem cells differentiate after being modified.

[0066] In some aspects, the stem cells differentiate into corneal cells or corneal endothelial cells.

[0067] In an embodiment of the invention, there is provided a method of treating Fuchs' endothelial corneal dystrophy (FECD) in a human subject, said method comprising delivering to said subject any of the compositions described herein or transplanting any of the modified cells described herein into the cornea of ​​said human subject.

[0068] In some embodiments, the composition is delivered to cells of the patient's cornea in vivo.

[0069] In some embodiments, the composition is introduced into the cell by a lentivirus-like particle (LVLP).

[0070] In some embodiments, the modified cells are delivered ex vivo to the patient's cornea.

[0071] In an embodiment of the invention, there is provided a pharmaceutical composition for use in excising an intronic trinucleotide CTG repeat expansion in an allele of TCF4 in a cell, the pharmaceutical composition comprising any of the compositions described herein, the composition being delivered to the cell.

[0072] In embodiments of the invention, there is provided the use of any of the compositions described herein or any of the modified cells described herein in the treatment of FECD, comprising delivering said composition or said modified cells to a subject suffering from or at risk of suffering from FECD.

[0073] In embodiments of the invention, a medicament for use in treating FECD is provided that contains any of the compositions described herein or any of the modified cells described herein, and the medicament delivers the composition or the modified cells to a subject suffering from or at risk of suffering from FECD.

[0074] In an embodiment of the invention, a kit is provided for excising an intronic trinucleotide CTG repeat expansion from an allele of TCF4 in a cell, comprising any of the compositions described herein and instructions for delivering the composition to a cell.

[0075] In some embodiments, the composition is delivered to cells ex vivo.

[0076] In embodiments of the invention, kits are provided for treating FECD in a subject, comprising any of the compositions described herein or any of the modified cells described herein and instructions for delivering the composition or modified cells to a subject suffering from or at risk of suffering from FECD.

[0077] In embodiments of the invention, there is provided any of the compositions described herein or any of the modified cells described herein for use in treating FECD, comprising delivering said composition or said modified cells to a subject suffering from or at risk of suffering from FECD.

[0078] In some embodiments, the first or second RNA molecule in the methods and compositions described herein may be modified in its guide sequence portion to contain 1, 2, 3, 4, or 5 nucleotide mismatches to the target sequence, which in some embodiments provide greater target specificity to the CRISPR nuclease-RNA molecule complex than would be provided by a guide sequence portion that is highly complementary to the target sequence.

[0079] In some embodiments, a gene editing composition is provided that includes an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1 to 2325. In some embodiments, the RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease. In some embodiments, the sequence that binds to a CRISPR nuclease is a tracrRNA sequence.

[0080] In some embodiments, the RNA molecule further comprises a portion having a tracr mate sequence.

[0081] In some embodiments, the RNA molecule may further comprise one or more linker moieties.

[0082] In embodiments of the invention, the length of the RNA molecule may be at most 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleotides. Each possibility represents a separate embodiment. In embodiments of the invention, the length of the RNA molecule may be 17 to at most 300 nucleotides, 100 to at most 300 nucleotides, 150 to at most 300 nucleotides, 100 to at most 500 nucleotides, 100 to at most 400 nucleotides, 200 to at most 300 nucleotides, 100-200 nucleotides, or 150 to at most 250 nucleotides. Each possibility is a separate aspect.

[0083] In some embodiments of the invention, the composition further comprises a tracrRNA molecule.

[0084] Some embodiments of the invention provide methods for excising an intronic trinucleotide CTG repeat expansion from an allele of transcription factor 4 (TCF4) in a cell, the method comprising delivering to the cell a CRISPR nuclease and a composition containing an RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1216-2325, and a second RNA molecule comprising a guide sequence portion having 17 to 50 contiguous nucleotides within any of SEQ ID NOs: 1-1215 and a CRISPR nuclease.

[0085] In embodiments of the invention, at least one CRISPR nuclease and an RNA molecule, or the RNA molecules, are delivered to a subject and / or cell at substantially the same time or at different times.

[0086] In some embodiments, the tracrRNA molecule is delivered to a subject and / or cell at substantially the same time or at different times as the CRISPR nuclease and the RNA molecule, or as the RNA molecule.

[0087] The compositions and methods of this disclosure can be used to treat Fuchs endothelial corneal dystrophy (FECD).

[0088] Any one or a combination of the above strategies for inactivating TCF4 expression can be used in the present invention.

[0089] Embodiments of the compositions described herein include at least one CRISPR nuclease, an RNA molecule, and a tracrRNA molecule that are simultaneously effective in a subject or cell. The at least one CRISPR nuclease, the RNA molecule, and the tracrRNA can be delivered substantially simultaneously, or can be delivered at different times and simultaneously effective. For example, this includes delivering the CRISPR nuclease to a subject or cell before the RNA molecule and / or the tracrRNA are substantially present in the subject or cell.

[0090] In some embodiments, the cells are corneal cells, hi some embodiments, the cells are corneal endothelial cells, hi some embodiments, the cells are stem cells, or fibroblasts, blood cells, hepatocytes, keratinocytes, or other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs).

[0091] TCF4 editing strategy to excise CTG expanded repeats The TCF4 gene encodes transcription factor 4, a basic helix-loop-helix transcription factor. The encoded protein recognizes the Ephrussi box (E-box) binding site (CANNTG), a motif originally identified in immunoglobulin enhancers. This gene is widely expressed and may play an important role in nervous system development. Defects in this gene are the cause of Pitt-Hopkins syndrome. Multiple alternatively spliced ​​transcript variants encoding distinct proteins have been reported.

[0092] Furthermore, the CTG repeats in the TCF4 intron, which normally range from 10 to 37 repeats, can increase to more than 50 repeats and cause disease. Specifically, the causative mutation of Fuchs endothelial corneal dystrophy is a heterozygous trinucleotide repeat expansion (CTG)n in the TCF4 intron. Because the CTG repeats are transcribed bidirectionally and cause disease, treatment strategies include excision of the CTG repeats from the TCF4 allele.

[0093] Thus, the present invention provides a method for excising a CTG trinucleotide repeat (TNR) from at least one TCF4 allele using two guide molecules. The repeat is located in intron 3 of TCF4, starting at 18:55587044 and ending at 18:55585353.

[0094] One strategy is to perform biallelic excision of the TCF4 repeat. This can be achieved by utilizing guides flanking the expanded repeat in intron 3 of TCF4. For example, one guide can target the region hg38_chr18:55585922 to 55586155 (downstream of the repeat), and a second guide can target the region hg38_chr18:55586227 to 55586483 (upstream of the repeat) to excise the expanded TCF4 repeat.

[0095] Excision of one allele of the TCF4 expanded repeat can be achieved using a first guide that targets a SNP located upstream or downstream of the expanded repeat in intron 3, and a second, non-discriminatory guide that targets a sequence within intron 3. For example, excision of the TCF4 expanded repeat may be carried out using a first guide that targets the SNP position rs34071688 located upstream of the repeat, and a second, non-discriminatory guide that targets a sequence located downstream of the repeat.

[0096] Excision of one allele of the TCF4 expanded repeat can also be achieved by knocking out the allele with the expanded repeat and simultaneously excising the expanded repeat. This can be done using guides targeting SNPs located in various regions of the gene, such as exons, introns, promoter regions, or intergenic regions downstream or upstream of the gene. To enable excision of the gene fragment containing the CTG repeat, a non-discriminatory guide should be located in intron 3. This strategy leads to knockout of the allele with the expanded repeat by excision of a large fragment that blocks its transcription, destabilizes the transcript, and generates a premature stop codon, resulting in nonsense-mediated degradation or a truncated protein.

[0097] In some embodiments, one guide molecule targets upstream of the TNR and the other guide molecule targets downstream of the TNR. Excision of a TNR without altering normal expression of TCF4 can be achieved, for example, by excising a CTG TNR with two guides that target up to 350 nucleotides upstream and 250 nucleotides downstream of the TNR.

[0098] In some embodiments, the target cells are corneal cells. In some embodiments, the cells are corneal endothelial cells. In some embodiments, the delivery of the guide molecule is performed in vivo. In some embodiments, the delivery is performed using lentivirus-like particles (LVLPs).

[0099] Alternatively, cells such as iPS-derived endothelial cells can be edited ex vivo and transplanted into the cornea, or iPS cells can be edited, differentiated, and transplanted into the cornea.

[0100] CRISPR nucleases and PAM recognition In some embodiments, the sequence-specific nuclease is selected from a CRISPR nuclease or a functional variant thereof. In some embodiments, the sequence-specific nuclease is an RNA-guided DNA nuclease. In such embodiments, an RNA sequence that guides the RNA-guided DNA nuclease (e.g., Cpf1) binds to and / or directs the RNA-guided DNA nuclease to all TCF4 alleles in the cell. In some embodiments, the CRISPR complex does not further comprise a tracrRNA. One of skill in the art will appreciate that RNA molecules can be engineered to bind to selected targets in the genome using methods commonly known in the art.

[0101] As used herein, the term "PAM" refers to a nucleotide sequence in a target DNA that is located adjacent to the target DNA sequence and recognized by a CRISPR nuclease complex. PAM sequences may vary depending on the type of nuclease. In addition, there are CRISPR nucleases that can target almost any PAM. In some embodiments of this invention, the CRISPR system utilizes one or more RNA molecules containing a guide sequence portion that guides the CRISPR nuclease to the target DNA site by forming Watson-Crick base pairs between the guide sequence portion and the protospacer of the target DNA site adjacent to the protospacer adjacent motif (PAM), an additional requirement for target recognition. The CRISPR nuclease then cleaves the target DNA site, creating a double-stranded break within the protospacer. In a non-limiting example, a Type II CRISPR system utilizes a mature crRNA:tracrRNA complex that guides a CRISPR nuclease (e.g., Cas9) to a target DNA by forming Watson-Crick base pairs between the guide sequence portion of the crRNA and a protospacer on the target DNA adjacent to a protospacer adjacent motif (PAM). One of skill in the art will understand that the engineered RNA molecules of the present invention are further designed to bind to a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM), e.g., a PAM corresponding to a sequence associated with the CRISPR nuclease being utilized.The PAM may be, for example, but not limited to, NGG or NAG (where N is any nucleobase) for Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Jejuni Cas9 WT; NGAN or NGNG for SpCas9-VQR variants; NGCG for SpCas9-VRER variants; NGAG for SpCas9-EQR variants; NRRH for SpCas9-NRRH variants (where N is any nucleobase, R is A or G, and H is A, C, or T); NRTH for SpCas9-NRTH variants (where N is any nucleobase, R is A or G, and H is A, C, or T); or SpCas9-NRCH variants (where N is any nucleobase, R is A or G, and H is A, C, or T). NRCH (where N is any nucleobase, R is A or G, and H is A, C, or T) for the SpG variant of SpCas9; NG (where N is any nucleobase); NG or NA (where N is any nucleobase) for the SpCas9-NG variant of SpCas9; NR, NRN, or NYN (where N is any nucleobase, R is A or G, and Y is C or T) for the SpRY variant of SpCas9; and Streptococcus canis (Streptococcus canis Cas9 variant (ScCas9), NNG (where N is any nucleobase); NNNRRT (where N is any nucleobase and R is A or G) for Staphylococcus aureus SaKKH-Cas9 variant (SaCas9); NNNNGATT (where N is any nucleobase) for Neisseria meningitidis (NmCas9); TTN (where N is any nucleobase) for Alicyclobacillus acidophilus Cas12b (AacCas12b); or TTTV (where V is A, C, or G) for Cpfl. The RNA molecules of the invention are each designed to form a complex with one or more different CRISPR nucleases and to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0102] In some embodiments, RNA-guided DNA nucleases (e.g., CRISPR nucleases) may be used to create double- or single-stranded DNA breaks at desired locations in a cell's genome. While the most commonly used RNA-guided DNA nucleases are derived from CRISPR systems, other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. See, e.g., U.S. Patent Application Publication No. 2015 / 0211023, which is incorporated herein by reference.

[0103] There are a variety of CRISPR systems that can be used to practice the present invention. The CRISPR system may be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl These include Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.

[0104] In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease derived from a type II CRISPR system (e.g., Cas9). CRISPR nucleases have been shown to inhibit the expression of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, and Streptosporangium roseum. roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas species sp.), Crocosphaera watsonii, Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.CRISPR nucleases may be derived from Bacillus subtilis, Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or any species encoding a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria may also be used in the present invention (see Burstein et al., Nature, 2017). Variants of CRISPR proteins with known PAM sequences, such as the SpCas9 D1135E variant, SpCas9 VQR variant, SpCas9 EQR variant, or SpCas9 VRER variant, may also be used in the present invention.

[0105] Therefore, RNA-guided DNA nucleases of the CRISPR system, such as Cas9 protein or modified Cas9, or homologs or orthologs of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems, such as Cpf1 and its homologs and orthologs, may be used in the compositions of the invention. Other CRISPR nucleases, such as those described in WO2020 / 223514 and WO2020 / 223553 (incorporated herein by reference), may also be used.

[0106] In certain embodiments, the CRISPR nuclease may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that shares qualitative biological properties with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence polypeptides and their fragments that share biological activity with the corresponding native sequence polypeptide. The contemplated biological activity is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of the polypeptide and covalently modified and fusion forms thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include, but are not limited to, mutants, fusions, and covalently modified forms of the Cas protein or a fragment thereof. Derivatives include, but are not limited to, CRISPR nickases, catalytically inactive or "dead" CRISPR nucleases, and fusions of CRISPR nucleases or derivatives thereof with other enzymes, such as base editors or retrotransposons. See, for example, Anzalone et al. (2019) and PCT International Patent Application No. PCT / US2020 / 037560.

[0107] In some embodiments, the CRISPR nuclease or its derivative may be fused to a protein having enzymatic activity. In some embodiments, the enzymatic activity modifies target DNA. In some embodiments, the enzymatic activity is nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity. In some embodiments, the enzymatic activity is nuclease activity. In some embodiments, the enzymatic activity introduces double-strand breaks in target DNA. In some embodiments, the enzymatic activity modifies a target polypeptide associated with target DNA. In some cases, the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, or a demyristoylating activity. In some cases, the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, or a deubiquitinating activity.

[0108] Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, may be obtainable from cells, may be chemically synthesized, or may be obtained by a combination of these methods. The cells may be cells that naturally produce Cas proteins, or may be cells that naturally produce Cas proteins and have been genetically engineered to produce higher expression levels of endogenous Cas proteins or to produce Cas proteins from introduced exogenous nucleic acids encoding the same or different Cas proteins as the endogenous Cas. In some cases, the cells do not naturally produce Cas proteins but are genetically engineered to produce Cas proteins.

[0109] In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease that utilizes a T-rich protospacer adjacent motif. Cpf1 makes staggered DNA double-strand breaks. Two Cpf1 enzymes from Acidaminococcus and Lachnospiraceae have been shown to efficiently edit genomes in human cells (see Zetsche et al., 2015).

[0110] Thus, RNA-guided DNA nucleases of Type II CRISPR systems, such as the Cas9 protein or modified Cas9, or homologs, orthologs or variants of Cas9, or other RNA-guided DNA nucleases belonging to other CRISPR systems, such as Cpfl and its homologs, orthologs or variants, may be used in the present invention.

[0111] In some embodiments, the guide molecule comprises one or more chemical modifications that confer new or improved properties (e.g., stability against degradation, hybridization energy, binding to RNA-guided DNA nucleases). Suitable chemical modifications include, but are not limited to, modified bases, modified sugars, or modified internucleoside linkages. Non-limiting examples of suitable chemical modifications include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methylpseudouridine, β,D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methyladenosine, 1-methylguano ... Chirpseudouridine, 1-methylguanosine, 1-methylinosine, "2,2-dimethylguanosine", 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, "β,D-mannosylqueosine", 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine Uridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid methyl ester, uridine-5-oxyacetic acid, wybutoxocin, queusine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine Examples of uridine include uridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine, 2'-O-methyl-5-methyluridine, 2'-O-methyluridine, wybutosine, "3-(3-amino-3-carboxypropyl)uridine, (acp3)u", 2'-O-methyl (M), 3'-phosphorothioate (MS), 3'-thioPACE (MSP), pseudouridine, or 1-methylpseudouridine.Each realization of the invention is a separate embodiment.

[0112] In addition to targeting TCF4 alleles with RNA-guided CRISPR nucleases, other means of blocking TCF4 expression in target cells include, but are not limited to, the use of gapmers, shRNAs, siRNAs, customized TALENs, meganucleases, zinc finger nucleases, small molecule inhibitors, and other methods known in the art to reduce or eliminate gene expression in target cells (see, for example, U.S. Patent Nos. 6,506,559; 7,560,438; 8,420,391; 8,552,171; 7,056,704; 7,078,196; 8,362,231; 8,372,968; 9,045,754; WO 2004 / 067736; WO 2006 / 097853). No. 2003 / 087341; No. 2000 / 041566; No. 2003 / 080809; No. 2010 / 079430; No. 2010 / 079430; No. 2011 / 072246; No. 2018 / 057989 and No. 2017 / 164230 (the entire contents of which are incorporated herein by reference).

[0113] Advantageously, the guide RNA molecules provided herein, when complexed with CRISPR nucleases in cells, improve TCF4 knockout efficiency compared to other guide RNA molecules. These specially designed sequences may also be useful for identifying target sites in TCF4 for other nucleotide-targeting-based gene editing or gene silencing methods (e.g., siRNA, TALEN, meganucleases, or zinc finger nucleases).

[0114] Cellular delivery The compositions described herein may be delivered to target cells by any suitable means. The RNA molecule compositions of the present invention may be targeted to cells containing and / or expressing TCF4 alleles, such as corneal endothelial cells or stem cells. Delivery to cells may be in vivo, ex vivo, or in vitro. In some embodiments, the compositions are delivered using lentivirus-like particles (LVLPs).

[0115] Additionally, the nucleic acid compositions described herein may be delivered to a cell as one or more of a DNA molecule, an RNA molecule, a ribonucleoprotein (RNP), a nucleic acid vector, or a combination thereof.

[0116] In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS) or 2'-O-methyl, 3' thio PACE (MSP), pseudouridine, and 1-methylpseudouridine. Each realization of this invention is a separate embodiment.

[0117] Nucleic acid compositions (e.g., compositions of RNA molecules of the invention) may be delivered using an appropriate viral vector system. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into target tissues. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy. Non-viral vector delivery systems include naked nucleic acid and nucleic acid complexed with a delivery vehicle (e.g., liposomes, poloxamers). For reviews of gene therapy, see Anderson (1992); Nabel & Felgner (1993); Mitani & Caskey (1993); Dillon (1993); Miller (1992); Van Brunt (1988); Vigne (1995); Kremer & Perricaudet (1995); Haddada et al. (1995), and Yu et al. (1994).

[0118] Non-viral methods of nucleic acid and / or protein delivery include electroporation, lipofection, microinjection, biolistic bombardment, particle gun acceleration, uptake of nucleic acids by virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycation or lipid:nucleic acid conjugates, artificial virions, and facilitators, or nucleic acids and / or proteins can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizobium meliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus, cassava vein mosaic virus) (see, e.g., Chung et al., J. Immunol. 2004, 103:111-114). (See Zuris et al. (2015); Coelho et al. (2013); Judge et al. (2006); and Basha et al. (2011). Sonoporation, for example using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. Cationic lipid delivery of proteins and / or nucleic acids is also contemplated for in vivo, ex vivo, or in vitro delivery (see Zuris et al. (2015); Coelho et al. (2013); see also Judge et al. (2006); and Basha et al. (2011)).

[0119] Non-viral vectors, such as transposon-based systems (e.g., recombinant Sleeping Beauty transposon system, recombinant PiggyBac transposon system), may be delivered to target cells and used to transpose the polynucleotide sequences of or encoding the molecules of the composition in the target cells.

[0120] Other representative nucleic acid delivery systems include those offered by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and Lipofectamine® RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO 91 / 17424 and WO 91 / 16024. Delivery to cells (ex vivo administration) or target tissues (in vivo administration) is possible.

[0121] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (see, e.g., Crystal, Science (1995); Blaese et al., (1995); Behr et al., (1994); Remy et al. (1994); Gao and Huang (1995); Ahmad and Allen (1992); U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0122] Another method of delivery involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). The EDV is specifically delivered to the target tissue using a bispecific antibody, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody carries the EDV to the surface of the target cell, where it is transported into the cell by endocytosis. After entering the cell, the contents are released (see MacDiarmid et al., 2009).

[0123] The use of RNA or DNA viral systems for viral delivery of nucleic acids takes advantage of the highly evolved methods that viruses use to target specific cells in the body and transport their viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or can be used to engineer cells in vitro and the modified cells are then administered to patients (ex vivo). Traditional viral systems for nucleic acid delivery include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia viral, and herpes simplex viral vectors for gene transfer.

[0124] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and are capable of packaging up to 6-10 kb of foreign sequence. A minimal set of cis-acting long terminal repeats is sufficient for vector replication and packaging, allowing therapeutic genes to be integrated into target cells and persistently express the transgene. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchschacher et al. (1992); Johann et al. (1992); Sommerfelt et al. (1990); Wilson et al. (1989); Miller et al. (1991); WO 94 / 26877).

[0125] At least six viral vectors are currently available for gene transfer in clinical trials, which utilize an approach involving complementation of a defective vector with a gene inserted into a helper cell line to prepare the transducing agent.

[0126] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (see Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317 / pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al., 1995). Transduction efficiencies of MFG-S packaging vectors were greater than 50% (Ellem et al., (1997); Dranoff et al., 1997).

[0127] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus and AAV, and Psi-2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are typically generated by producer cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences required for packaging and (if applicable) subsequent integration into the host, with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeats (ITRs) from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line that lacks other AAV genes, i.e., ITR sequences, but contains a helper plasmid encoding rep and cap. The cell line is also infected with adenovirus as a helper. The helper virus promotes AAV vector replication and AAV gene expression from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Furthermore, AAV can be produced on a clinical scale using the baculovirus system (see U.S. Pat. No. 7,479,554).

[0128] In many gene therapies, it is desirable for gene therapy vectors to be delivered with high specificity to specific tissues. Therefore, viral vectors can be engineered to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present in the target cell. For example, Han et al. (1995) reported that Moloney murine leukemia virus can be engineered to express human heregulin fused to gp70 and that the recombinant virus infects specific human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell combinations, where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB, Fv) with specific binding affinity for virtually any selected cellular receptor. While this discussion applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences that favor uptake by specific target cells.

[0129] Gene therapy vectors can be delivered in vivo by administration to an individual patient, for example, by systemic administration (eg, intravitreal, intravenous, intraperitoneal, intramuscular, subcutaneous, intracranial injection) or by local application.

[0130] Alternatively, vectors can be delivered ex vivo to cells, e.g., cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into the patient after selection of cells that have incorporated the vector. Non-limiting exemplary ex vivo methods may include removing tissue (e.g., peripheral blood, bone marrow, spleen) from the patient for culture, transferring the nucleic acid into the cultured cells (e.g., hematopoietic stem cells), and then transplanting the cells into the patient's target tissue (e.g., bone marrow, spleen). In some embodiments, the stem cells or hematopoietic stem cells may be further treated with a survival-enhancing agent.

[0131] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., by re-infusion of the transfected cells into a host) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid composition, and re-infused into the subject (e.g., patient). A variety of cells suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney, Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications (6th edition, 2010) and references cited therein in the discussion of methods for isolating and culturing cells from patients).

[0132] Alternatively, vectors (e.g., retroviruses, liposomes) carrying therapeutic nucleic acid compositions can be administered directly to an organism for transduction of cells in vivo. Administration is by routes typically used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application (e.g., eye drops, creams), and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while multiple routes of administration for a particular composition can be used, certain routes often provide a more rapid and effective response than others. In some embodiments, the composition is delivered by IV injection.

[0133] Suitable vectors for introducing transgenes into cells include non-integrating lentiviral vectors, see, e.g., U.S. Patent Application Publication No. 2009 / 0117617.

[0134] Pharmaceutically acceptable carriers are determined in part by the composition being administered, as well as by the method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions available, for example, as described in Remington's Pharmaceutical Sciences, 17th ed., 1989.

[0135] Example of an RNA guide sequence that specifically targets an allele of the TCF4 gene While numerous guide sequences can be designed to target the TCF4 gene, the nucleotide sequences shown in Table 1 and identified by SEQ ID NOs: 1-2325 were specifically selected to effectively carry out the methods described herein.

[0136] Guide sequences designed for use in the previously described embodiments to link TCF4 target sequences are shown in Table 1. Each engineered guide molecule is further designed to bind to a target genomic DNA sequence of interest adjacent to a protospacer adjacent motif (PAM) (e.g., a PAM corresponding to the sequence NGG or NAG (where N is any nucleobase)). The guide sequences are designed to work with one or more different CRISPR nucleases, including, for example, SpCas9WT (PAM sequence: NGG), SpCas9.VQR.1 (PAM sequence: NGAN), SpCas9.VQR.2 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.3 (PAM sequence: NGGG), SpCas9.VQR.4 (PAM sequence: NGGG), SpCas9.VQR.5 (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.6 (PAM sequence: NGGG), SpCas9.VQR.7 (PAM sequence: NGGG), SpCas9.VQR.8 (PAM sequence: NGGG), SpCas9.VQR.9 (PAM sequence: NGGG), SpCas9.VQR.10 (PAM sequence: NGAN), SpCas9.VQR.11 (PAM sequence: NGAN), SpCas9.VQR.12 (PAM sequence: NGNG), SpCas9.EQR (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT (PAM sequence: NNGRRT), SpCas9.VQR.13 (PAM sequence: NGAG), SpCas9.VRER (PAM sequence: NGCG), SpCas9WT ( Examples of CRISPR nucleases include, but are not limited to, NmCas9WT (PAM sequence: NNNNGATT), Cpf1 (PAM sequence: TTTV), JeCas9WT (PAM sequence: NNNVRYM), OMNI-50 (PAM sequence: NGG), OMNI-79 (PAM sequence: NGG), OMNI-103 (PAM sequence: NNRACT), OMNI-159 (NNNNCMAN), or OMNI-124 (PAM sequence: NNGNRMNN). The RNA molecules of the present invention are each designed to form a complex with one or more different CRISPR nucleases and are designed to target a polynucleotide sequence of interest using one or more different PAM sequences corresponding to the CRISPR nucleases.

[0137] OMNI nucleases are further described in WO 2023 / 019269, WO 2022 / 170199, WO 2023 / 107946, U.S. Pat. No. 11,666,641, WO 2020 / 223514, WO 2022 / 098693, WO 2023 / 019263, U.S. Patent Application Publication No. 2023 / 0122086, WO 2021 / 248016, WO 2023 / 102407, WO 2022 / 087135, and WO 2022 / 226215, the contents of which are incorporated herein by reference.

[0138] In this specification, the following nucleotide identifiers are used to represent nucleotide bases:

[0139] [Table B]

[0140] [Table 1]

[0141] Examples are presented to facilitate a more complete understanding of the present invention. The following examples illustrate representative modes of making and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only. [Example]

[0142] Experiment details Example 1 Target analysis of the TCF4 guide sequence portion Guide sequences containing 17 to 50 consecutive nucleotides within any of SEQ ID NOs: 1 to 2325 are screened for high on-target activity in human cells using CRISPR nucleases. On-target activity is determined by DNA capillary electrophoresis.

[0143] Example 2 Excision of TCF4 Expanded Repeats The trinucleotide repeat CTG18.1 is located in intron 3 of the TCF4 gene. Up to 75% of FECD patients have at least one TCF4 allele with a CTG18.1 expansion (more than 50 copies of "CTG"). To excise the repeat for FECD treatment, we designed four sgRNAs upstream and downstream of the repeat (see Figure 1), and tested the activity of each sgRNA using SpCas9 in U2OS cells.

[0144] In simple terms, 2 x 10 5 Cells were mixed with a preassembled RNP consisting of 105 pmol of SpCas9 protein (Alt-R® Sp Cas9 Nuclease V3, 1081059, IDT), 124 pmol of sgRNA (Alt-Rs™ CRISPR-Cas9 sgRNA, IDT), and 100 pmol of electroporation enhancer (Alt-R® Cas9 Electroporation Enhancer, 1075916). Cells were then electroporated using the SE cell 4D-nucleofecuor X Kit S (V4XC-1032, Lonza) with the DN-100 program. Cell fractions were harvested 72 hours after electroporation, and genomic DNA was extracted and analyzed by next-generation sequencing (NGS) to measure on-target activity. NGS analysis showed that all eight guides showed very high activity, with editing observed in over 90% of cells (Figure 2).

[0145] To confirm that the removal of the repeat region does not affect TCF4 expression, we performed excision of the repeat region with different guide combinations (upstream and downstream of the repeat) in U2OS cells and tested TCF4 expression by qRT-PCR and Western blot. 5Cells were mixed with pre-assembled RNPs consisting of 105 pmol of SpCas9 protein (Alt-R® Sp Cas9 Nuclease V3, 1081059, IDT) and 124 pmol of each sgRNA (Alt-R™ CRISPR-Cas9 sgRNA, IDT). These RNP combinations were mixed with 100 pmol of electroporation enhancer (Alt-R® Cas9 Electroporation Enhancer, 1075916) at the time of RNP formation and electroporated using the SE cell 4D-nucleofecuor X Kit S (V4XC-1032, Lonza) with the DN-100 program. Cells were grown for at least 7 days to obtain enough cells for genomic DNA, RNA, and protein extraction. After RNA extraction, cDNA was synthesized and qRT-PCR was performed using fast SYBR primers and primers specific for the exon 11-exon 12 junction of TCF4 (Figure 3). For Western blot analysis, cells were lysed in RIPA buffer, and 40 μg of total protein was separated by SDS-PAGE. TCF4 protein levels were detected using a specific TCF4 antibody (ab217668, Abcam). GAPDH was used as a loading control (Figure 4).

[0146] As shown, only excision by gRNA2+gRNA5 and gRNA2+gRNA7 reduced TCF4 mRNA and protein levels.

[0147] To verify the level of excision, we designed a ddPCR assay (QX200, BioRad) using a probe specific for the g4 + g6 excision pattern so that only excised fragments showed a positive signal (FAM). RPP30 was used as an endogenous control (HEX). The results showed that the excision rate of the g4 + g6 combination was 42% (Figure 5).

[0148] A summary of the TCF4 guide sequence segments tested is shown below.

[0149] [Table C]

[0150] Example 3 Screening of guide sequence portion Using several OMNI CRISPR nucleases, we screened different guides in HeLa cells, providing proof-of-concept that the guides can make double-strand breaks upstream and downstream of the TCF4 repeat expansion.

[0151] A summary of the TCF4 guide sequence segments tested is shown below. Briefly, the sgRNA activity is shown targeting TCF4 intron 3 around the expanded repeat CTG18.1 locus. To test the activity of each guide with the indicated nucleases, HeLa cells were transfected with the nucleases and sgRNAs, and activity was measured by next-generation sequencing (NGS) 3 days after transfection.

[0152] [Table D-1]

[0153] [Table D-2]

[0154] A table listing the amino acid sequences of the CRISPR nucleases and sgRNA scaffolds used is provided below:

[0155] [Table E]

[0156] Example 4 Evaluation of Removal - Transfection in HeLa Cells To measure the excision level of the CTG18.1 expansion repeat, two excision compositions were examined by droplet digital PCR (ddPCR). Briefly, HeLa cells were transfected with α-nuclease and two sgRNAs using a JetOptimus transfection system. To assess the activity of each guide in this particular experiment, each sgRNA was also transfected individually. Cells were harvested 3 days after transfection for next-generation sequencing (NGS) (Figure 6A) and 10 days after transfection for genome extraction and ddPCR (Figure 6B). mCherry was used as a reporter of transfection efficiency and quantified by flow cytometry. Excision was measured using the EvaGreen "gain of signal" assay (QX200, BioRad) with primers surrounding the excision region. In the absence of excision, the amplified amplicon would be too long (>200 bp) to be detected by ddPCR, and a signal would only appear after excision of an amplified sequence less than 200 bp. Signals were normalized to the RPP30 housekeeping gene. With OMNI-103 nuclease, sgRNA10 and sgRNA13 resulted in 34.77% and 50.89% editing and 8.5% excision, respectively. With OMNI-110 nuclease, sgRNA183 and sgRNA189 resulted in 39.33% and 36.05% editing and 4.89% excision, respectively.

[0157] Because TCF4 is a transcription factor centrally controlling cellular functions, we aimed to excise the expanded repeat without affecting TCF4 expression. To test whether excision affected TCF4 expression, we measured TCF4 protein levels after excision with the indicated compositions 14 days after transfection. For Western blot analysis, cells were lysed in RIPA buffer, and 40 μg of total protein was loaded onto SDS-PAGE. A specific antibody (ab217668, Abcam) was used to detect TCF4 protein levels, and GAPDH was used as a loading control. No effect on TCF4 expression was observed (Figure 7).

[0158] Example 5 Evaluation of Excision - Infection with LVLP in U2OS Cells We also tested excision using lentivirus-like particles (LVLPs), which are a viable delivery system for corneal endothelial cells. LVLPs are viral particles containing a lentiviral envelope but lack viral genetic material. They contain only sgRNAs that bind to structural viral proteins via aptamer-binding proteins (ABPs) and nuclease proteins recruited by guides. Upon infection of target cells, the RNPs contained in the particles are released, resulting in transient expression and activity of the RNPs (Lyu et al., Nucleic Acids Research, 2019).

[0159] OMNI-50 nuclease is known to be highly active as an RNP and has been shown to be efficiently packaged into LVLPs. To test the activity of OMNI-50, we packaged either the upstream or downstream RNP components into LVLPs and tested the editing levels of each sgRNA in U2OS cells (Figure 8A). Three days after infection, sgRNA4 (downstream) and sgRNA195 (upstream) showed 53.0% and 84.43%, respectively (Figure 8B).

[0160] To measure the level of excision of the expanded repeat region, cells were infected with a mixture of the upstream and downstream LVLP compositions or with these compositions packaged in the same LVLP product (all-in-one). In both cases, the level of excision was 15% at 18 days postinfection (Fig. 8B).

[0161] Twenty-one days after infection, RNA was extracted from the excised cells, cDNA was synthesized, and qRT-PCR was performed using fast SYBR and primers specific for the exon 11-exon 12 junction of TCF4 (Fig. 9). No significant effect on TCF4 mRNA levels was observed after excision.

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Claims

1. 1. A method for excising an intronic trinucleotide CTG repeat expansion from an allele of transcription factor 4 (TCF4) in a cell, comprising: at least one CRISPR nuclease or a nucleotide molecule encoding a CRISPR nuclease; a first RNA molecule comprising a first guide sequence portion, or a nucleotide molecule encoding said first RNA molecule; and a second RNA molecule comprising a second guide sequence portion, or a nucleotide molecule encoding said second RNA molecule; introducing into said cells a composition comprising a complex of the CRISPR nuclease and the first RNA molecule that makes a double-strand break upstream of the intron trinucleotide CTG repeat expansion, and a complex of the CRISPR nuclease and the second RNA molecule that makes a double-strand break downstream of the intron trinucleotide CTG repeat expansion; wherein the first guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1216-2325, and the second guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1-1215.

2. 2. The method of claim 1, wherein the first guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1216-2325, the second guide sequence portion comprises 17 to 50 contiguous nucleotides within a sequence set forth in any of SEQ ID NOs: 1-1215, and at least one TCF4 allele in the cell has a heterozygous SNP at position rs34071688.

3. 2. The method of claim 1, wherein the first guide sequence portion comprises 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 46145-47292, and the second guide sequence portion comprises 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 45057-45092, and at least one TCF4 allele in the cell has a heterozygous SNP at any position selected from the group consisting of rs746872826, 18:5558615, rs879522127, and rs1268568114.

4. The first guide sequence portion comprises 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1216 to 2325, and the second guide sequence portion comprises 17 to 50 contiguous nucleotides within the sequence set forth in any of SEQ ID NOs: 1 to 1215, and at least one of the TCF4 alleles in the cell is selected from the group consisting of rs71670792, rs1261085, rs1261084, rs11431395, rs373174214, rs35555522, rs66807288, rs141970461, rs71674214, rs8766, rs1022136 2, rs2276195, rs569385112, rs398041379, rs72925008, rs8090085, rs5825130, rs56887277, rs397836157, rs55755941, rs781691419, rs11409023, r s10221357, rs11661961, rs11660565, rs61656112, rs60565673, rs72925018, rs1261073, rs1261076, rs748718974, rs13381800, rs34773632, rs1942 265, rs7241077, rs62092440, rs1893431, rs3838898, rs1893430, 18:55251930_G_GTTTT, rs771385941, rs4800988, rs1942264, rs1261093, rs15399 50, rs1539951, rs62092442, rs62092444, rs11662842, rs11664992, rs1261134, rs1261114, rs1788027, rs397858367, rs113662542, rs1261118, rs10 701336, rs397809469, rs150323043, rs1038226655, rs149454001, rs796169498, rs9955026, rs1046741326, rs754435392, rs867471715, rs77760825 6, rs762153709, rs1153636, rs1153637, rs5825134, rs35480166, rs893946, rs374155330, rs781053385, rs899293868, rs996440450, rs1349129287,rs780424692、rs34702622、rs11428164、rs1660235、rs1440473、rs1788026、rs11332509、rs1660237、rs1631486、rs1025804、rs753933037、rs1660233、rs200650987、rs71951255、rs368762262、rs751007744、rs780342991、rs1660241、rs796749696、rs61468075、rs777518462、rs1660242、rs1440476、rs1011392、18:55374871_T_TA、rs1788030、rs1623427、rs1621581、rs1788025、rs1788023、rs1348047、rs1788019、rs9950000、rs9958125、rs9320010、rs3794891、rs757629087、rs772409228、rs12607679、rs3794889、rs4801149、rs12605773、rs2872041、rs4801150、rs1020169、rs7238888、rs7235757、rs2958178、rs2958165、rs2958171、rs1328839434、rs796792902、rs2958175、rs11309751、rs2958186、rs2919446、rs2958161、rs2860511、rs2958162、rs2919451、rs2919450、rs201657057、rs2958163、rs1440477、rs2958166、rs377458803、rs2958169、rs8098843、rs11412305、rs386387765、18:55423173_T_TA、rs781071274、rs4374254、rs370693034、rs761981780、rs8084308、rs77540208、rs9320016、rs4524013、rs1025639279、rs4500831、rs7229456、rs12967143、rs12963334、rs12963463、rs398100891、rs12958048、18:55434419_C_CTTT、rs375388593、rs140134419、rs4801153、rs4801154, rs745460290, rs527450659, rs4341827, rs4468713, rs7228159, rs145330990, rs7231748, rs34577882, rs34578042, rs1452789, rs14527 88, rs12606995, rs188225813, rs732779, rs11385247, rs9966430, rs2924321, rs151196106, rs3760600, rs2924328, rs1377243, rs2924329, rs58251 42、rs199707137、18:55468891_C_CCCA、rs11338618、rs149728054、rs112 98284、rs7233312、rs2924331、18:55480276_C_CAA、rs2958182、rs2958183 rs2958184、rs2924332、rs2924333、rs2060889、rs2958187、rs2924335、r s138885827、rs2924336、rs59413482、rs796565215、18:55496896_C_CAA、r s4801157、rs2958188、rs2958189、rs2060886、rs3017183、rs2958158、rs2 924338、rs12956276、rs55812411、rs776881842、rs1452791、rs9957668、rs 9954890, rs9964328, rs67387556, rs1491335073, 18:55511330_T_TAAA, rs751932079, rs2957261, 18:55511331_T_TAAAA, rs8090106, rs140221855 rs17089851, rs398032944, rs1341922999, rs624244, rs627685, rs9948513, rs9965067, rs9965195, rs35371867, rs11441646, rs9949107, rs7240986 rs4801158, rs72627231, rs11412432, rs33938531, rs4800990, rs4458089, rs4572488, rs12968271, rs9636107, rs2123389, rs9947814, rs713522072. The method of claim 1, wherein the patient has a heterozygous SNP at any of the SNP positions selected from the group consisting of rs1452787, rs2123392, rs2123393, 18:55559041_T_TA, rs34935191, rs74182105, rs139870092, rs76053687, rs150848781, rs564960433, rs41396445, and rs34232463.

5. The method of any one of claims 1 to 4, wherein the composition is introduced into cells of a subject or into cells in culture.

6. The method according to any one of claims 1 to 5, wherein the cells are corneal cells or corneal endothelial cells.

7. The method of any one of claims 1 to 6, wherein the composition is introduced into the cells in vivo.

8. The method of claim 7, wherein the composition is introduced into the cells by a lentivirus-like particle (LVLP).

9. 6. The method of claim 5, wherein the cells are stem cells, fibroblasts, blood cells, hepatocytes, keratinocytes, other cells that can be reprogrammed into induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPS cells), iPS-derived cells, or iPS-derived corneal endothelial cells.

10. The method of claim 9 , wherein the composition is introduced into the cells ex vivo.

11. The method of any one of claims 1 to 10, wherein the CRISPR nuclease, the first RNA molecule and the second RNA molecule are introduced into the cell substantially simultaneously or at different times.

12. 12. The method of claim 1, wherein the first or second RNA molecule is a crRNA molecule or an sgRNA molecule.

13. The method of any one of claims 1 to 12, wherein the composition further comprises at least one tracrRNA molecule.

14. A composition comprising an RNA molecule having a guide sequence portion comprising 17 to 50 consecutive nucleotides within the sequence set forth in any of SEQ ID NOs: 1 to 2325.

15. 15. The composition of claim 14, further comprising a second RNA molecule, wherein the first RNA molecule has a guide sequence portion comprising 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1216 to 2325, and the second RNA molecule has a guide sequence portion comprising 17 to 50 contiguous nucleotides within a sequence set forth in any one of SEQ ID NOs: 1 to 1215.

16. The composition of claim 14 or 15, further comprising a CRISPR nuclease.

17. The composition of any one of claims 14 to 16, further comprising a tracrRNA molecule.

18. A cell modified by the method of any one of claims 1 to 13 or modified using the composition of any one of claims 14 to 17.

19. The modified cell of claim 18, wherein the cell is a corneal cell or a corneal endothelial cell.

20. 19. The modified cell of claim 18, wherein the cell is a stem cell or a fibroblast, a blood cell, a hepatocyte, a keratinocyte, or other cell that can be reprogrammed into an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), an iPS-derived cell, or an iPS-derived corneal endothelial cell.

21. 21. The modified cell of claim 20, wherein the stem cell differentiates after being modified.

22. 22. The modified cells of claim 20 or 21, wherein the stem cells differentiate into corneal cells or corneal endothelial cells.

23. 26. A method of treating Fuchs' endothelial corneal dystrophy (FECD) in a human subject, comprising delivering to the subject a composition of any one of claims 14-17 or transplanting modified cells of any one of claims 21-25 into the cornea of ​​the human subject.

24. 24. The method of claim 23, wherein the composition is delivered to cells of the cornea of ​​a patient in vivo.

25. 25. The method of claim 23 or 24, wherein the composition is introduced into the cells by a lentivirus-like particle (LVLP).

26. 26. The method of any one of claims 23 to 25, wherein the modified cells are delivered to cells of the cornea of ​​a patient ex vivo.

27. A medicament for use in excising an intronic trinucleotide CTG repeat expansion in an allele of TCF4 in a cell, comprising the composition of any one of claims 14 to 17, wherein the composition of any one of claims 14 to 17 is delivered to the cell.

28. 22. Use of a composition according to any one of claims 14 to 17 or a modified cell according to any one of claims 18 to 22 in the treatment of FECD, comprising delivering the composition according to any one of claims 14 to 17 or the modified cell according to any one of claims 18 to 22 to a subject suffering from or at risk of suffering from FECD.

29. A medicament for use in treating FECD, comprising the composition of any one of claims 14 to 17 or the modified cells of any one of claims 18 to 22, wherein the composition of any one of claims 14 to 17 or the modified cells of any one of claims 18 to 22 is delivered to a subject suffering from or at risk of suffering from FECD.

30. 18. A kit for excising an intronic trinucleotide CTG repeat expansion from an allele of TCF4 in a cell, comprising the composition of any one of claims 14 to 17 and instructions for delivering the composition to the cell.

31. The kit of claim 30 , wherein the composition is delivered to the cells ex vivo.

32. A kit for treating FECD in a subject, comprising a composition described in any one of claims 14 to 17 or a modified cell described in any one of claims 18 to 22, and instructions for delivering the composition or modified cell to a subject suffering from or at risk of suffering from FECD.

33. A composition according to any one of claims 14 to 17 or a modified cell according to any one of claims 18 to 22 for use in treating FECD, comprising delivering the composition according to any one of claims 14 to 17 or the modified cell according to any one of claims 18 to 22 to a subject suffering from or at risk of suffering from FECD.

34. A composition comprising at least one CRISPR nuclease or a nucleotide molecule encoding a CRISPR nuclease; and a means for excising an intronic trinucleotide CTG repeat expansion from an allele of transcription factor 4 (TCF4) in a cell.

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