Ribonucleic acid (RNA) site-directed editing by utilizing artificially constructed RNA editing enzyme and related application

Abstract

The invention provides RNA site-directed editing by utilizing an artificially constructed RNA editing enzyme and related application. According to the present invention, an RNA recognition domain for binding RNA and a functional utility domain are fused to form a novel functional protein, and the novel functional protein specifically targets the target RNA through the recognition domain and uses the utility domain to perform RNA editing.

Description

[0001] This application is a divisional application of a Chinese patent application filed on March 30, 2020, with application number 202080000996.X, and invention name “Using artificially constructed RNA editing enzymes for site-specific RNA editing and related applications”. Technical Field

[0002] The present invention belongs to the biological field, and specifically, the present invention relates to RNA site-specific editing using artificially constructed RNA editing enzymes and related applications. Background Art

[0003] DNA is the main genetic material in organisms. Currently, a large proportion of known human diseases are caused by genetic mutations, and single-base mutations are the largest type of them. Therefore, developing a method to change the sequence of a single base in the genome and efficiently and accurately repair pathogenic single-base mutations is very meaningful for the study and treatment of genetic diseases.

[0004] The current gene therapy strategy for single-base mutation diseases is mainly to treat the disease by directly repairing or replacing the mutant gene at the DNA or RNA level. The main methods are the CRISPR-based DNA base editing systems ABE and CBE. These technologies can perform base editing to a certain extent, but there are still many shortcomings:

[0005] (1) The current CRISPR-mediated base editing system lacks precision and has low efficiency in single-base editing. There is generally an editing window, which will introduce additional mutations when editing the target site.

[0006] (2) The CRISPR system is very large, and current gene introduction technology makes it difficult to package the entire CRISPR system in the same system for delivery at one time, resulting in inefficient gene introduction and low editing efficiency during the treatment process.

[0007] (3) Due to the size limitation of the transgene, some genomic sites are difficult to transfer.

[0008] (4) Immune response and toxicity caused by Cas proteins as bacterial proteins.

[0009] (5) Mutations that occur during gene insertion or some unexpected events that occur during integration.

[0010] Of particular importance is that, since changes in genomic DNA persist throughout the life of the cell, concerns about the long-term safety of genomic DNA editing have always been a major issue.

[0011] Therefore, those skilled in the art are in urgent need of developing a method that can effectively edit genes so as to perform effective gene therapy for single base mutation diseases. Summary of the invention

[0012] The purpose of the present invention is to provide a method and application thereof that can effectively edit genes.

[0013] In a first aspect of the present invention, an RNA editing enzyme is provided, the RNA editing enzyme comprising:

[0014] (a) an RNA recognition domain, wherein the RNA recognition domain is used to recognize an RNA recognition sequence of an RNA sequence to be edited and bind to the RNA recognition sequence;

[0015] (b) a utility domain, wherein the utility domain is used to perform nucleotide editing on the RNA sequence to be edited;

[0016] Wherein, the RNA recognition domain and the utility binding domain are operably connected.

[0017] In another preferred embodiment, the utility domain is selected from the following group: a deamination catalytic domain of an ADAR family member, a deamination catalytic domain of an APOBEC family member, an RNA methylase, an RNA demethylase, an uracil synthetase, or a combination thereof.

[0018] In another preferred embodiment, the RNA recognition domain contains n recognition units, each of which is used to recognize an RNA base, wherein n is a positive integer of 5-30.

[0019] In another preferred embodiment, n is 6-24, more preferably 8-20, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

[0020] In another preferred embodiment, each recognition unit is an α-helical repeating sequence, and three amino acids at specific positions on the repeating sequence are responsible for binding to RNA bases.

[0021] In another preferred embodiment, the n recognition units are connected in series to form an RNA base recognition region.

[0022] In another preferred embodiment, the RNA recognition domain further comprises optional protection zones located on both sides of the RNA base recognition region, for protecting the RNA base recognition region.

[0023] In another preferred embodiment, the RNA recognition domain is derived from PUF protein.

[0024] In another preferred embodiment, the RNA recognition domain does not include the domain located upstream of the RNA base recognition region in the PUF protein (except the protection zone), but may or may not include the domain located downstream of the RNA base recognition region in the PUF protein (except the protection zone).

[0025] In another preferred embodiment, the protection zone is a non-functional repeat sequence at both ends of the RNA base recognition region.

[0026] In another preferred embodiment, the RNA editing enzyme further comprises one or more elements selected from the following group: a connecting peptide, a tag sequence, a signal peptide sequence, a positioning peptide sequence, or a combination thereof.

[0027] In another preferred embodiment, the localization peptide sequence includes a nuclear localization signal sequence, a mitochondrial localization signal sequence, or a combination thereof.

[0028] In another preferred embodiment, the one or more elements are operably linked to the RNA recognition domain and the utility domain.

[0029] In another preferred embodiment, the one or more elements are independently located at the N-terminus and / or C-terminus and / or the middle of the RNA editing enzyme.

[0030] In another preferred embodiment, the signal peptide is located at the N-terminus of the RNA editing enzyme.

[0031] In another preferred embodiment, the connecting peptide, tag sequence, and / or positioning peptide signal are each independently located between the recognition domain and the utility domain.

[0032] In another preferred embodiment, the RNA is single-stranded.

[0033] In another preferred example, the RNA editing enzyme includes an RNA recognition domain and a utility domain, and optionally a connecting peptide, a tag sequence, a signal peptide sequence and / or a positioning peptide sequence.

[0034] In another preferred embodiment, the structure of the RNA editing enzyme is shown in any one of the following formulas I to IV:

[0035] D-L2-A-L1-B(I);

[0036] D-L2-B-L1-A(II);

[0037] A-L1-B-L2-D(III);

[0038] B-L1-A-L2-D(IV);

[0039] In the formula, each "-" is independently a connecting peptide or a peptide bond;

[0040] A is the RNA recognition domain;

[0041] B is the utility domain;

[0042] L1 and L2 are each independently no or a linker peptide;

[0043] D is no or localized peptide.

[0044] In another preferred embodiment, the localization peptide is located at the N-terminus, C-terminus or middle of the RNA editing enzyme.

[0045] In another preferred embodiment, the RNA recognition domain is a domain that can recognize and bind to RNA, and is preferably derived from an RNA binding protein.

[0046] In another preferred embodiment, the RNA recognition domain is a PUF protein fragment.

[0047] In another preferred embodiment, the PUF protein fragment is a fragment of the PUF protein domain that can recognize and bind to RNA.

[0048] In another preferred embodiment, the PUF protein includes PUF protein and homologous proteins thereof.

[0049] In another preferred embodiment, the PUF protein is from a mammal, preferably from a primate, and more preferably from a human.

[0050] In another preferred embodiment, the RNA recognition domain has an amino acid sequence as shown in SEQ ID NO.:1.

[0051] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIQLKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDGHVLKCVKDQNGNHVVQKCIECVQPQSLQFIIDAFKGQVFALSTH PYGCRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG(SEQ ID NO.:1)

[0052] In another preferred embodiment, the ADAR family members include: dADAR, ADAR1, ADAR2, TadA, or a combination thereof.

[0053] In another preferred embodiment, the ADAR family member is derived from humans, fruit flies or bacteria.

[0054] In another preferred embodiment, the ADAR family members include: Drosophila ADAR, human ADAR1, human ADAR2, Escherichia coli TadA, or a combination thereof.

[0055] In another preferred embodiment, the ADAR1 includes natural ADAR1 and ADAR1 mutants.

[0056] In another preferred embodiment, the ADAR1 mutant is mutated at position 1008 in the amino acid sequence corresponding to natural ADAR1; preferably, the glutamic acid (E) at position 1008 is mutated to glutamine (Q).

[0057] In another preferred embodiment, the effector domain is from ADAR1 and has an amino acid sequence shown in SEQ ID NO.: 2:

[0058] KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGET VNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPV ESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKT KETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV(SEQ ID NO.:2)

[0059] Alternatively, the effector domain is an ADAR1-E1008Q effector domain, the amino acid sequence of which is the same as SEQ ID No.: 2, but position 211 in SEQ ID No.: 2 is mutated from E to Q:

[0060] KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGET VNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPV ESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKT KETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV(SEQ ID No.: 2, in which position 211 is mutated from E to Q).

[0061] In another preferred embodiment, the ADAR2 mutant undergoes a mutation at position 488 in the amino acid sequence corresponding to natural ADAR2; preferably, the glutamic acid (E) at position 488 is mutated to glutamine (Q).

[0062] In another preferred embodiment, the effector domain has an amino acid sequence shown in SEQ ID NO.: 2, 3 or 4 or a derivative sequence thereof.

[0063] In another preferred embodiment, the derivative sequence includes: SEQ ID No.: 2, and the 211th position is mutated from E to Q); SEQ ID No.: 3, wherein the 227th position is mutated from E to Q; SEQ ID No.: 4, wherein the 187th position is mutated from E to Q.

[0064] In another preferred embodiment, the effector domain is selected from the following group:

[0065] (i) having an ADAR2-isoform1 effector domain having the amino acid sequence shown in SEQ ID NO.: 3

[0066] (ii) an ADAR2-isoform1-E488Q effector domain having the amino acid sequence shown in SEQ ID NO.: 3 and a mutation from E to Q at position 227; and

[0067] (iii) an ADAR2-isoform2 effector domain having the amino acid sequence shown in SEQ ID NO.: 4;

[0068] (iv) an ADAR2-isoform2-E488Q effector domain having the amino acid sequence shown in SEQ ID NO.: 4 and a mutation from E to Q at position 187;

[0069] In another preferred embodiment, the amino acid sequence of the ADAR2-isoform1 effector domain is as follows:

[0070] DQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTG

[0071] TKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPC

[0072] GDARIFSPHEPILEGSRSYTQAGVQWCNHGSLQPRPPGLLSDPSTSTFQGAGTTEPADRHPNRKARGQLRTKIESGE

[0073] GTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRIS

[0074] NIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGK

[0075] VPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP(SEQ IDNO.:3)

[0076] In another preferred embodiment, the amino acid sequence of the ADAR2-isoform1-E488Q effector domain is as follows:

[0077] DQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLN NKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEGSRSYTQAGVQWCNHGSLQPRPPGLLSDPSTSTFQGAGTTEPADRHPNRKARGQLRTKIESGQGTIP VRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP (the yellow amino acid is the amino acid mutation site at position 488) (SEQ ID No.: 3, wherein position 227 is mutated from E to Q)

[0078] In another preferred embodiment, the amino acid sequence of the ADAR2-isoform2 effector domain is as follows:

[0079] DQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTG

[0080] TKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPC

[0081] GDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGI

[0082] QGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVG

[0083] DSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKA

[0084] GLGAWVEKPTEQDQFSLTP(SEQ ID NO.:4)

[0085] In another preferred embodiment, the amino acid sequence of the ADAR2-isoform2-E488Q effector domain is as follows:

[0086] DQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRF LYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLL TMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP (yellow amino acid is the 488th amino acid mutation site) (SEQ ID No.: 4, the 187th position is mutated from E to Q)

[0087] In another preferred embodiment, the APOBEC family members include: Apobec1, Apobec3A, Apobec3G, or a combination thereof.

[0088] In another preferred embodiment, the APOBEC family member is derived from human or mouse, preferably from human.

[0089] In another preferred embodiment, the APOBEC family member is selected from the following group: human Apobec1, human Apobec3A, human Apobec3B, human Apobec3C, human Apobec3D, human Apobec3F, human Apobec3G, human Apobec3H, human AID, mouse Apobec1, mouse Apobec3A, mouse AID, rat Apobec1, rat Apobec3A, rat AID or a combination thereof.

[0090] In another preferred embodiment, the APOBEC3A has the amino acid sequence (full-length amino acid sequence) shown in SEQ ID NO.:5.

[0091] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAEL RFDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQV SIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN(SEQ ID NO.:5).

[0092] In another preferred embodiment, the RNA methylase includes: METTL3, METTL14, or a combination thereof.

[0093] In another preferred embodiment, the RNA demethylase is α-ketoglutarate-dependent dioxygenase FTO.

[0094] In another preferred embodiment, the uracil synthetase is pseudouridine7.

[0095] In another preferred embodiment, the editing window of the RNA editing enzyme is positions 7-14, preferably positions 8-13, and more preferably positions 9-11, starting from the first position at the 5' end of the PUF binding site (ie, position 1).

[0096] In another preferred embodiment, the RNA editing enzyme does not contain RNA and / or DNA.

[0097] In another preferred embodiment, the RNA recognition domain and the utility domain in the RNA editing enzyme are connected in a head-to-tail, head-to-head, tail-to-head, or tail-to-tail manner.

[0098] In another preferred embodiment, the RNA recognition domain and the utility domain in the RNA editing enzyme are directly connected or connected through a connecting peptide.

[0099] In another preferred embodiment, the C-terminus or N-terminus of the RNA editing enzyme also includes an NLS sequence and an MLS sequence.

[0100] NLS nuclear localization signal sequence: PKKKRKV (SEQ ID No.: 13).

[0101] MLS mitochondrial localization signal sequence: MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQNKVQ (SEQ ID No.: 14).

[0102] In another preferred embodiment, the connecting peptide is a non- or flexible peptide.

[0103] In another preferred embodiment, the connecting peptide is selected from the following group: Linker2, Linker7, XTEN, Linker20, Linker40, or a combination thereof.

[0104] In another preferred embodiment, the length of the connecting peptide is 0-40 aa, preferably 2-20 aa.

[0105] In another preferred embodiment, the connecting peptide has an amino acid sequence shown in any one of SEQ ID NO.: 6-9:

[0106] Linker2:EF

[0107] Linker7: EFTGNGS (SEQ ID NO.: 6)

[0108] XTEN:SGSETPGTSESATPES(SEQ ID NO.:7)

[0109] Linker20:DQTPSRQPIPSEGLQLHLPQ(SEQ ID NO.:8)

[0110] Linker41:KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGS(SEQ ID NO.:9)

[0111] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the RNA editing enzyme described in the first aspect of the present invention.

[0112] In the third aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide described in the second aspect of the present invention.

[0113] In another preferred embodiment, the vector includes DNA and RNA.

[0114] In another preferred embodiment, the vector is selected from the following group: plasmid, viral vector, transposon, or a combination thereof.

[0115] In another preferred embodiment, the vector includes a DNA virus or a retroviral vector.

[0116] In another preferred embodiment, the vector is selected from the following group: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

[0117] In another preferred embodiment, the vector is a lentiviral vector.

[0118] In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, intron, transcription termination signal, polyadenylation sequence, replication origin, selective marker, nucleic acid restriction site, and / or homologous recombination site.

[0119] In another preferred embodiment, the vector is a vector containing or inserted with the polynucleotide described in the second aspect of the present invention.

[0120] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention, or the exogenous polynucleotide described in the second aspect of the present invention is integrated into the chromosome, or expresses the RNA editing enzyme described in the first aspect of the present invention.

[0121] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0122] In another preferred embodiment, the host cell is a human cell or a non-human mammalian cell.

[0123] In the fifth aspect of the present invention, a preparation is provided, characterized in that the preparation comprises the RNA editing enzyme described in the first aspect of the present invention, or the polynucleotide described in the second aspect, or the vector described in the third aspect, and a pharmaceutically acceptable carrier or excipient.

[0124] In another preferred embodiment, the preparation is a liquid preparation.

[0125] In a sixth aspect of the present invention, a use of the RNA editing enzyme according to the first aspect of the present invention, or the polynucleotide according to the second aspect, or the host cell according to the fourth aspect of the present invention is provided, characterized in that it is used to prepare

[0126] (a) drugs or agents for gene therapy; and / or

[0127] (b) Reagents for editing RNA.

[0128] In another preferred embodiment, the editing of RNA includes mutating A to G and / or mutating C to U in the RNA.

[0129] In a seventh aspect of the present invention, a method for editing RNA is provided, the method comprising the steps of:

[0130] (1) providing RNA to be edited and the RNA editing enzyme described in the first aspect of the present invention; and

[0131] (2) Editing the RNA using the RNA editing enzyme according to claim 1.

[0132] In another preferred embodiment, the method is in vitro or in vivo.

[0133] In another preferred embodiment, the method is for non-diagnostic and non-therapeutic purposes.

[0134] In another preferred embodiment, the editing RNA includes mutating A to G and / or mutating C to U in the RNA.

[0135] In an eighth aspect of the present invention, a method for preparing the RNA editing enzyme of the first aspect of the present invention is provided, the method comprising the steps of:

[0136] Under conditions suitable for expression, culturing the host cell described in the fourth aspect of the present invention to express the RNA editing enzyme; and isolating the RNA editing enzyme.

[0137] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0138] The ninth aspect of the present invention provides a method for treating a disease, the method comprising: administering to a subject in need thereof the RNA editing enzyme as described in the first aspect of the present invention, or the polynucleotide as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention, or the preparation as described in the fifth aspect of the present invention.

[0139] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 Construction of different A→G RNA editing enzymes is shown.

[0141] Figure 2 Editing of target RNA using PARSEs is shown.

[0142] Figure 3It was shown that replacing the positions of PUF and ADAR produced an enzyme that was unable to edit the target RNA.

[0143] Figure 4 The efficiency and off-target rate analysis of PARSEs editing of target RNA are shown.

[0144] Figure 5 Repair of aberrant RNA editing events in the GRIA2 gene by ePARSE1 is shown.

[0145] Figure 6 The use of ePARSE2 to repair pathogenic point mutations in related genes is shown.

[0146] Figure 7 It was shown that extending the PUF recognition domain in the PARSE system can significantly improve the RNA editing accuracy of the PARSE system.

[0147] Figure 8 Construction of APRSEs to edit target RNA is shown.

[0148] Fig. 9 The use of APRSE to repair pathogenic point mutations in related genes is shown.

[0149] Fig.10 It was shown that extending the PUF recognition domain in the APRSE system can significantly improve the RNA editing accuracy of the APRSE system.

[0150] Fig.11 A schematic diagram of the structure of the PUF element is shown. DETAILED DESCRIPTION

[0151] After extensive and in-depth research, the inventors have developed a uniquely structured RNA editing enzyme for the first time. The inventors unexpectedly discovered that a new RNA editing enzyme based on the recognition domain and utility domain of RNA binding can very effectively target specific RNA regions and perform efficient and accurate RNA editing. The RNA editing enzyme of the present invention can not only perform RNA editing efficiently and accurately, but also effectively prevent reverse mutations, and therefore has the advantages of being more flexible, safe, and efficient. On this basis, the inventors completed the present invention.

[0152] Terminology

[0153] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0154] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0155] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0156] PUF protein (Pumilio homolog 1)

[0157] PUF protein is a sequence-specific RNA binding protein with a conserved RNA binding domain. It regulates the stability or translation efficiency of mRNA by binding to the 3'-UTR of the target mRNA. The typical PUF binding domain contains 8 α-helical repeat sequences, each of which is 36 amino acids and is responsible for recognizing and binding to 1 base. At both ends of the binding domain, there is a non-functional repeat sequence to protect the 8 repeat sequences in the middle. The three amino acids at specific positions on the α-helical repeat sequence are responsible for binding to RNA bases. Among them, the side chain amino acids at positions 12 and 16 bind to the RNA base through hydrogen bonds, and the amino acid at position 13 plays an auxiliary binding role. Different amino acid combinations are responsible for recognizing different bases (see Fig.11 ). After the modification and design of each repeating sequence of the PUF protein, the newly designed PUF of the present application can recognize and bind to any 8-base RNA sequence. After modification, the number of PUF repeating sequences can be increased or decreased to expand the binding ability of PUF to RNA, so that the PUF of the present application can recognize 6 to 16 bases, and further can recognize 20 bases. In different species, PUF protein has many homologous proteins with similar sequence characteristics and similar functions as PUF protein.

[0158] In a preferred embodiment of the present invention, human PUM1 is selected as a tool for identifying RNA.

[0159] ADAR

[0160] ADAR (Double-stranded RNA-specific adenosine deaminase) protein is a class of RNA deaminases that act on double-stranded RNA. It catalyzes the hydrolysis and deamination of adenosine in double-stranded RNA to form inosine (adenosine-to-inosine, A-to-I), which is called A-to-I RNA editing. Inosine (I) is recognized as guanosine (G) during translation, achieving A-to-G RNA editing. The main members of this protein family are ADAR1, ADAR2 and ADAR3. The gene editing produced by this enzyme will affect the expression and function of genes, including changing the amino acid sequence of proteins by changing the mRNA translation of codons; pre-mRNA splicing by changing the splicing site recognition sequence; achieving RNA stability by changing the sequence involved in nuclease recognition; RNA virus genomes achieve genetic stability by changing the sequence during viral RNA replication and regulate some structure-dependent RNA metabolic activities, such as microRNA production, targeting or protein-RNA interaction.

[0161] Representative ADARs include ADAR1, ADAR2-isoform1 and ADAR2-isoform1, or homologous proteins thereof.

[0162] A representative full-length amino acid sequence of ADAR1 is as follows:

[0163] MNPRQGYSLSGYYTHPFQGYEHRQLRYQQPGPGSSPSSFLKQIEFLKGQLPEAPVIGKQTPSLPPSLPGLRP

[0164] RFPVLLASSTRGRQVDIRGVPRGVHLRSQGLQRGFQHPSPRGRSLPQRGVDCLSSHFQELSIYQDQEQRILKFLEEL

[0165] GEGKATTAHDLSGKLGTPKKEINRVLYSLAKKGKLQKEAGTPPLWKIAVSTQAWNQHSGVVRPDGHSQGAPNSDPSL

[0166] EPEDRNSTSVSEDLLEPFIAVSAQAWNQHSGVVRPDSHSQGSPNSDPGLEPEDSNSTSALEDPLEFLDMAEIKEKIC

[0167] DYLFNVSDSSALNLAKNIGLTKARDINAVLIDMERQGDVYRQGTTPPIWHLTDKKRERMQIKRNTNSVPETAPAAIP

[0168] ETKRNAEFLTCNIPTSNASNNMVTTEKVENGQEPVIKLENRQEARPEPARLKPPVHYNGPSKAGYVDFENGQWATDD

[0169] IPDDLNSIRAAPGEFRAIMEMPSFYSHGLPRCSPYKKLTECQLKNPISGLLEYAQFASQTCEFNMIEQSGPPHEPRF

[0170] KFQVVINGREFPPAEAGSKKVAKQDAAMKAMTILLEEAKAKDSGKSEESSHYSTEKESEKTAESQTPTPSATSFFSG

[0171] KSPVTTLLECMHKLGNSCEFRLLSKEGPAHEPKFQYCVAVGAQTFPSVSAPSKKVAKQMAAEEAMKALHGEATNSMA

[0172] SDNQPEGMISESLDNLESMMPNKVRKIGELVRYLNTNPVGGLLEYARSHGFAAEFKLVDQSGPPHEPKFVYQAKVGG

[0173] RWFPAVCAHSKKQGKQEAADAALRVLIGENEKAERMGFTEVTPVTGASLRTMLLLSRSPEAQPKTLPLTGSTFHDQ

[0174] IAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFI

[0175] RFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQ

[0176] GKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQG

[0177] HLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRN

[0178] ELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV*(SEQ ID No.:10)

[0179] A representative full-length amino acid sequence of ADAR2-isoform1 is as follows:

[0180] MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKT

[0181] PGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASE

[0182] AHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPP

[0183] FPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFNLHLDQT

[0184] PSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGE

[0185] YMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFS

[0186] PHEPILEGSRSYTQAGVQWCNHGSLQPRPPGLLSDPSTSTFQGAGTTEPADRHPNRKARGQLRTKIESGEGTIPVRS

[0187] NASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPP

[0188] LYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLR

[0189] SKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP*(SEQ ID No.:11)

[0190] A representative full-length amino acid sequence of ADAR2-isoform2 is as follows:

[0191] MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFNLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP*(SEQ ID No.:12)

[0192] APOBEC proteins

[0193] APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) is a class of evolutionarily conserved cytidine deaminases that act on the single-stranded region of DNA / RNA, catalyzing the hydrolytic deamination of cytidine to form uridine (cytosine-to-uracil, C-to-U) on single-stranded DNA / RNA, thereby achieving C-to-U DNA / RNA editing. The main members of this protein family include AID, Apobec1, Apobec2, Apobec3A, Apobec3B, Apobec3C, Apobec3D, Apobec3F, Apobec3G, Apobec3H and Apobec4.

[0194] RNA editing enzymes

[0195] As used herein, "RNA editing enzyme of the present invention", "artificial RNA editing enzyme", "fusion protein" or "polypeptide of the present invention" all refer to the RNA editing enzyme described in the first aspect of the present invention.

[0196] The RNA editing enzyme of the present invention comprises:

[0197] (a) an RNA recognition domain, wherein the RNA recognition domain is used to recognize an RNA recognition sequence of an RNA sequence to be edited and bind to the RNA recognition sequence;

[0198] (b) a utility domain, wherein the utility domain is used to perform nucleotide editing on the RNA sequence to be edited;

[0199] Wherein, the RNA recognition domain and the utility binding domain are operably connected.

[0200] As used herein, "operably connected" or "operably connected" refers to a parallel relationship in which the elements are in a relationship that allows them to function in an expected manner. For example, if the RNA recognition domain is connected to the utility domain (directly, or through a connecting element, or through other functional elements located between the two), then as long as the RNA recognition domain and the utility domain can each perform their functions, that is, the RNA recognition domain recognizes and binds to a predetermined RNA recognition sequence, and the utility domain can perform nucleotide editing on the RNA sequence to be edited, then the two are operably connected. Similarly, if a promoter can cause a coding sequence to be transcribed or expressed, then the promoter is operably connected to the coding sequence.

[0201] As used herein, the term "RNA editing enzyme of the present invention" also includes variant forms of sequences having the above-mentioned activities. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, preferably 1) amino acids, and addition or deletion of one or several (usually within 3, preferably within 2, and more preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. In addition, the term also includes polypeptides of the present invention in monomeric and polymeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0202] The present invention also includes active fragments, derivatives and analogs of the above-mentioned RNA editing enzymes. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the function or activity of the RNA editing enzyme of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0203] A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of Formula I. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0204] Table A

[0205] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0206] The present invention also provides analogs of the RNA editing enzyme of the present invention. The difference between these analogs and the polypeptides shown in SEQ ID NO.: 8, 9 or 13 may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0207] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0208] Coding sequence

[0209] The present invention also relates to polynucleotides encoding the RNA editing enzyme according to the present invention.

[0210] The polynucleotide of the present invention can be in the form of DNA or RNA. DNA can be a coding strand or a non-coding strand. The full-length nucleotide sequence of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0211] The present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells produced by genetic engineering using the vectors or polypeptide coding sequences of the present invention. The above polynucleotides, vectors or host cells may be isolated.

[0212] As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0213] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0214] The present invention also relates to variants of the above-mentioned polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change its function of encoding the RNA editing enzyme of the present invention.

[0215] The full-length nucleotide sequence encoding the fusion protein of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0216] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0217] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0218] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The DNA / RNA fragments amplified can be separated and purified by conventional methods such as by gel electrophoresis.

[0219] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method for expressing the RNA editing enzyme of the present invention on the NK cells by recombinant technology.

[0220] The polynucleotide sequence of the present invention can be used to obtain NK cells expressing the RNA editing enzyme of the present invention by conventional recombinant DNA technology, which generally includes the steps of: transducing the first expression cassette and / or the second expression cassette of the present invention into NK cells, thereby obtaining the NK cells.

[0221] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the RNA editing enzyme of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0222] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0223] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0224] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: bacterial cells of Escherichia coli, Bacillus subtilis, Streptomyces; fungal cells such as Pichia pastoris, Saccharomyces cerevisiae cells; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, NS0, COS7, or 293 cells, etc.

[0225] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0226] The obtained transformant can be cultured by conventional methods to express the protein encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the promoter selected is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0227] The protein in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0228] Carrier

[0229] The present invention also provides vectors containing the polynucleotides of the present invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentivirus vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0230] Briefly summarized, usually by operably connecting the expression cassette of the present invention or nucleic acid sequence to a promoter and incorporating it into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0231] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0232] The expression cassette or nucleic acid sequence can be cloned into many types of vectors. For example, the expression cassette or nucleic acid sequence can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0233] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0234] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject cell in vivo or in vitro. Many retroviral systems are known in the art.

[0235] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0236] An example of a suitable promoter is an early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be a part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0237] The expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0238] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0239] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, for example, mammals (such as human T cells), bacteria, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0240] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0241] Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0242] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0243] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0244] preparation

[0245] The present invention provides a preparation as described in the fifth aspect of the present invention. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection.

[0246] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0247] The technical solution of the present invention has the following beneficial effects:

[0248] (1) The RNA editing enzyme of the present invention is a single-component protease that does not contain any RNA components and is composed of endogenous human protein sequences. Therefore, these engineered proteins have lower immunogenicity and system complexity than the CRISPR-Cas system in gene therapy. Moreover, the system is more flexible and safer than DNA editing.

[0249] (2) The RNA editing enzyme of the present invention has a low off-target rate and a high editing efficiency.

[0250] (3) The RNA editing enzyme of the present invention has high editing accuracy and can achieve single-base gene editing.

[0251] (4) The RNA editing enzyme of the present invention is a protease that can be targeted to an organelle or a nucleus through an organelle localization signal to exert its function. For example, a nuclear localization signal NLS is connected to the N-terminus or C-terminus of the RNA editing enzyme to localize the editing enzyme to the nucleus to exert its function, or a mitochondrial localization signal MLS is connected to localize the enzyme to the mitochondria to exert its editing function.

[0252] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0253] Experimental methods

[0254] The deamination catalytic domains of members of the RNA adenosine deaminase ADAR family and members of the RNA cytidine deaminase APOBEC family were cloned and fused with the RNA binding protein PUF to form two different RNA editing enzymes PARSE and APRSE. According to the above design, PARSE and APRSE RNA editing enzymes were verified at the cellular level to edit RNA. The editing efficiency and accuracy of these enzymes were tested, and the off-target effects of these enzymes at the transcriptome level were analyzed. The constructed RNA editing enzymes were used to repair some single-base pathogenic point mutations, which were used to repair pathogenic point mutations in GRIA2, COL3A1, DMD, EZH2, SCN1A and SCN5A genes.

[0255] Specifically, the steps include:

[0256] 1. Based on the existing RNA binding protein PUF, the dADAR, ADAR1, and ADAR2 genes encoding ADAR proteins were cloned separately using molecular cloning technology, and then the two genes were spliced ​​together to form a new fusion gene. This new fusion gene encodes the PARSE protein to construct different A-to-I RNA site-directed editing enzymes. Different RNA cytidine deaminases (human apobec1, human apobec3a, human3b, human3c, human3d, human3g, human3h, humanAID, mouse APOBEC1, mouse APOBEC3A, mouse AID, RatAPOBEC1) were fused to PUF using molecular cloning technology to construct different C-to-U RNA site-directed editing enzymes.

[0257] 2. Detect the editing activity of these new RNA editing enzymes at the cellular level. Transfer the PARSE RNA editing enzyme and GFP reporter gene plasmid into the cells at the same time. After 48 hours, collect the RNA of the GFP reporter gene and sequence it to detect RNA editing events.

[0258] 3. To detect the editing activity of these new RNA editing enzymes at the cellular level, the APRSE RNA editing enzyme and the GFP reporter gene plasmid were simultaneously transferred into the cells. After 48 hours, the RNA of the GFP reporter gene was collected and sequenced to detect RNA editing events.

[0259] 4. Use RNA seq high-throughput sequencing technology to detect and analyze the efficiency, accuracy and off-target rate of RNA editing enzymes.

[0260] 5. Use the constructed RNA editing enzyme to repair some single-base pathogenic mutations, and use PARSE to repair pathogenic point mutations such as GRIA2, COL3A1 and DMD.

[0261] 6. Use the constructed RNA editing enzyme to repair some single-base pathogenic mutations, and use APRSE to repair pathogenic point mutations such as EZH2, SCN1A and SCN5A genes.

[0262] Example 1

[0263] ADAR is an adenosine editing enzyme that can catalyze the deamination of RNA adenosine to form inosine (A-to-I). The ADAR catalytic domains from three different sources were cloned and fused with the linker and RNA-binding protein PUF to develop three different artificial RNA editing enzymes (RNA editases). The system was named artificial PUF- A DAR R NA s equence e ditors(PARSE-d,PARSE1,PARSE2)( Figure 1 , A-D)

[0264] The editing activities of three RNA editing enzymes, PARSE-d, PARSE1 and PARSE2, were detected at the cellular level. The PARSE RNA editing enzyme and GFP reporter gene plasmid were simultaneously transferred into the cells. After 48 hours, the RNA of the GFP reporter gene was collected and sequenced to detect RNA editing events, proving that PARSE-d, PARSE1 and PARSE2 all have the ability to efficiently edit target RNA ( Figure 2 A). By constructing a GFP mRNA containing a premature stop codon, PARSE was used to edit the A to G at a specific site, thereby restoring the expression of the GFP gene, demonstrating that PARSE has high-precision site-specific editing capabilities ( Figure 2 B).

[0265] The ADAR catalytic domains from three different sources were cloned and fused with the RNA binding protein PUF through a linker. The ADAR was placed at the N-terminus of the newly designed fusion protein, and the PUF was placed at the C-terminus of the newly designed fusion protein, forming a new RNA editing enzyme with the ADAR catalytic domain in front and the PUF RNA binding protein in the back. This newly synthesized RNA editing enzyme was used to edit the target RNA, and no editing events were detected ( Figure 3 ). This proves that the RNA editing enzyme that depends on ADAR has strict requirements on the location of RNA binding protein, and the RNA editing enzyme of the present invention has excellent RNA editing activity.

[0266] Example 2

[0267] The editing efficiency of target RNA by ADAR was improved by optimizing the ADAR catalytic domain, and the efficiency, accuracy and off-target rate of RNA editing enzymes were detected and analyzed by RNA seq high-throughput sequencing technology. A new point mutation was introduced in the catalytic domain of ADAR1 and ADAR2 to improve the editing efficiency of ADAR ( Figure 4 A) Using RNA seq high-throughput sequencing technology, we detected and analyzed the efficiency and accuracy of RNA editing enzymes and found that PARSE1, ePARSE1, PARSE2, and ePARSE2 can edit target RNA with efficiencies of 42%, 65%, 67%, and 78%, respectively. Figure 4B). Analysis of RNA-seq results showed that PARSE editing had a certain degree of off-target rate, but compared with the editing efficiency of the target site, the off-target efficiency was lower. The off-target rate could be reduced by reducing the transfection amount of PARSE during the later optimization process ( Figure 4 C).

[0268] The constructed ePARSE1 was used to repair the pathogenic editing site of the GRIA2 gene. GRIA2 is a subunit of a calcium channel protein. A mutation in the 607th amino acid of the protein will cause the calcium channel protein to fail to close, resulting in a pathogenic phenotype. The results of using ePARSE1 to perform site-specific repair on the site showed that the repair efficiency was 68%, which means that the repair of the pathogenic point mutation of GRIA2 (p.Q607R) can be completed at the cellular level, providing a powerful tool for further treatment of this gene mutation ( Figure 5 A), and the off-target efficiency of this site is low ( Figure 5 B), showing promising application prospects of ePARSE1 in treating this disease.

[0269] The constructed ePARSE2 was used to repair the pathogenic point mutations of COL3A1 and DMD genes. The results showed that the repair efficiency was 33%, 25%, and 30%, respectively. That is, the pathogenic point mutations of COL3A1 and DMD genes can be repaired at the cellular level, providing a powerful tool for further treatment of the gene mutation. Figure 6 A, 6B, 6C), showing good application prospects for treating this type of disease through ePARSE2.

[0270] Example 3

[0271] The RNA binding protein PUF was optimized to improve the accuracy of PARSE editing RNA and reduce the off-target rate. By optimizing the PUF domain, PUF8 that recognizes 8 bases was optimized to PUF10 that recognizes and binds to 10 bases. Figure 7 , AC), this optimized design did not reduce the editing efficiency of the RNA target site, and with the increase in the number of bases that PUF can recognize and bind to, this strategy reduced the off-target rate of PARSE by 10 times, effectively reducing the off-target effect, and has the potential for further optimization. PUF can be optimized to recognize and bind to 12 bases, 16 bases, or even longer, which can greatly expand the application range of PARSE.

[0272] Example 4

[0273] APOBECs can catalyze cytidine-to-uridine (C-to-U) nucleotide editing. The catalytic domains of RNA cytidine deaminases (human apobec1, human apobec3a, human3b, human3c, human3d, human3g, human3h, humanAID, mouse APOBEC1, mouse APOBEC3A, mouseAID, Rat APOBEC1) from various APOBEC families were cloned and fused with linker and RNA binding protein PUF to form a variety of new C to U RNA site-directed editing enzymes. This system is named artificial A POBEC- P UF R NA s equence e ditors (APRSE), and is further divided into APRSE-NLS that can enter the cell nucleus and APRSE expressed in the cytoplasm. The figure below uses Apobec3A as an example to illustrate ( Figure 8 A).

[0274] The editing activities of APRSE-NLS and APRSE were detected at the cellular level. The APRSE RNA editing enzyme and GFP reporter gene plasmid were simultaneously transferred into cells. After 48 hours, the RNA of the GFP reporter gene was collected and sequenced to detect RNA editing events, proving that APRSE-NLS and APRSE both have the ability to efficiently edit target RNA ( Figure 8 B, 8C), the results showed that APRSE has high-precision site-specific editing capabilities, and can directly edit the second base downstream of the APRSE binding site with high precision and high efficiency. The editing efficiency of different editing sites is between 30% and 80%.

[0275] The constructed APRSE was used to repair the pathogenic point mutations of EZH2, SCN1A and SCN5A genes. The results showed that the repair efficiency was 39%, 23% and 12%, respectively. That is, the pathogenic point mutations of EZH2, SCN1A and SCN5A genes can be repaired at the cellular level, providing a powerful tool for further treatment of the gene mutations. Fig. 9 A, 9B, 9C), showing the good application prospect of APRSE in treating this type of disease.

[0276] Example 5

[0277] The RNA binding protein PUF was optimized to improve the accuracy of APRSE editing RNA and reduce off-target efficiency. By optimizing the PUF domain, PUF8 that recognizes 8 bases was optimized to PUF10 that recognizes and binds to 10 bases. Fig.10 , AC), this optimized design did not reduce the editing efficiency of the RNA target site, and with the increase in the number of bases that PUF can recognize and bind, this strategy reduced the off-target effect of APRSE by 12 times, effectively reducing the off-target effect, and has the potential for further optimization. PUF can be optimized to recognize and bind 12 bases, 16 bases, or even longer, which can greatly expand the application scope of APRSE.

[0278] discuss

[0279] Compared with direct specific editing and manipulation of DNA, specific manipulation of RNA is reversible and has greater flexibility. Gene therapy programs targeting RNA can effectively avoid the shortcomings of DNA gene therapy. Therefore, gene manipulation at the RNA level has better controllability and safety, making this type of gene therapy more conducive to the transformation of basic research into clinical practice.

[0280] The current RNA-level base editing mainly includes REPAIR based on CRISPR-Cas13 and the method of recruiting endogenous cellular RNA adenosine deaminase ADAR for RNA editing through oligonucleotide fragments. REPAIR based on CRISPR-Cas13 also has the same problems as the above-mentioned DNA editing, including the system is too large, the editing efficiency and editing accuracy are low, and it is easy to cause immune response. The method based on oligonucleotide fragments to recruit endogenous cellular RNA adenosine deaminase for RNA editing currently has too small a scope of application, and also has the problems of delivery difficulties, relatively single efficacy, and a relatively complex system.

[0281] The inventors fuse the RNA recognition domain and the effector domain to form a new functional protein, which specifically targets the target RNA through the recognition domain and uses the effector domain to edit RNA, and eliminates pathogenic RNA by correcting the wrong point mutations of DNA. The artificially constructed RNA editing enzyme is a single-component protease that does not contain any RNA component and is composed of endogenous human protein sequences. Therefore, these engineered proteins have lower immunogenicity and system complexity than the CRISPR-Cas system in gene therapy. Moreover, the system has more flexible and safe features compared to DNA editing.

[0282] In addition, since artificial RNA editing enzymes based on a single protein can connect to different cell localization sequences to locate in different subcellular structures (including the nucleus, cytoplasm, mitochondria, chloroplasts, etc.), artificial RNA editing enzymes can specifically edit RNA in different subcellular structures. Taking mitochondria as an example, there is currently no effective editing method for mitochondrial genes, so artificial RNA editing enzymes can play an irreplaceable role in mitochondrial gene manipulation.

[0283] Therefore, constructing modular artificial RNA-binding proteins through synthetic biology, fusing RNA editing proteins to RNA-binding proteins, and thus specifically manipulating the editing of targeted RNA is a new therapeutic approach for targeting RNA. Since the artificially designed PUF factor can be reprogrammed to recognize almost any 8-nucleotide sequence, it can theoretically be used to edit any given RNA transcript. It is expected that by applying this system, some disease-related mutations can be targeted for editing. This system provides a useful tool for targeted editing of RNA in human cells, and it is promising to treat some diseases caused by nucleic acid mutations. Moreover, the artificially constructed PUF-Factor is a simple enzyme that does not contain any RNA components and is composed of endogenous human protein sequences. These engineered proteins may be a simpler and more practical alternative to the CRISPR-Cas system in gene therapy.

[0284] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An RNA editing enzyme, characterized in that The RNA editing enzymes include: (a) an RNA recognition domain, wherein the RNA recognition domain is used to recognize an RNA recognition sequence of an RNA sequence to be edited and bind to the RNA recognition sequence; (b) a utility domain, wherein the utility domain is used to perform nucleotide editing on the RNA sequence to be edited; Wherein, the RNA recognition domain and the utility binding domain are operably connected.

2. The RNA editing enzyme according to claim 1, characterized in that The utility domain is selected from the group consisting of a deamination catalytic domain of an ADAR family member, a deamination catalytic domain of an APOBEC family member, an RNA methylase, an RNA demethylase, an uracil synthetase, or a combination thereof.

3. The RNA editing enzyme according to claim 1, characterized in that The RNA recognition domain contains n recognition units, each of which is used to recognize an RNA base, wherein n is a positive integer of 5-30.

4. The RNA editing enzyme according to claim 1, characterized in that The RNA editing enzyme further comprises one or more elements selected from the following group: a connecting peptide, a tag sequence, a signal peptide sequence, a positioning peptide sequence, or a combination thereof.

5. The RNA editing enzyme according to claim 1, characterized in that The RNA editing enzyme includes an RNA recognition domain and a utility domain, and optionally a connecting peptide, a tag sequence, a signal peptide sequence and / or a localization peptide sequence.

6. The RNA editing enzyme according to claim 1, characterized in that The structure of the RNA editing enzyme is shown in any one of Formula I to Formula IV below: D-L2-A-L1-B(I); D-L2-B-L1-A(II); A-L1-B-L2-D(III); B-L1-A-L2-D(IV); In the formula, each "-" is independently a connecting peptide or a peptide bond; A is the RNA recognition domain; B is the utility domain; L1 and L2 are each independently no or a linker peptide; D is no or localized peptide.

7. The RNA editing enzyme according to claim 2, characterized in that The ADAR family members include: dADAR, ADAR1, ADAR2, TadA, or a combination thereof.

8. An isolated polynucleotide, characterized in that The polynucleotide encodes the RNA editing enzyme of claim 1.

9. A carrier, characterized in that The vector comprises the polynucleotide of claim 8.

10. A use of the RNA editing enzyme according to claim 1, the polynucleotide according to claim 8, or the host cell according to the vector according to claim 9, characterized in that: For preparation (a) drugs or agents for gene therapy; and / or (b) Reagents for editing RNA.

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