Protein mutant and application thereof in treatment of HBB gene mutation related diseases

Abstract

The invention discloses a protein mutant of a sequence with 80% or more of identity compared with SEQ ID NO.1, gRNA used in cooperation with the protein mutant, a gene editing system, an expression vector, a modified cell, a pharmaceutical composition and application of the protein mutant in treatment of thalassemia or sickle-type anemia related to HBB gene mutation, and the gRNA, the gene editing system, the expression vector, the modified cell, the pharmaceutical composition and the application of the protein mutant in treatment of HBB gene mutation related thalassemia or sickle-type anemia. The protein mutant has substitution at one or more positions in the 549 , 555 , 559 or 606 positions of SEQ ID NO. 1.

Description

Protein mutants and their application in treating diseases related to HBB gene mutation Technical Field

[0001] The present invention relates to the field of gene therapy, and specifically to protein mutants, gRNA used in conjunction with protein mutants, gene editing systems, expression vectors, modified cells, methods for preparing modified cells, kits, pharmaceutical compositions, and applications in treating diseases associated with HBB gene mutations. Background Art

[0002] Thalassemia is a typical inherited blood disorder, primarily divided into two types: alpha thalassemia and beta thalassemia. Patients with beta thalassemia have a mutation in the HBB (hemoglobin, beta) gene on chromosome 11. Abnormal cells resulting from the HBB gene mutation produce excessive amounts of alpha protein chains, which then bind to the membranes of red blood cells, causing damage. If the concentration of alpha protein chains is too high, they can form toxic aggregates. Thalassemia can be classified as recessive, mild, or severe. Severe cases require regular lifelong blood transfusions and medication.

[0003] Before birth, the protein produced by the HBG (hemoglobin, gama) gene primarily binds to the α-protein chain, performing normal hemoglobin function. After birth, HBG gene expression is gradually suppressed. Meanwhile, the HBB gene gradually replaces HBG's function, and the HBG and HBB genes have a good ability to replace each other.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are a gene editor and a natural immune mechanism in most bacteria and archaea. Analysis of the flanking sequences of the CRISPR cluster revealed the presence of a polymorphic family gene nearby that functions in conjunction with the CRISPR region, hence the name CRISPR-associated (Cas). Therefore, gene editing can be used to disrupt sites where HBG gene expression is suppressed, reactivating HBG expression and restoring normal red blood cell function.

[0005] The CRISPR / Cas system is a commonly used gene editing system that has been successfully applied to precise genome editing in animals and plants. This system uses RNA-mediated targeting to recognize specific sites on the DNA double strand and cleaves them using nucleases. The most widely used nucleases are Cas9 and Cpf1. Cas9 and Cpf1 use RNA-mediated targeting to recognize specific sites on the DNA double strand and cleave them, creating double-strand breaks. Cells then repair these breaks through NHEJ (nonhomologous end joining) or HR (homologous recombination) pathways, achieving site-specific modification of the target gene. A widely used commercial Cas9 nuclease is SpCas9, which recognizes the PAM sequence NGG located 3' to the target sequence and cleaves 3 bp from the PAM sequence, creating blunt ends. LbCpf1 is a widely used commercial Cpf1 nuclease that recognizes the PAM sequence TTTN located 5' to the target sequence and cleaves distally, creating sticky ends.

[0006] Both SpCas9 and LbCpf1 have relatively strict PAM sequences, which restrict the design of target sites. The SpCas9 and LbCpf1 proteins, composed of 1368 and 1228 amino acids, respectively, are too large to be packaged and delivered using AAV, which to some extent limits their application in animal cells. Furthermore, the SpCas9 targeting sequence is 20 base pairs, making it prone to similar sequences appearing throughout the genome, resulting in off-target effects.

[0007] Currently, there is an urgent need to find new available Cas proteins and obtain gene editing tools that can effectively edit genes at the loci of hereditary blood diseases.

[0008] Summary of the Invention

[0009] The present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.

[0010] In one aspect, the disclosed embodiments provide a protein mutant comprising a sequence having greater than 80% identity with SEQ ID NO: 1, wherein the protein mutant has a substitution at one or more of positions 549, 555, 559 or 606 of SEQ ID NO: 1.

[0011] In some embodiments, the sequence having 80% or greater identity to SEQ ID NO: 1 comprises 1 to 100 conservative substitutions, deletions, or additions of amino acids relative to SEQ ID NO: 1.

[0012] In some embodiments, the protein mutant has a sequence as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0013] In some embodiments, the protein mutant is a Cas protein.

[0014] In some embodiments, the PAM recognition sequence of the Cas9 protein is 5'-YN, wherein Y is C or T, and N is A, T, C or G;

[0015] Preferably, the PAM recognition sequence is TCTA or CTTG.

[0016] In another aspect, the present disclosure provides an isolated nucleic acid, wherein the nucleic acid has a nucleotide sequence encoding the protein mutant described in the above aspects,

[0017] Preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO: 46.

[0018] On the other hand, the presently disclosed embodiments provide a gRNA for use in conjunction with the protein mutant described in any one of the above embodiments, wherein the gRNA has a sequence as shown in any one of SEQ ID NOs: 7-18.

[0019] In some embodiments, based on the protein mutant shown in SEQ ID NO: 2, the gRNA has a sequence as shown in any one of SEQ ID NOs: 7-12.

[0020] In some embodiments, based on the protein mutant shown in SEQ ID NO: 3, the gRNA has a sequence as shown in any one of SEQ ID NOs: 13-18.

[0021] In some embodiments, based on the gRNA shown in SEQ ID NOs:7-12, the gRNA recognizes the target sequence shown in SEQ ID NOs:22-27.

[0022] In some embodiments, based on the gRNA as shown in SEQ ID NOs: 13-18, the gRNA recognizes the target sequence shown in SEQ ID NOs: 28-33.

[0023] On the other hand, the present disclosure provides a gene editing system, including the protein mutant described in the above embodiment and the gRNA used in conjunction with the protein mutant described in the above embodiment.

[0024] Optionally, it further comprises crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA.

[0025] In another aspect, the present disclosure provides an expression vector comprising a first nucleic acid encoding the protein mutant described in the above embodiment, and a second nucleic acid encoding the gRNA used in conjunction with the protein mutant described in the above embodiment.

[0026] Optionally, further comprising a third nucleic acid expressing a homologous recombination sequence,

[0027] Optionally, further comprising a regulatory element operably linked to the first nucleic acid and configured to express the first nucleic acid, the second nucleic acid, and the third nucleic acid.

[0028] In some embodiments, the expression vector is a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus.

[0029] In another aspect, the present disclosure provides a modified cell, wherein the modified cell is modified to release the inhibition of HBG protein expression or activate the expression of HBG protein, preferably the cell is a hematopoietic stem cell.

[0030] In some embodiments, the modified cell comprises the gene editing system described in the above embodiments, or comprises the expression vector described in the above embodiments.

[0031] In yet another aspect, the presently disclosed embodiments provide a method for preparing modified cells, wherein the method comprises introducing the gene editing system described in the above embodiments into the cells to be modified, or transfecting the expression vector described in the above embodiments into the cells to be modified.

[0032] In yet another aspect, the present disclosure provides a kit comprising the expression vector described in the above embodiment or the modified cell described in the above embodiment, or the modified cell prepared by the method described in the above embodiment.

[0033] In yet another aspect, the presently disclosed embodiments provide a pharmaceutical composition comprising the protein mutant, gRNA, gene editing system, expression vector, or modified cells as described in the above embodiments, and a pharmaceutically acceptable carrier.

[0034] In yet another aspect, the presently disclosed embodiments provide for the use of the protein mutants, gRNA, gene editing systems, expression vectors, or modified cells described in the above embodiments in the preparation of a drug for treating a disease associated with HBB gene mutation in a subject.

[0035] In some embodiments, the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia.

[0036] In some embodiments, treating the disease associated with HBB gene mutation comprises releasing the inhibition of HBG protein expression or activating the expression of HBG protein in the subject.

[0037] On the other hand, the embodiments of the present disclosure provide for the use of the protein mutants, gRNA, gene editing systems, expression vectors, or modified cells as described in the above embodiments as drugs.

[0038] In another aspect, the present disclosure provides that the protein mutants, gRNA, gene editing systems, expression vectors, modified cells, or pharmaceutical compositions described in the above embodiments are used to treat diseases associated with HBB gene mutations in subjects.

[0039] In some embodiments, the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia.

[0040] In some embodiments, treating the disease associated with HBB gene mutation comprises releasing the inhibition of HBG protein expression or activating the expression of HBG protein in the subject.

[0041] In yet another aspect, the present disclosure provides a method for treating a disease associated with HBB gene mutation, comprising administering a therapeutically effective amount of the protein mutant, gRNA, gene editing system, expression vector, modified cell, or pharmaceutical composition described in the above embodiments to a subject in need thereof.

[0042] In some embodiments, the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia.

[0043] In some embodiments, treating the disease associated with HBB gene mutation comprises releasing the inhibition of HBG protein expression or activating the expression of HBG protein in the subject.

[0044] In yet another aspect, the present disclosure provides a method for treating thalassemia or sickle cell anemia associated with HBB gene mutation, comprising the steps of:

[0045] (1) Gene editing of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia to obtain gene-edited hematopoietic stem cells,

[0046] wherein the gene editing is performed by using the gene editing system or expression vector as described in the above embodiment;

[0047] (2) identifying the function of the gene-edited hematopoietic stem cells; and

[0048] (3) Returning the gene-edited hematopoietic stem cells that have restored hematopoietic function to the patient.

[0049] In some embodiments, the gene editing in step (1) comprises:

[0050] Introducing the gene editing system described in the above embodiment into the hematopoietic stem cells obtained from the patient; or

[0051] The expression vector described in the above example is transfected into the hematopoietic stem cells obtained from the patient.

[0052] In yet another aspect, the present disclosure provides a system for treating thalassemia or sickle cell anemia associated with HBB gene mutation, comprising:

[0053] A gene editing device configured to edit the genes of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia,

[0054] wherein the gene editing is performed by using the gene editing system or expression vector as described in the above embodiment;

[0055] an identification device configured to identify the function of the gene-edited hematopoietic stem cell; and

[0056] A reinfusion device is configured to reinfuse the gene-edited hematopoietic stem cells that have restored hematopoietic function into the patient.

[0057] In the embodiments of the present disclosure, novel gene editing systems such as CRISPR / Cas editing systems containing novel Cas proteins BES4-R and BES4-RVR, novel Cas protein (BES4-R and BES4-RVR) expression vectors and pharmaceutical compositions and kits containing the same are proposed, as well as the use of the novel gene editing system for the treatment of thalassemia or sickle cell anemia associated with HBB gene mutations.

[0058] Compared with the gene editing system SpCas9 in the related art, the novel gene editing system provided by the present disclosure has fewer amino acids (the existing SpCas9 protein is 1368 amino acids, and the BES4-R and BES4-RVR disclosed in the present disclosure are both 1245 amino acids), which can be easily loaded into the corresponding gene delivery vector, making it easier to be delivered to the target cell for editing. In addition, compared with SpCas9, the novel gene editing system provided by the embodiments of the present disclosure has a less specific PAM recognition sequence and more editable target sites.

[0059] Compared with the existing gene editing system containing BES4 protein, the novel gene editing system containing BES4-R protein and BES4-RVR protein provided by the present disclosure has different PAMs to choose from, so it can identify more target gene sites and is less likely to be off-target.

[0060] Therefore, the novel gene editing system provided by the embodiments of the present disclosure has the advantages of more editable target sites, higher gene editing efficiency, lower off-target rate and easy delivery to target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 is a schematic diagram showing the PAM recognition sequences of BES4, BES4-R, and BES4-RVR.

[0062] FIG2 is a schematic diagram showing the targeting sequences of BES4, BES4-R, BES4-RVR, and SpCas9.

[0063] FIG3 is a schematic diagram showing the gel electrophoresis results of BES4-HBG-g3 and spCas9-117 gene editing.

[0064] FIG4 is a schematic diagram showing the gel electrophoresis results of BES4-R, BES4-RVR, and SpCas9 gene editing.

[0065] FIG5 is a schematic diagram showing a comparison of gene editing efficiencies at three target sites of BES4-RVR.

[0066] Figure 6 is a schematic diagram showing the comparison of gene editing efficiency of BES4-RVR, BES4-R, SpCas9, and BES4, from left to right are BES4-RVR, BES4-R, SpCas9, and BES4 respectively. DETAILED DESCRIPTION

[0067] The embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present disclosure, but should not be understood as limiting the present disclosure.

[0068] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0069] The present disclosure is based on the following discoveries and facts of the inventors: a CRISPR-Cas system containing the BES4 protein (hereinafter referred to as BES4) derived from Eubacterium coprostanoligenes, wherein the BES4 protein is 1245 aa in length and the corresponding PAM sequence is TTTN. The inventors engineered the BES4 protein to obtain its mutants BES4-R and BES4-RVR. By designing gRNAs corresponding to BES4-R and BES4-RVR, a novel gene editing system was used to perform gene cutting at sites in the HBG gene promoter region that were suppressed, achieving good cutting efficiency, providing an ideal target and an effective gene editing system for the treatment of blood diseases such as thalassemia or sickle cell anemia.

[0070] The BES4-R and BES4-RVR mutant proteins of the new gene editing system are both composed of 1245 amino acids. The number of amino acids is relatively small and can be easily loaded into the corresponding gene delivery vector, making it easier to be delivered to the target cells for editing.

[0071] The PAM recognition sequence of BES4-R and BES4-RVR is 5'-YN, which allows them to recognize a wider range of target sites. The novel gene editing systems BES4-R and BES4-RVR exhibit improved gene editing efficiency and reduced off-target rates.

[0072] In the relevant technology, the gene therapy schemes for type B (β) thalassemia are roughly divided into three types: (1) using gene editing to perform site-specific repair on the mutated HBB gene to restore the function of the HBB gene in the genome; (2) introducing the normal HBB gene expression sequence into the genome through the means of lentiviral vectors, and the expressed normal HBB protein can restore the function of blood cells to a certain extent; (3) by relieving the inhibitory expression of HBG protein, the expression of normal HBG is restarted in the cells, and the normal HBG protein replaces the abnormal HBB protein to restore the function of blood cells, mainly through gene editing to knock out the BCL11A enhancer or destroy the inhibitor binding site region upstream of the HBG gene.

[0073] Regarding the above treatment options, option (1) is theoretically feasible, but since HBB pathogenic mutations are not single and the efficiency of site-specific repair is low, it is difficult to form a universal gene therapy program; option (2) is difficult to control the gene insertion site and copy number, and has a high treatment risk; option (3) currently mainly uses the SpCas9 system for gene editing, and its overall system is too large to be loaded into transport vectors such as AAV. At the same time, its PAM recognition sequence also limits its site selection and the SpCas9 system has a high off-target rate.

[0074] The present disclosure mainly optimizes and improves the above-mentioned scheme (3). The inventors unexpectedly obtained a new CIRSPR-Cas gene editing system containing BES4-R and BES4-RVR mutant proteins. The new gene editing system is used for gene editing, which avoids the shortcomings of the above-mentioned schemes (1) and (2). Compared with scheme (3), it has the advantages of more editable target sites, higher gene editing efficiency, lower off-target rate and easy delivery to target cells.

[0075] The disclosed embodiments provide a protein mutant comprising a sequence having greater than 80% identity with SEQ ID NO: 1, wherein the protein mutant has a substitution at one or more of positions 549, 555, 559 or 606 of SEQ ID NO: 1.

[0076] In an embodiment of the present disclosure, the protein mutant is a Cas protein.

[0077] In an embodiment of the present disclosure, the protein mutant has a sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0078] In the embodiments of the present disclosure, the Cas protein having the amino acid sequence shown in SEQ ID NO: 1 refers to the Cas protein named "BES4".

[0079]

[0080] In the embodiments of the present disclosure, the Cas protein having the amino acid sequence shown in SEQ ID NO: 2 refers to the Cas protein named "BES4-R".

[0081]

[0082]

[0083] In the embodiments of the present disclosure, the Cas protein having the amino acid sequence shown in SEQ ID NO: 3 refers to the Cas protein named "BES4-RVR".

[0084]

[0085] The Cas protein provided in the embodiments of the present disclosure consists of 1245 amino acids, which has a relatively small number of amino acids and can be easily loaded into the corresponding gene delivery vector, making it easier to be delivered to the target cell for gene editing.

[0086] The term "having more than 80% identity" includes 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. The stated values ​​are inclusive and include ranges of numbers between two values. Amino acid sequences having more than 80% identity to the sequences provided herein are included in the present disclosure. All amino acid sequences described herein may contain amino acid substitutions, e.g., conservative substitutions, that do not adversely affect the function of the protein comprising the amino acid sequence.

[0087] In some embodiments, the protein mutant having 80% or greater identity to SEQ ID NO: 1 comprises 1 to 100 conservative substitutions, deletions, or additions of amino acids relative to SEQ ID NO: 1, and the protein mutant has nuclease activity and exhibits in vitro cleavage activity and intracellular editing activity. For example, the protein mutant comprises 1 to 10 conservative substitutions, deletions, or additions of amino acids relative to the sequence of SEQ ID NO: 1.

[0088] More specifically, compared to SEQ ID NO: 1, the protein mutants have substitutions at one or more of positions 549, 555, 559, or 606 of SEQ ID NO: 1. For example, BES4-R has mutations at N549R and T606P compared to the wild-type BES4 sequence; and BES4-RVR has mutations at N549R, K555V, C559R, and T606P compared to the wild-type BES4 sequence. By substituting one or more of positions 549, 555, 559, or 606, the specificity of the PAM recognition sequence of the Cas protein is reduced, thereby enabling recognition of more target gene sites and reducing off-target effects.

[0089] In some embodiments, the PAM recognition sequence of the protein mutant is 5'-YN, wherein Y is C or T, and N is A, T, C or G. In some embodiments, the PAM recognition sequence of the protein mutant is TCTA or CTTG.

[0090] In this document, when representing bases, unless otherwise specified, the bases represented by the letters N and Y have the common meanings in the art, that is, N represents a random or arbitrary base A, T, C or G, and Y represents a random or arbitrary base C or T.

[0091] More specifically, the PAM recognition sequence of BES4, which has the amino acid sequence set forth in SEQ ID NO:1, is 5'-TTTN. The PAM recognition sequence of BES4-R or BES4-RVR, which has the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3, is 5'-YN. Compared to BES4, the BES4-R and BES4-RVR mutant proteins recognize a more abundant PAM sequence and have lower PAM sequence specificity, thereby enhancing cleavage efficiency and reducing off-target rates.

[0092] In an embodiment of the present disclosure, an isolated nucleic acid is provided, wherein the nucleic acid has a nucleotide sequence encoding a protein mutant described in the above embodiments. Specifically, the nucleotide sequence set forth in SEQ ID NO:39 encodes a BES4-R mutant protein having an amino acid sequence set forth in SEQ ID NO:2. The nucleotide sequence set forth in SEQ ID NO:46 encodes a BES4-RVR mutant protein having an amino acid sequence set forth in SEQ ID NO:3.

[0093] In an embodiment of the present disclosure, a gRNA is provided for use in conjunction with the protein mutant of any one of the above embodiments, wherein the gRNA has a sequence as shown in any one of SEQ ID NOs: 4-18.

[0094] In some embodiments, based on the Cas protein shown in SEQ ID NO: 1, the gRNA has a sequence as shown in any one of SEQ ID NOs: 4-6.

[0095] BES4_site1 gRNA:

[0096] 5'-AAUUUCUACUAUUGUAGAUGCCAGCCUUGCCUUGACCAAUAG-3'(SEQ ID NO:4)

[0097] BES4_site2 gRNA:

[0098] 5'-AAUUUCUACUAUUGUAGAUACCAAUAGCCUUGACAAGGCAAA-3'(SEQ ID NO:5)

[0099] BES4_site3 gRNA:

[0100] 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3'(SEQ ID NO:6)

[0101] In some embodiments, based on the protein mutant shown in SEQ ID NO: 2, the gRNA has a sequence as shown in any one of SEQ ID NOs: 7-12.

[0102] BES4-R_site1 gRNA:

[0103] 5'-AAUUUCUACUAUUGUAGAUGCAUUGAGAUAGUGUGGGGAAGG-3'(SEQ ID NO:7)

[0104] BES4-R_site2 gRNA:

[0105] 5'-AAUUUCUACUAUUGUAGAUUGCAUUGAGAUAGUGUGGGGAAG-3'(SEQ ID NO:8)

[0106] BES4-R_site3 gRNA:

[0107] 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3'(SEQ ID NO:9)

[0108] BES4-R_site4 gRNA:

[0109] 5'-AAUUUCUACUAUUGUAGAUGCCUUGACAAGGCAAACUUGACC-3'(SEQ ID NO:10)

[0110] BES4-R_site 5 gRNA:

[0111] 5'-AAUUUCUACUAUUGUAGAUUUGCCUUGACCAAUAGCCUUGAC-3'(SEQ ID NO:11)

[0112] BES4-R_site6 gRNA:

[0113] 5'-AAUUUCUACUAUUGUAGAUAGACUAUUGGUCAAGUUUGCCUU-3'(SEQ ID NO:12)

[0114] In some embodiments, based on the protein mutant shown in SEQ ID NO: 3, the gRNA has a sequence as shown in any one of SEQ ID NOs: 13-18.

[0115] BES4-RVR_site1 gRNA:

[0116] 5'-AAUUUCUACUAUUGUAGAUGCCAGCCUUGCCUUGACCAAUAG-3'(SEQ ID NO:13)

[0117] BES4-RVR_site2 gRNA:

[0118] 5'-AAUUUCUACUAUUGUAGAUCUCUUGGGGGCCCCUUCCCCACA-3'(SEQ ID NO:14)

[0119] BES4-RVR_site3 gRNA:

[0120] 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3'(SEQ ID NO:15)

[0121] BES4-RVR_site4 gRNA:

[0122] 5'-AAUUUCUACUAUUGUAGAUAGAUAGUGUGGGGAAGGGGCCCC-3'(SEQ ID NO:16)

[0123] BES4-RVR_site5 gRNA:

[0124] 5'-AAUUUCUACUAUUGUAGAUCAUUGAGAUAGUGUGGGGAAGGG-3'(SEQ ID NO:17)

[0125] BES4-RVR_site6 gRNA:

[0126] 5'-AAUUUCUACUAUUGUAGAUUCAAGGCUAUUGGUCAAGGCAAG-3'(SEQ ID NO:18)

[0127] In some embodiments, based on the gRNA shown in SEQ ID NOs: 4-6, the gRNA recognizes the target sequence shown in SEQ ID NOs: 19-21.

[0128] BES4_site1:5'-GCCAGCCTTGCCTGACCAATAG-3'(SEQ ID NO:19)

[0129] BES4_site2:5'-ACCAATAGCCTTGACAAGGCAAA-3'(SEQ ID NO:20)

[0130] BES4_site3:5'-CCTTGTCAAGGCTATTGGTCAAG-3'(SEQ ID NO:21)

[0131] In some embodiments, based on the gRNA shown in SEQ ID NOs:7-12, the gRNA recognizes the target sequence shown in SEQ ID NOs:22-27.

[0132] BES4-R_site1: 5'-GCATTGAGATAGTGTGGGGAAGG-3' (SEQ ID NO: 22)

[0133] BES4-R_site2: 5'-TGCATTGAGATAGTGTGGGGAAG-3' (SEQ ID NO: 23)

[0134] BES4-R_site3:5'-CCTTGTCAAGGCTATTGGTCAAG-3'(SEQ ID NO:24)

[0135] BES4-R_site4:5'-GCCTTGACAAGGCAAACTTGACC-3'(SEQ ID NO:25)

[0136] BES4-R_site5: 5'-TTGCCTTGACCAATAGCCTTGAC-3' (SEQ ID NO: 26)

[0137] BES4-R_site6:5'-AGACTATTGGTCAAGTTTGCCTT-3'(SEQ ID NO:27)

[0138] In some embodiments, based on the gRNA as shown in SEQ ID NOs: 13-18, the gRNA recognizes the target sequence shown in SEQ ID NOs: 28-33.

[0139] BES4-RVR_site1:5'-GCCAGCCTTGCCTGACCAATAG-3'(SEQ ID NO:28)

[0140] BES4-RVR_site2: 5'-CTCTTGGGGGCCCCTTCCCCACA-3' (SEQ ID NO: 29)

[0141] BES4-RVR_site3:5'-CCTTGTCAAGGCTATTGGTCAAG-3'(SEQ ID NO:30)

[0142] BES4-RVR_site4: 5'-AGATAGTGTGGGGAAGGGGCCCC-3' (SEQ ID NO: 31)

[0143] BES4-RVR_site5: 5'-CATTGAGATAGTGTGGGGAAGGG-3' (SEQ ID NO: 32)

[0144] BES4-RVR_site6:5'-TCAAGGCTATTGGTCAAGGCAAG-3'(SEQ ID NO:33)

[0145] In an embodiment of the present disclosure, a gene editing system is provided, comprising a protein mutant as described in the above embodiment and a gRNA used in conjunction with the protein mutant. In some embodiments, the gene editing system further comprises crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA.

[0146] Herein, the gene editing system refers to the Crispr-Cas system. The terms "Crispr," "crispr," or "CRISPR" all refer to clustered regularly interspaced short palindromic repeats. Capitalization, lowercase, or capitalized are commonly used in this field. Accordingly, the Crispr-Cas system may be described differently depending on capitalization.

[0147] Cas enzymes cut at target DNA sites, typically identifying the target site in the following way: an RNA molecule called Crispr RNA (crRNA) uses a portion of its sequence to bind to an RNA molecule called tracrRNA through base pairing, forming a chimeric RNA (tracrRNA / crRNA). The crRNA's guide RNA then base-pairs with the target DNA site, guiding the Cas protein to bind to the target site and cut. Crispr's targeting specificity is determined by two factors: base pairing between the guide RNA and the target DNA sequence, and the Cas protein and a short DNA sequence at the 3' end of the target DNA, known as the PAM (protospacer adjacent motif) recognition sequence.

[0148] In an embodiment of the present disclosure, an expression vector is provided, comprising a first nucleic acid encoding the protein mutant described in any one of the above embodiments, and a second nucleic acid encoding the gRNA used in conjunction with the protein mutant described in any one of the above embodiments.

[0149] In some embodiments, the expression vector further comprises a third nucleic acid expressing a homologous recombination sequence. In some embodiments, the expression vector further comprises a regulatory element operably linked to the first nucleic acid and configured to express the first nucleic acid, the second nucleic acid, and the third nucleic acid.

[0150] In some embodiments, the expression vector is a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus. A first nucleic acid encoding the protein mutant described in any of the above embodiments and a second nucleic acid encoding the gRNA used in conjunction with the protein mutant described in any of the above embodiments are loaded into the lentivirus without affecting the packaging and activity of the lentivirus, thereby providing a gene editing tool for in vivo gene editing.

[0151] In an embodiment of the present disclosure, a modified cell is provided, wherein the modified cell is modified to relieve the inhibition of HBG protein expression or to activate the expression of HBG protein. In some embodiments, the cell is a hematopoietic stem cell. The hematopoietic stem cell is modified to obtain a modified hematopoietic stem cell. The modified hematopoietic stem cell comprises the gene editing system described in the above embodiment, or comprises the expression vector described in the above embodiment. The modified hematopoietic stem cell can restore hematopoietic function and express hemoglobin. After the modified hematopoietic stem cell is reinfused into the body, hematopoietic stem cell engraftment and hematopoietic reconstruction can be successfully achieved.

[0152] In an embodiment of the present disclosure, a method for preparing modified cells is provided, comprising introducing the gene editing system described in the above embodiments into the cells to be modified, or transfecting the expression vector described in the above embodiments into the cells to be modified. In some embodiments, the cells are hematopoietic stem cells. The hematopoietic stem cells are modified to obtain modified hematopoietic stem cells. The modified hematopoietic stem cells can restore hematopoietic function and express hemoglobin.

[0153] In an embodiment of the present disclosure, a kit is provided, comprising the expression vectors described in the above embodiments, or the modified cells described in the above embodiments, or the modified cells prepared by the methods described in the above embodiments. In some embodiments, the cells are hematopoietic stem cells. The hematopoietic stem cells are modified to obtain modified hematopoietic stem cells. The modified hematopoietic stem cells are capable of restoring hematopoietic function and expressing hemoglobin. The kit is used to treat blood diseases associated with HBB gene mutations, such as thalassemia or sickle cell anemia.

[0154] In an embodiment of the present disclosure, a pharmaceutical composition is provided, comprising the protein mutant described in the above embodiments, the gRNA described in the above embodiments, the gene editing system described in the above embodiments, the expression vector described in the above embodiments, or the modified cell described in the above embodiments, and a pharmaceutically acceptable carrier.

[0155] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more of the active ingredients described herein, such as protein mutants, guide RNA used in conjunction with the protein mutants, gene editing systems, expression vectors, and modified cells, with other components, such as physiologically / pharmaceutically acceptable carriers, excipients, diluents, adjuvants, vehicles, and additional therapeutic agents for treating blood disorders such as thalassemia or sickle cell anemia. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.

[0156] As used herein, the term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance disclosed herein, but does not interfere with the biological activity of the active substance and has no toxic side effects on the subject or patient, wherein the active substance is a protein mutant, gRNA, gene editing system, expression vector, or modified cell as described in the above embodiments. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents, etc. The carrier can be a liquid, semisolid (e.g., a paste), or a solid carrier. Examples of carriers include water, saline solutions or other buffers (e.g., phosphate, citrate buffer), oils, alcohols, proteins (e.g., serum albumin, gelatin), carbohydrates (e.g., monosaccharides, disaccharides, and other carbohydrates, including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrin), gels, lipids, liposomes, resins, porous matrices, adhesives, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents; salts form counterions or combinations thereof.

[0157] In an embodiment of the present disclosure, a method for treating a disease associated with an HBB gene mutation is also provided, comprising administering to a subject in need thereof a therapeutically effective amount of the protein mutant described in the above embodiments, the gRNA described in the above embodiments, the gene editing system described in the above embodiments, the expression vector described in the above embodiments, the modified cell described in the above embodiments, or the pharmaceutical composition described in the above embodiments.

[0158] In some embodiments, the disease associated with the HBB gene mutation is a blood disease. In some embodiments, the blood disease is thalassemia or sickle cell anemia. Treating the disease associated with the HBB gene mutation includes relieving inhibition of HBG protein expression or initiating HBG protein expression in the subject. The expressed HBG protein binds to the excess α-protein chains caused by the HBB gene mutation, reducing the concentration of α-protein chains and preventing damage to the cell membranes of red blood cells caused by the excess α-protein chains, thereby restoring hematopoietic stem cells in patients with thalassemia or sickle cell anemia and expressing hemoglobin.

[0159] As used herein, the term "treating" refers to ameliorating a disease or condition (i.e., slowing, arresting, or alleviating the development of a disease or condition or at least one of its clinical symptoms). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, "treating" refers to delaying the onset, occurrence, or worsening of a disease or condition.

[0160] As used herein, the term "therapeutically effective amount" refers to an amount of the active ingredient sufficient to effectively treat a disease when administered to a subject. The amount of the active ingredient may vary depending on the active ingredient, the disease and its severity, and the condition, age, weight, and sex of the subject being treated. The active ingredient in this disclosure refers to the protein mutants, gRNAs, gene editing systems, expression vectors, or modified cells described in the above examples.

[0161] The term "subject" refers to an animal. Typically, the animal is a mammal. In some embodiments, the subject is a primate (e.g., a human, including male or female), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In some embodiments, the subject is a human. In some embodiments, the subject is a human suffering from a disease or at risk of developing a disease.

[0162] In an embodiment of the present disclosure, a method for treating thalassemia or sickle cell anemia associated with HBB gene mutation is also provided, comprising the steps of:

[0163] (1) Gene editing of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia to obtain gene-edited hematopoietic stem cells,

[0164] wherein the gene editing is performed by using the gene editing system described in the above embodiment or the expression vector described in the above embodiment;

[0165] (2) identifying the function of the gene-edited hematopoietic stem cells; and

[0166] (3) Returning the gene-edited hematopoietic stem cells that have restored hematopoietic function to the patient.

[0167] In some embodiments, the gene editing in step (1) comprises:

[0168] Introducing the gene editing system described in the above embodiment into the hematopoietic stem cells obtained from the patient; or

[0169] The expression vector described in the above examples is transfected into the hematopoietic stem cells obtained from the patient.

[0170] In an embodiment of the present disclosure, a system for treating thalassemia or sickle cell anemia associated with HBB gene mutation is also provided, comprising:

[0171] A gene editing device configured to edit the genes of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia,

[0172] wherein the gene editing is performed by using the gene editing system described in the above embodiment or the expression vector described in the above embodiment;

[0173] an identification device configured to identify the function of the gene-edited hematopoietic stem cell; and

[0174] A reinfusion device is configured to reinfuse the gene-edited hematopoietic stem cells that have restored hematopoietic function into the patient.

[0175] In the embodiments of the present disclosure, the protein mutants described in the above embodiments, the gRNA described in the above embodiments, the gene editing systems described in the above embodiments, the expression vectors described in the above embodiments, or the modified cells described in the above embodiments are provided for use as drugs.

[0176] In the embodiments of the present disclosure, the protein mutants described in the above embodiments, the gRNA described in the above embodiments, the gene editing systems described in the above embodiments, the expression vectors described in the above embodiments, the modified cells described in the above embodiments, or the pharmaceutical compositions described in the above embodiments are also provided for use in treating diseases associated with HBB gene mutations in subjects in need thereof.

[0177] In some embodiments, the disease associated with the HBB gene mutation is a blood disease, such as thalassemia or sickle cell anemia. Treating the disease associated with the HBB gene mutation includes relieving inhibition of HBG protein expression or initiating HBG protein expression in the subject. The expressed HBG protein binds to excess α-protein chains caused by the HBB gene mutation, reducing the concentration of α-protein chains and preventing damage to the red blood cell membranes caused by the excess α-protein chains. This allows the hematopoietic stem cells of patients with thalassemia or sickle cell anemia to restore hematopoietic function and express hemoglobin.

[0178] In the embodiments of the present disclosure, the use of the protein mutants described in the above embodiments, the gRNA described in the above embodiments, the gene editing system described in the above embodiments, the expression vector described in the above embodiments, or the modified cells described in the above embodiments in the preparation of drugs is also provided. The drugs are used to treat diseases associated with HBB gene mutations in subjects in need thereof.

[0179] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0180] Experiments targeting gene editing in the HBG promoter region using gene editing systems containing BES4, BES4-R, or BES4-RVR were performed as follows.

[0181] Example 1 - Design and synthesis of gene editing plasmids

[0182] The PAM identification information of BES4, BES4-R, and BES4-RVR has been obtained, as shown in Figure 1.

[0183] Targeted gene locus sequence location: GRCh38_hg38-Chr11 5271140:5271311.

[0184] Targeted gene locus sequence:

[0185]

[0186] Comparative Example (1) - Wild-type BES4

[0187] The BES4Cas protein has the following nucleic acid sequence and amino acid sequence.

[0188] The nucleic acid sequence of BES4Cas protein after optimization of human cell expression:

[0189]

[0190]

[0191]

[0192] Amino acid sequence of BES4Cas protein:

[0193]

[0194] Based on the sequence of the targeted gene site and the PAM sequence recognized by the BES4 editing system, three target sequences were identified:

[0195] BES4_site1: 5'-GCCAGCCTTGCCTGACCAATAG-3' (SEQ ID NO: 19)

[0196] BES4_site2: 5'-ACCAATAGCCTTGACAAGGCAAA-3' (SEQ ID NO: 20)

[0197] BES4_site3: 5'-CCTTGTCAAGGCTATTGGTCAAG-3' (SEQ ID NO: 21)

[0198] The corresponding gRNA DNA sequence is:

[0199] BES4_site1 gRNA template: 5'-AATTTCTACTATTGTAGATGCCAGCCTTGCCTTGACCAATAG-3' (SEQ ID NO: 36)

[0200] BES4_site2 gRNA template: 5'-AATTTCTACTATTGTAGATACCAATAGCCTTGACAAGGCAAA-3' (SEQ ID NO: 37)

[0201] BES4_site3 gRNA template: 5'-AATTTCTACTATTGTAGATCCTTGTCAAGGCTATTGGTCAAG-3' (SEQ ID NO: 38)

[0202] The corresponding gRNA sequence is:

[0203] BES4_site1 gRNA: 5'-AAUUUCUACUAUUGUAGAUGCCAGCCUUGCCUUGACCAAUAG-3' (SEQ ID NO: 4)

[0204] BES4_site2 gRNA: 5'-AAUUUCUACUAUUGUAGAUACCAAUAGCCUUGACAAGGCAAA-3' (SEQ ID NO: 5)

[0205] BES4_site3 gRNA: 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3' (SEQ ID NO: 6)

[0206] According to the above BES4 Cas nucleic acid sequence, BES4_site1 gRNA nucleic acid sequence and three target sequences, three plasmids, namely BES4-HBG-g1, BES4-HBG-g2 and BES4-HBG-g3, were synthesized by gene synthesis for these three target sequences by Beijing Liuhe BGI Genomics Co., Ltd. These three plasmids are used to express the gene editing system BES41 for cutting the target sequence shown in SEQ ID NO: 19, the gene editing system BES4 2 for cutting the target sequence shown in SEQ ID NO: 20, and the gene editing system BES4 3 for cutting the target sequence shown in SEQ ID NO: 21.

[0207] Experimental Example (1)—Mutant BES4-R

[0208] The nucleic acid sequence and amino acid sequence of the BES4-R Cas protein are as follows.

[0209] The nucleic acid sequence of BES4-R Cas protein after optimization for human cell expression:

[0210]

[0211]

[0212]

[0213] Amino acid sequence of BES4-R Cas protein:

[0214]

[0215] Based on the targeted gene locus sequence and the PAM sequence recognized by the BES4-R system, six target sequences were identified:

[0216] BES4-R_site1: 5'-GCATTGAGATAGTGTGGGGAAGG-3' (SEQ ID NO: 22)

[0217] BES4-R_site2: 5'-TGCATTGAGATAGTGTGGGGAAG-3' (SEQ ID NO: 23)

[0218] BES4-R_site3: 5'-CCTTGTCAAGGCTATTGGTCAAG-3' (SEQ ID NO: 24)

[0219] BES4-R_site4: 5'-GCCTTGACAAGGCAAACTTGACC-3' (SEQ ID NO: 25)

[0220] BES4-R_site5: 5'-TTGCCTTGACCAATAGCCTTGAC-3' (SEQ ID NO: 26)

[0221] BES4-R_site6: 5'-AGACTATTGGTCAAGTTTGCCTT-3' (SEQ ID NO: 27)

[0222] The DNA sequence recognized by the corresponding gRNA of BES4-R is:

[0223] BES4-R_site1 gRNA template: 5'-AATTTCTACTATTGTAGATGCATTGAGATAGTGTGGGGAAGG-3' (SEQ ID NO: 40)

[0224] BES4-R_site2 gRNA template: 5'-AATTTCTACTATTGTAGATTGCATTGAGATAGTGTGGGGAAG-3'

[0225] (SEQ ID NO: 41)

[0226] BES4-R_site3 gRNA template: 5'-AATTTCTACTATTGTAGATCCTTGTCAAGGCTATTGGTCAAG-3'

[0227] (SEQ ID NO: 42)

[0228] BES4-R_site4 gRNA template: 5'-AATTTCTACTATTGTAGATGCCTTGACAAGGCAAACTTGACC-3'

[0229] (SEQ ID NO: 43)

[0230] BES4-R_site5 gRNA template: 5'-AATTTCTACTATTGTAGATTTGCCTTGACCAATAGCCTTGAC-3'

[0231] (SEQ ID NO: 44)

[0232] BES4-R_site6 gRNA template: 5'-AATTTCTACTATTGTAGATAGACTATTGGTCAAGTTTGCCTT-3' (SEQ ID NO: 45)

[0233] The corresponding gRNA sequence for BES4-R is:

[0234] BES4-R_site1 gRNA: 5'-AAUUUCUACUAUUGUAGAUGCAUUGAGAUAGUGUGGGGAAGG-3' (SEQ ID NO: 7)

[0235] BES4-R_site2 gRNA: 5'-AAUUUCUACUAUUGUAGAUUGCAUUGAGAUAGUGUGGGGAAG-3' (SEQ ID NO: 8)

[0236] BES4-R_site3 gRNA: 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3' (SEQ ID NO: 9)

[0237] BES4-R_site4 gRNA: 5'-AAUUUCUACUAUUGUAGAUGCCUUGACAAGGCAAACUUGACC-3' (SEQ ID NO: 10)

[0238] BES4-R_site5 gRNA: 5'-AAUUUCUACUAUUGUAGAUUUGCCUUGACCAAUAGCCUUGAC-3' (SEQ ID NO: 11)

[0239] BES4-R_site6 gRNA: 5'-AAUUUCUACUAUUGUAGAUAGACUAUUGGUCAAGUUUGCCUU-3' (SEQ ID NO: 12)

[0240] Based on the above BES4-R nucleic acid sequence, the nucleic acid sequence of BES4-R_site1 to site6 gRNA and the six target sequences, six plasmids, namely BES4-R-HBG-g1, BES4-R-HBG-g2, BES4-R-HBG-g3, BES4-R-HBG-g4, BES4-R-HBG-g5 and BES4-R-HBG-g6, were synthesized by gene synthesis by Beijing Liuhe BGI Genomics Co., Ltd. These six plasmids are used to express the gene editing system BES4-R1 for cutting the target sequence shown in SEQ ID NO: 22, the gene editing system BES4-R2 for cutting the target sequence shown in SEQ ID NO: 23, the gene editing system BES4-R3 for cutting the target sequence shown in SEQ ID NO: 24, the gene editing system BES4-R4 for cutting the target sequence shown in SEQ ID NO: 25, the gene editing system BES4-R5 for cutting the target sequence shown in SEQ ID NO: 26, or the gene editing system BES4-R6 for cutting the target sequence shown in SEQ ID NO: 27.

[0241] Experimental Example (2)—Mutant BESR-RVR

[0242] The nucleic acid sequence and amino acid sequence of the BES4-RVR Cas protein are as follows.

[0243] The nucleic acid sequence of the BES4-RVR Cas protein after optimization for expression in human cells:

[0244]

[0245]

[0246] Amino acid sequence of the BES4-RVR Cas protein:

[0247]

[0248]

[0249] Based on the targeted gene site sequence and the PAM sequence recognized by the BES4-RVR system, six target sites were identified:

[0250] BES4-RVR_site1: 5'-GCCAGCCTTGCCTGACCAATAG-3' (SEQ ID NO: 28)

[0251] BES4-RVR_site2: 5'-CTCTTGGGGGCCCCTTCCCCACA-3' (SEQ ID NO: 29)

[0252] BES4-RVR_site3: 5'-CCTTGTCAAGGCTATTGGTCAAG-3' (SEQ ID NO: 30)

[0253] BES4-RVR_site4: 5'-AGATAGTTGTGGGGAAGGGGCCCC-3' (SEQ ID NO: 31)

[0254] BES4-RVR_site5: 5'-CATTGAGATAGTGTGGGGAAGGG-3' (SEQ ID NO: 32)

[0255] BES4-RVR_site6: 5'-TCAAGGCTATTGGTCAAGGCAAG-3' (SEQ ID NO: 33)

[0256] The DNA sequence recognized by the gRNA corresponding to BES4-RVR is:

[0257] BES4-RVR_site1 gRNA template: 5'-AATTTCTACTATTGTAGATGCCAGCCTTGCCTTGACCAATAG-3' (SEQ ID NO: 47)

[0258] BES4-RVR_site2 gRNA template: 5'-AATTTCTACTATTGTAGATCTCTTGGGGGCCCCTTCCCCACA-3' (SEQ ID NO: 48)

[0259] BES4-RVR_site3 gRNA template: 5'-AATTTCTACTATTGTAGATCCTTGTCAAGGCTATTGGTCAAG-3' (SEQ ID NO: 49)

[0260] BES4-RVR_site4 gRNA template: 5'-AATTTCTACTATTGTAGATAGATAGTGTGGGGAAGGGGCCCC-3' (SEQ ID NO: 50)

[0261] BES4-RVR_site5 gRNA template: 5'-AATTTCTACTATTGTAGATCATTGAGATAGTGTGGGGAAGGG-3' (SEQ ID NO: 51)

[0262] BES4-RVR_site6 gRNA template: 5'-AATTTCTACTATTGTAGATTCAAGGCTATTGGTCAAGGCAAG-3' (SEQ ID NO: 52)

[0263] The corresponding gRNA sequence for BES4-RVR is:

[0264] BES4-RVR_site1 gRNA: 5'-AAUUUCUACUAUUGUAGAUGCCAGCCUUGCCUUGACCAAUAG-3' (SEQ ID NO: 13)

[0265] BES4-RVR_site2 gRNA: 5'-AAUUUCUACUAUUGUAGAUCUCUUGGGGGCCCCUUCCCCACA-3' (SEQ ID NO: 14)

[0266] BES4-RVR_site3 gRNA: 5'-AAUUUCUACUAUUGUAGAUCCUUGUCAAGGCUAUUGGUCAAG-3' (SEQ ID NO: 15)

[0267] BES4-RVR_site4 gRNA: 5'-AAUUUCUACUAUUGUAGAUAGAUAGUGUGGGGAAGGGGCCCC-3' (SEQ ID NO: 16)

[0268] BES4-RVR_site5 gRNA: 5'-AAUUUCUACUAUUGUAGAUCAUUGAGAUAGUGUGGGGAAGGG-3' (SEQ ID NO: 17)

[0269] BES4-RVR_site6 gRNA: 5'-AAUUUCUACUAUUGUAGAUUCAAGGCUAUUGGUCAAGGCAAG-3' (SEQ ID NO: 18)

[0270] Based on the above BES4-RVR nucleic acid sequence, BES4-RVR_site1 to site6 gRNA nucleic acid sequence and six target sequences, six plasmids were synthesized by gene synthesis by Beijing Liuhe BGI Genomics Co., Ltd. for these six target sequences, namely

[0271] BES4-RVR-HBG-g1, BES4-RVR-HBG-g2, BES4-RVR-HBG-g3, BES4-RVR-HBG-g4, BES4-RVR-HBG-g5, and BES4-RVR-HBG-g6. These six plasmids are used to express BES4-RVR 1, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 28, BES4-RVR 2, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 29, BES4-RVR 3, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 30, BES4-RVR 4, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 31, BES4-RVR 5, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 32, or BES4-RVR 6, a gene editing system for cleavage of the target sequence set forth in SEQ ID NO: 33, respectively.

[0272] In addition, SpCas9 was used as a control example (2), and three plasmids, PX458-117, PX458-200, and PX458-114, were used. These three plasmids were from ADDGENE.

[0273] Example 2-Extraction and Preparation of Gene Editing Plasmids

[0274] The plasmid designed in Example 1 was prepared according to the following steps:

[0275] Take 15 mL of LB liquid medium (sterilized in advance at high temperature and high pressure, room temperature), add 15 μL of 1000X Amp antibiotic, use a 10 μL pipette tip to pick up the punctured strain containing the target plasmid (produced by Beijing Liuhe BGI Genomics Co., Ltd.), place it in the culture medium, and culture at 37°C, 200 rpm for 12-16 hours;

[0276] The cultured and expanded bacterial solution was centrifuged at 8000 rpm for 3 min and the culture medium was discarded;

[0277] According to the operating procedures described in the instructions, the target plasmid was extracted using an endotoxin-free kit (from Tiangen Biochemical Technology (Beijing) Co., Ltd., China); and

[0278] After extraction, the DNA concentration was quantified using Nanodrop (purchased from Thermo Fisher Scientific, USA) and stored at -20°C.

[0279] Example 3 - Transfection of gene editing plasmids

[0280] The plasmid extracted in Example 2 was transfected into cells for gene editing according to the following steps.

[0281] The day before transfection, use a pipette to remove the original culture medium of the HEK293T cells (~90% confluence) in the seed plate, slowly add about 2mL of preheated DPBS (Gibco) along the wall to wash the cell surface, and then add 1mL of preheated digestion solution (TrypLE Express, Gibco) to digest. After about 3 minutes, add an appropriate amount of preheated DMEM medium containing serum to terminate the digestion and re-suspend by pipetting. Take a small amount of cell suspension and gently pipette to mix with an equal proportion of trypan blue dye (Solarbio). Take about 20μL of the mixture and add it to a cell counting plate (Countstar) and use a cell analyzer (Countstar Rigel S2) to count the live cells. Finally, use a 12-well cell culture plate for plating and culture, with about 0.5 to 1*10 per well. 6 cells.

[0282] Once the cells reach 50% to 70% confluency, use the Lipofectamine 3000 kit (Invitrogen) to transfect the cells with the target plasmid (2 μg of plasmid and 2.4 μL of Lipofectamine 3000 Reagent per well). In principle, change the culture medium as needed 6 hours after transfection. For detailed transfection procedures, refer to the manufacturer's instructions.

[0283] After transfection, cells need to be cultured for 2-3 days to allow sufficient gene editing to occur.

[0284] After cell culture is complete, calculate the transfection efficiency and harvest the cells. Follow the same cell digestion and resuspension steps as above (using the same reagent dosage as the cell culture area). Transfer approximately 20 μL of the cell suspension to a cell counting plate and analyze the cell using the green fluorescence (GFP) channel to calculate the plasmid transfection efficiency. Transfer the remaining cells to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute. Remove the supernatant and harvest the cells.

[0285] Example 4 - Identification of genome editing activity

[0286] Sequences near the targeted region:

[0287]

[0288] After harvesting the cells, genome extraction and T7E1 enzyme digestion experiments were performed to preliminarily detect the effect of gene editing. The steps are as follows.

[0289] (1) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., China). The genomic (gDNA) concentration was quantified using Nanodrop and stored at −20°C. For detailed steps, refer to the instruction manual.

[0290] (2) Target region PCR: A high-fidelity amplification enzyme (PrimeSTAR GXL DNA Polymerase, TAKARA, Japan) was used to amplify the target site region from gDNA. The amplification primers are shown in Table 1 below. The primers used were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. After a clear and single target band was observed under a gel imager by 1% agarose gel (TAE) electrophoresis (7.5 V / cm, 30 min), the gel was cut and the target band was purified and recovered using a PCR purification and gel extraction kit (NucleoSpin Extract, MN, Germany). The concentration of the recovered target band was measured using Nanodrop.

[0291] Table 1 PCR amplification primers

[0292]

[0293] (3) Denaturation and Annealing: The modified DNA and control reaction systems were mixed according to Table 2 and subjected to heat denaturation and annealing. The program settings for the PCR instrument (purchased from Bio-Rad Laboratories, Inc., USA) are shown in Table 3.

[0294] Table 2 Reaction system

[0295] Component input: DNA 200ng 10*TE7I Buffer 2μL ddH2O to 19.7μL

[0296] Table 3 Denaturation and annealing conditions

[0297] Temperature time 95℃ 3 minutes -1℃ / second 70 seconds 25℃ hold

[0298] (4) T7E1 enzyme digestion: Add 0.3 μL of T7EI nuclease to the reaction system in step (3), totaling 20 μL of reaction system, and place the reaction system in a 37°C water bath for 20 minutes of enzyme digestion reaction.

[0299] (5) Activity detection: After the reaction was completed, 4 μL of 6*Gel Loading Dye (NEB) was added and the bands were detected by agarose gel electrophoresis (160 V for 30 min).

[0300] The results are shown in Figures 3 and 4. The experimental results indicate that the gene editing systems BES4, BES4-R, and BES4-RVR all have gene editing activity targeting the repressed sites in the promoter region of the HBG gene.

[0301] Example 5 - Amplicon library construction and sequencing

[0302] The target site region was amplified using the genome after gene editing in Example 4 as a template. The editing efficiency of SpCas9, BES4, BES4-R, and BES4-RVR was compared and evaluated by sequencing the amplicon library.

[0303] Primers were designed for the target sites of BES4-g3, BES4-g6, BES4-RVR-g3, BES4-RVR-g6, BES4-RVR-g7, BES4-R-3, BES4-R-6, PX458-114, PX458-117, and PX458-200, and amplification reactions were performed. The target site of BES4-RVR-g7 is the same as that of BES4-R-g6. The sequencing results are shown in Figure 5.

[0304] The specific operations are as follows:

[0305] (1) Use Primer3 (https: / / primer3.ut.ee / ) to output primers for the above target sites.

[0306] (2) Primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0307] Two rounds of PCR amplification were performed. The amplification conditions are shown in Table 4:

[0308] In the first round of amplification, primers containing adapters at their 5' ends were used for amplification. The sequences of the primers (excluding the adapter) and the amplified products are shown in Table 5.

[0309] In the second round of amplification, according to the corresponding order of samples and labels (barcodes) in Table 6, amplification was performed using primers containing barcodes and the product recovered after the first round of amplification purified as a template. Finally, the target amplification product with the adapter and barcode was obtained.

[0310] (3) DNB library construction and sequencing: Purify the target amplification product obtained above and use the purified amplification mixture to construct the library. For detailed steps and procedures, please refer to the MGI PCR-Free DNA Library Preparation Kit. Sequencing was performed using the National Gene Bank (CNGB) DNBSEQ-T1 & T5 platform PE100.

[0311] (4) Crispresso2 (https: / / github.com / pinellolab / CRISPResso2) was used to analyze the amplicon sequencing data.

[0312] Table 4 Amplification conditions

[0313] 98℃ for 3 minutes, 98℃ for 20 seconds, 55℃ for 20 seconds, 68℃ for 20 seconds, 68℃ for 5 minutes, 12℃ for 32 cycles, first round PCR, 20 cycles, second round PCR

[0314] Table 5 Primer sequences used for target site amplification

[0315]

[0316]

[0317] Table 6: Adapter and Barcode sequence information used in library construction

[0318]

[0319]

[0320] Sequencing results showed that the same target region was cut out by gene editing of BES4-g3, BES4-g6, BES4-RVR-g3, BES4-RVR-g6, BES4-RVR-g7, BES4-R-3, BES4-R-6, PX458-114 and PX458-117.

[0321]

[0322] Gene editing with PX458-200 to excise the targeted region:

[0323]

[0324] A comparison of the gene editing efficiencies of the experimental groups BES4-RVR and BES4-R, and the control groups SpCas9 and BES4, is shown in Figure 6. Figure 6 shows that BES4-RVR achieved an editing efficiency of 29.3% at the g3 site, significantly exceeding the 19.4% editing efficiency of SpCas9 (PX458-117). BES4-RVR achieved editing efficiencies of 29.3% and 13.9% at the g3 and g7 sites, respectively, while BES4-R achieved editing efficiencies of 20.7% and 14.8% at the g3 and g6 sites, respectively, surpassing the 14.2% and 3.8% editing efficiencies of wild-type BES4 at the g3 and g6 sites, respectively.

[0325] In the present disclosure, the term "comprising" is an open expression, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0326] The terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory. All embodiments of the present disclosure can be implemented alone or in combination with other embodiments, and are all deemed to be within the scope of protection claimed by the present disclosure.

[0327] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0328] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

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Claims

1. A protein mutant comprising a sequence having more than 80% identity with SEQ ID NO: 1, wherein the protein mutant has a substitution at one or more of positions 549, 555, 559 or 606 of SEQ ID NO:

1. 2 . The protein mutant according to claim 1 , wherein the protein mutant having 80% or greater identity to SEQ ID NO: 1 comprises 1 to 100 conservative substitutions, deletions or additions of amino acids relative to SEQ ID NO:

1.

3. The protein mutant according to claim 1 or 2, wherein the protein mutant has a sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. The protein mutant according to any one of claims 1 to 3, wherein the protein mutant is a Cas protein.

5. The protein mutant according to claim 4, wherein the PAM recognition sequence of the Cas protein is 5'-YN, Wherein Y is C or T, and N is A, T, C or G; Preferably, the PAM recognition sequence is TCTA or CTTG.

6. An isolated nucleic acid, wherein the nucleic acid has a nucleotide sequence encoding the protein mutant according to claim 1, Preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO:

46.

7. A gRNA for use with the protein mutant according to any one of claims 1 to 5, wherein the gRNA has a sequence as shown in any one of SEQ ID NOs: 7-18.

8. The gRNA according to claim 7, wherein Based on the protein mutant shown in SEQ ID NO: 2, the gRNA has a sequence as shown in any one of SEQ ID NOs: 7-12; or based on the protein mutant shown in SEQ ID NO: 3, the gRNA has a sequence as shown in any one of SEQ ID NOs: 13-18.

9. The gRNA according to claim 7 or 8, wherein Based on the gRNAs shown in SEQ ID NOs: 7-12, the gRNAs recognize the target sequences shown in SEQ ID NOs: 22-27; or Based on the gRNAs shown in SEQ ID NOs: 13-18, the gRNAs recognize the target sequences shown in SEQ ID NOs: 28-33.

10. A gene editing system comprising the protein mutant according to any one of claims 1 to 5 and the gRNA used in conjunction with the protein mutant according to any one of claims 7 to 9, Optionally, it further comprises crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA.

11. An expression vector comprising a first nucleic acid encoding the protein mutant according to any one of claims 1 to 5, and a second nucleic acid encoding the gRNA used in conjunction with the protein mutant according to any one of claims 7 to 9, Optionally, further comprising a third nucleic acid expressing a homologous recombination sequence, Optionally, further comprising a regulatory element operably linked to the first nucleic acid and configured to express the first nucleic acid, the second nucleic acid, and the third nucleic acid. 12 . The expression vector according to claim 11 , wherein the expression vector is a retrovirus, a lentivirus, an adenovirus or an adeno-associated virus.

13. A modified cell, wherein the modified cell is modified to release the inhibition of HBG protein expression or activate the expression of HBG protein, preferably the cell is a hematopoietic stem cell.

14. The modified cell of claim 13, wherein the modified cell comprises the gene editing system of claim 10, or comprises the expression vector of claim 11 or 12.

15. A method for preparing the modified cell of claim 13 or 14, wherein the method comprises introducing the gene editing system of claim 10 into the cell to be modified, or transfecting the expression vector of claim 11 or 12 into the cell to be modified. 16 . A kit comprising the expression vector according to claim 11 or 12 or the modified cell according to claim 13 or 14, or the modified cell prepared by the method according to claim 15.

17. A pharmaceutical composition comprising the protein mutant of any one of claims 1 to 5, the gRNA of any one of claims 7 to 9, the gene editing system of claim 10, the expression vector of claim 11 or 12, or the modified cell of claim 13 or 14, and a pharmaceutically acceptable carrier.

18. Use of the protein mutant of any one of claims 1 to 5, the gRNA of any one of claims 7 to 9, the gene editing system of claim 10, the expression vector of claim 11 or 12, or the modified cell of claim 13 or 14 in the preparation of a medicament for treating a disease associated with an HBB gene mutation in a subject.

19. The use according to claim 18, wherein the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia. 20 . The use according to claim 18 or 19 , wherein treating the disease associated with HBB gene mutation comprises releasing the inhibition of HBG protein expression or activating the expression of HBG protein in the subject.

21. The protein mutant of any one of claims 1 to 5, the gRNA of any one of claims 7 to 9, the gene editing system of claim 10, the expression vector of claim 11 or 12, or the modified cell of claim 13 or 14 is used as a drug.

22. The protein mutant of any one of claims 1 to 5, the gRNA of any one of claims 7 to 9, the gene editing system of claim 10, the expression vector of claim 11 or 12, the modified cell of claim 13 or 14, or the pharmaceutical composition of claim 17 is used for treating a disease associated with an HBB gene mutation in a subject.

23. The protein mutant, the gRNA, the gene editing system, the expression vector, the modified cell or the pharmaceutical composition used according to claim 22, wherein the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia.

24. The protein mutant, the gRNA, the gene editing system, the expression vector, the modified cell or the pharmaceutical composition for use according to claim 22 or 23, wherein treating the disease associated with the HBB gene mutation comprises relieving the inhibition of HBG protein expression or initiating the expression of HBG protein in the subject.

25. A method for treating a disease associated with an HBB gene mutation, comprising administering to a subject in need thereof a therapeutically effective amount of the protein mutant of any one of claims 1 to 5, the gRNA of any one of claims 7 to 9, the gene editing system of claim 10, the expression vector of claim 11 or 12, the modified cell of claim 13 or 14, or the pharmaceutical composition of claim 17.

26. The method according to claim 25, wherein the disease associated with HBB gene mutation is a blood disease, preferably thalassemia or sickle cell anemia. 27 . The method according to claim 25 or 26 , wherein treating the disease associated with HBB gene mutation comprises releasing the inhibition of HBG protein expression or activating the expression of HBG protein in the subject.

28. A method for treating thalassemia or sickle cell anemia associated with HBB gene mutation, comprising the steps of: (1) Gene editing of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia to obtain gene-edited hematopoietic stem cells, wherein the gene editing is performed by using the gene editing system of claim 10 or the expression vector of claim 11 or 12; (2) identifying the function of the gene-edited hematopoietic stem cells; and (3) Returning the gene-edited hematopoietic stem cells that have restored hematopoietic function to the patient.

29. The method of claim 28, wherein the gene editing in step (1) comprises: introducing the gene editing system according to claim 10 into the hematopoietic stem cells obtained from the patient; or The expression vector according to claim 11 or 12 is transfected into the hematopoietic stem cells obtained from the patient.

30. A system for treating thalassemia or sickle cell anemia associated with HBB gene mutation, comprising: A gene editing device configured to edit the genes of hematopoietic stem cells obtained from a patient with thalassemia or sickle cell anemia, wherein the gene editing is performed by using the gene editing system of claim 10 or the expression vector of claim 11 or 12; an identification device configured to identify the function of the gene-edited hematopoietic stem cell; and A reinfusion device is configured to reinfuse the gene-edited hematopoietic stem cells that have restored hematopoietic function into the patient.

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