WO2021136520A1 - 一种靶向编辑rna的新方法 - Google Patents

一种靶向编辑rna的新方法 Download PDF

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WO2021136520A1
WO2021136520A1 PCT/CN2020/142218 CN2020142218W WO2021136520A1 WO 2021136520 A1 WO2021136520 A1 WO 2021136520A1 CN 2020142218 W CN2020142218 W CN 2020142218W WO 2021136520 A1 WO2021136520 A1 WO 2021136520A1
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arrna
target
base
rna
adenosine deaminase
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PCT/CN2020/142218
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French (fr)
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袁鹏飞
易泽轩
刘能银
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博雅辑因（北京）生物科技有限公司
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Priority to AU2020417930A priority Critical patent/AU2020417930A1/en
Priority to US17/790,488 priority patent/US20230060517A1/en
Priority to CA3163281A priority patent/CA3163281A1/en
Priority to JP2022540984A priority patent/JP2023509178A/ja
Priority to EP20909741.9A priority patent/EP4085931A4/en
Priority to IL294336A priority patent/IL294336A/en
Priority to CN202080080529.2A priority patent/CN114728080A/zh
Priority to KR1020227026283A priority patent/KR20220122727A/ko
Publication of WO2021136520A1 publication Critical patent/WO2021136520A1/zh

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    • C12Y305/04004—Adenosine deaminase (3.5.4.4)

Definitions

  • This application belongs to the field of gene editing therapy. Specifically, this application creates a method for targeted editing of RNA called CUSPER (C to U Specific Programmable Editing of RNA), which includes the use of CUSPER technology to perform RNA editing from C to U The precise site editing of bases can be used to treat diseases caused by T to C mutations.
  • CUSPER C to U Specific Programmable Editing of RNA
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • CRISPR-based DNA editing technology requires the exogenous expression of Cas9 or other nucleases with similar functions, causing the following problems.
  • nucleases that require exogenous expression usually have a relatively large molecular weight, which makes the efficiency of delivering them into the body through viral vectors drastically reduced.
  • this method has potential for off-target nucleases, which will lead to potential carcinogenic risks in its applications.
  • exogenously expressed Cas9 and other similar nucleases are found in bacteria, rather than naturally occurring in humans or mammals, which makes it possible to cause an immune response in the patient's body, which may cause damage to the patient himself. On the other hand, it may also neutralize the exogenously expressed nuclease, thereby losing its proper activity and affecting the therapeutic effect.
  • RNA editing technology called REPAIR (RNA Editing for Programmable A to I Replacement), which expresses Cas13-ADAR fusion protein and single guide RNA ( Single guide RNA, sgRNA) can achieve the editing of target RNA from A to I2, but this method, like CRISPR technology, still requires the expression of foreign proteins. Unable to solve the problems caused by the expression of foreign proteins.
  • RESTORE Recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing, Merkle et al., 2019.
  • RESTORE can get rid of the dependence on foreign proteins, but the RESTORE technology needs to have high editing efficiency under the premise of the existence of IFN- ⁇ , and IFN- ⁇ is a key factor that determines the development and severity of autoimmunity9, which makes this technology The application in the medical field is greatly reduced.
  • a guide RNA is also used in the RESTORE technology, and the guide RNA it uses is a chemically synthesized oligonucleotide, and the synthesized oligonucleotide requires artificial introduction of a large number of chemical modifications to ensure its stability. .
  • these chemical modifications some of them are unnatural modifications, which make the oligonucleotide may be toxic or immunogenic; and some of the modifications will result in different conformations of the same base chain, making it possible for the same RNA sequence to have different conformations. Dozens of different conformational combinations, which increase the difficulty of its delivery into cells.
  • RNA editing technology Leveraging Endogenous ADAR for Programmable Editing of RNA.
  • this technology gets rid of the dependence on the overexpression of exogenous nucleases in principle, and it can be done by chemically synthesizing RNA, or it can be delivered to patients through vectors such as adeno-associated virus (AAV) and lentivirus.
  • AAV adeno-associated virus
  • this technology can only achieve adenosine A to creatinine I (creatinine I). I will be recognized as the editing of guanine G) during protein translation, making it impossible to deal with other mutations, such as the mutation from T to C.
  • this technology also requires a piece of RNA as a guide to recruit endogenous nucleases to the desired editing site.
  • the guide RNA is named arRNA (adar-recruiting RNA).
  • This application creates a new RNA editing technology CUSPER (C to U Specific Programmable Editing of RNA), which does not need to rely on the expression of bacterial-derived Cas13b, and expands the application range of RNA editing from A to I editing to C Edit to U.
  • CUSPER C to U Specific Programmable Editing of RNA
  • this application relates to:
  • composition or system for RNA editing comprising:
  • the domain has been modified to have the activity of catalyzing the deamination of cytidine
  • the arRNA comprises a complementary RNA sequence that hybridizes with the target RNA, and the arRNA recruits the adenosine deaminase protein or its catalytic domain to the target RNA to deaminate the target cytidine in the target RNA.
  • the construct expressing the modified adenosine deaminase protein or its catalytic domain and the construct comprising the coding sequence of the arRNA are the same construct. In some embodiments, the construct expressing the modified adenosine deaminase protein or its catalytic domain and the construct containing the coding sequence of the arRNA are separate constructs.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) that has cytidine deaminization activity by deleting, adding, or substituting one or more amino acids. protein.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) or its catalytic domain is cytidine deaminated by substituting one or more amino acids Active protein.
  • the modified adenosine deaminase protein of the present application includes the following mutation modification: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/ S495N/K418E/S661T, wherein the amino acid numbering modified by the mutation is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein of the present application includes other ADAR2 homologous proteins as a reference sequence and has E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L /D619G/S582T/V440I/S495N/K418E/S661T corresponding mutant protein.
  • the engineered composition or system for RNA editing of the present application comprises the catalytic domain of the modified adenosine deaminase protein described above.
  • the catalytic domain is the catalytic domain of the modified adenosine deaminase protein described above.
  • the engineered composition or system in item 3, wherein the mutation modification comprises: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/ K418E/S661T, wherein the amino acid numbering modified by the mutation is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein or its catalytic domain is a homologous protein of the NP_001103.1 protein, with mutations corresponding to the following mutations: E488Q/V351G/S486A/T375S/S370C/ P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T.
  • the arRNA contains unpaired nucleotides at one or more positions corresponding to the upstream, downstream, or upstream and downstream of the target base of the target RNA to form one of the upstream, downstream, or upstream and downstream positions of the target base. Or multiple nucleotide mismatches.
  • the length of the arRNA is >50nt, >55nt, >60nt, >65nt, >70nt, >75nt, >80nt, >85nt, >90nt,> 95nt, >100nt, >105nt, >110nt, >115nt, >120nt.
  • the arRNA is about 151-53 nt, 131-61 nt, 121-61 nt, 111-65, 101-71 nt, 91-71 nt, 81-71 nt in length.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • the length of the arRNA is any positive integer within the length range defined by this term.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt, 55nt- 45nt.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • a method for deaminating target cytosine in a target RNA in a cell comprising introducing the following 1) and 2) into the cell:
  • the modified adenosine deaminase protein or its catalytic domain is recruited to the arRNA of the target RNA or a construct comprising the arRNA or the coding sequence thereof, wherein the adenosine deaminase protein Or its catalytic domain is modified to have the activity of catalyzing the deamination of cytidine, the arRNA comprises a complementary RNA sequence that hybridizes with the target RNA, and the arRNA recruits the adenosine deaminase protein or its catalytic domain to The target RNA causes the target cytidine in the target RNA to be deaminated.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) that has cytidine deaminization activity by deleting, adding, or substituting one or more amino acids. protein.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) or its catalytic domain is cytidine deaminated by substituting one or more amino acids Active protein.
  • the modified adenosine deaminase protein of the present application includes the following mutation modification: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/ S495N/K418E/S661T, wherein the amino acid numbering modified by the mutation is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein of the present application includes other ADAR2 proteins as reference sequences and has E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G /S582T/V440I/S495N/K418 E/S661T corresponding mutant protein.
  • the engineered composition or system for RNA editing of the present application comprises the catalytic domain of the modified adenosine deaminase protein described above.
  • adenosine deaminase protein or its catalytic domain is ADAR2 protein or its homologous protein or its catalytic domain.
  • the mutation modification comprises: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T, wherein The amino acid numbering of the mutation modification is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein or its catalytic domain is a homologous protein of the NP_001103.1 protein, with mutations corresponding to the following mutations: E488Q/V351G/S486A/T375S/S370C/ P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T.
  • the arRNA contains unpaired nucleotides at one or more positions corresponding to the upstream, downstream, or upstream and downstream of the target base of the target RNA to form one of the upstream, downstream, or upstream and downstream positions of the target base. Or multiple nucleotide mismatches.
  • the target base triplet formed by the target base and its 5'and 3'adjacent bases is only on the target Mismatches are formed at bases, wherein the target base triplet is selected from: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU.
  • the length of the arRNA is >50nt, >55nt, >60nt, >65nt, >70nt, >75nt, >80nt, >85nt, >90nt, >95nt, >100nt, >105nt, >110nt, >115nt, >120nt.
  • the arRNA is about 151-53 nt, 1131-61 nt, 121-61 nt, 111-65, 101-71 nt, 91-71 nt, 81-71 nt in length.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15nt, 24nt-11nt.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt, 55nt-45nt.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • a method for treating diseases caused by a T to C mutation comprising using the method according to any one of items 21 to 39 to deaminate a target base C in a messenger RNA formed by transcription of the T to C mutation, To correct the mutation.
  • a modified adenosine deaminase protein wherein the adenosine deaminase protein is ADAR2, which comprises E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/ D619G/S582T/V440I/S495N/K418E/S661T mutation modification, wherein the amino acid number of the mutation modification is consistent with the amino acid number in NP_001103.1, and the ADAR2 protein has the activity of catalyzing the deamination of cytidine after the mutation modification .
  • ADAR2 comprises E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/ D619G/S582T/V440I/S495N/K4
  • the modified adenosine deaminase protein or its catalytic domain is a homologous protein of the NP_001103.1 protein, with mutations corresponding to the following mutations: E488Q/V351G/S486A/T375S/S370C/ P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T.
  • Figure 1 shows the BFP reporting system and target bases to be edited from C to U.
  • Figure 2 shows the selection of target bases when the target is a point U, and the design principles of the target base when the CUSPER and RESCUE technology design guide RNAs and the target is the adjacent base of the point.
  • Figure 3 shows the test of the CUSPER editing system. "/" in the figure means that no corresponding plasmid or arRNA was added, and only the same volume of water was added.
  • Figure 4 shows the preference of LEAPER technology for adjacent upstream and downstream bases of the target base.
  • the arrow in the figure shows the preference of LEAPER technology for the 5'adjacent upstream corresponding to Figure 3 when the 3'adjacent base of the target base is A.
  • Figure 5 shows the preference of RESCUE technology for adjacent upstream and downstream bases of the target base.
  • the arrow in the figure shows the preference of RESCUE technology for the 5'adjacent base corresponding to Figure 3 when the 3'adjacent base is A.
  • Figure 6 shows the repeated test results of the CUSPER editing system.
  • the "/" in the figure represents that no corresponding plasmid or arRNA was added but only the same volume of water was added.
  • Fig. 7 shows the sequence of mRNA and corresponding arRNA when target C corresponds to U and there is no mismatch between its adjacent bases.
  • Figure 8 shows the C to U RNA editing efficiency of all 16 combinations of 3’ and 5’ adjacent bases when the target C corresponds to U, and its adjacent bases are not mismatched
  • RNA editing technology C to U Specific Programmable Editing of RNA.
  • CUSPER expands the application range of RNA editing from A to I editing to C to U editing, and because the enzyme protein expressed by the mammal itself is used for editing, it avoids the introduction of heterogeneous foreign proteins into the cell, thereby improving safety And make the entire gene editing process more efficient and convenient.
  • CUSPER technology is the original technology in the technical solutions of this application, where CUSPER is the abbreviation of "C to U Specific Programmable Editing of RNA", that is, the specificity of "converting cytidine C to uridine U” Programming RNA editing".
  • This technology uses a short RNA that can complementary hybridize to the target RNA to recruit the modified adenosine deaminase protein or the protein containing its catalytic domain to the target RNA to deaminate the target cytidine and convert it into urine Glycosides.
  • the modified adenosine deaminase protein or the protein comprising its catalytic domain is modified to have the activity of catalyzing the deamination of cytosine.
  • the short RNA that can complementarily hybridize with the target RNA is arRNA.
  • arRNA adar-recruiting RNA
  • arRNA refers to a single-stranded RNA that can recruit adenosine deaminase protein or its catalytic domain to target RNA, and the arRNA recruits the adenosine deaminase protein Or its catalytic domain to the target RNA causes the target cytidine in the target RNA to deaminate.
  • the "complementarity" of a nucleic acid refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10). These are respectively about 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary” means that all consecutive residues of the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence.
  • substantially complementary means that within a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, at least about 70%, 75%, 80
  • base or a single nucleotide according to the Watson-Crick base pairing principle, when A is paired with T or U, and C is paired with G or I, it is called complement or match, and vice versa; and other bases Base pairing is called non-complementarity or mismatch.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of nucleotide residues.
  • the hydrogen bonding can occur through Watson Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • a sequence that can hybridize to a given sequence is called the "complementary sequence" of the given sequence.
  • the term “delivery” refers to the introduction of biological macromolecules such as nucleic acids and proteins into the cell membrane from outside the cell membrane through certain channels.
  • the “delivery” is, for example, electrotransfection, liposome transfection, lipid-nanoparticle delivery, virus delivery, exosomal delivery, and the like.
  • target RNA refers to the target RNA to be edited, which contains the cytidine to be edited.
  • the target RNA may be mature mRNA or mRNA precursor.
  • the cytidine to be edited is called “target base”, “target cytidine” or “target C”.
  • the base adjacent to the target cytidine at the 5'end of the target RNA is called the “5' adjacent base”; the base adjacent to the target cytidine at the 3'end of the target RNA is called the "3' adjacent base” Base”; the base triplet composed of the target base and its 3'and 5'adjacent bases is referred to herein as the "target base triplet".
  • target base When arRNA hybridizes with target RNA, the base opposite to the target base on arRNA is called “targeting base”, and the base adjacent to the target base at the 5'end of arRNA is called “5". 'Nearest neighbor base'; the base adjacent to the target base at the 3'end of arRNA is called “3' nearest neighbor base”; the target base and its 3'and 5'nearest neighbor base composition
  • target base triplet is referred to herein as the "targeted base triplet".
  • the length of the target base from the 3'end refers to the number of all bases from the 3'nearest neighbor of the target base to the 3'most terminal base; the target base is 5'from the 5'end. Length refers to the total number of bases from the 5'nearest base to the 5'most terminal base of the targeted base.
  • adenosine deaminase (Adenosine to inosine acting on RNA enzyme, ADAR) refers to a type of adenosine deaminase that is widely expressed in various tissues of eukaryotes (including humans and other mammals) , Can catalyze the conversion of adenosine A to inosine I in RNA molecules.
  • the E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T mutation refers to a series of mutations that occur in the ADAR2 protein.
  • the amino acid number of the mutation modification is consistent with the amino acid number in NP_001103.1, that is, NP_001103.1 is used as the reference sequence.
  • NP_001103.1 is used as the reference sequence.
  • the E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T mutations include the different ADAR2 protein reference sequences.
  • the mutant amino acid position corresponds to the mutation, and the ADAR2 protein is modified by the mutation to have the activity of catalyzing the deamination of cytidine.
  • the modified adenosine deaminase protein provided in this application includes NP_001103.1 as a reference sequence and includes E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G
  • the /S582T/V440I/S495N/K418E/S661T mutation-modified ADAR2 also covers ADAR2 that uses different ADAR2 proteins as the reference sequence and contains the corresponding mutation-modified ADAR2.
  • the term "construct” refers to a nucleic acid vector containing a certain nucleic acid sequence
  • the nucleic acid vector can be a linear nucleic acid molecule, a plasmid, a viral vector, or the like.
  • the nucleic acid molecule may be single-stranded or double-stranded.
  • the specific nucleic acid sequence may be a DNA sequence or an RNA sequence.
  • the nucleic acid sequence directly performs its function without being transcribed, translated, or expressed.
  • the nucleic acid sequence is a DNA sequence, which functions as an RNA molecule after being transcribed to form RNA.
  • the nucleic acid sequence is RNA, which functions as a polypeptide or protein after translation.
  • the nucleic acid sequence is DNA, which functions as a protein after forming a protein through the steps of transcription and translation.
  • the construct can enter the cell through packaging as a virus, lipid nanoparticles or exosomes, etc., or enter the cell through electrotransformation, microinjection, chemical transformation, and the like.
  • modification refers to changing the composition or structure of a nucleic acid or protein through chemical methods such as genetic engineering methods, thereby changing one or more of the characteristics or functions of the nucleic acid or protein.
  • the adenosine deaminase protein or its catalytic domain has been modified, such as adding, deleting and/or mutating one or more amino acids to have the effect of catalyzing the deamination of cytidine.
  • This application provides an engineered composition or system for RNA editing, which comprises:
  • the domain has been modified to have the activity of catalyzing the deamination of cytidine
  • the arRNA comprises a complementary RNA sequence that hybridizes with the target RNA, and the arRNA recruits the adenosine deaminase protein or its catalytic domain to the target RNA to deaminate the target cytidine in the target RNA.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) that has cytidine deaminization activity by deleting, adding, or substituting one or more amino acids. protein.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) or its catalytic domain is cytidine deaminated by substituting one or more amino acids Active protein.
  • the modified adenosine deaminase protein of the present application includes the following mutation modification: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/ S495N/K418E/S661T, wherein the amino acid numbering modified by the mutation is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein of the present application includes other ADAR2 homologous proteins as a reference sequence and has E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L /D619G/S582T/V440I/S495N/K418E/S661T corresponding mutant protein.
  • the engineered composition or system for RNA editing of the present application comprises the above-mentioned modified adenosine deaminase protein or its catalytic domain.
  • the catalytic domain is the catalytic domain of the modified adenosine deaminase protein described above.
  • the adenosine deaminase is mutated and modified at one or more sites so as to have the activity of deaminating cytidine to convert it into uridine.
  • the adenosine deaminase protein or its catalytic domain is an ADAR2 protein or its homologous protein or the catalytic domain of the ADAR2 protein or the catalytic domain of the ADAR2 protein homologous protein.
  • the mutation modification comprises: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T, wherein the mutation The modified amino acid numbering is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein or its catalytic domain is introduced into cells for expression through a construct, which is selected from any of linear nucleic acids, plasmids, and viral vectors.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the target base relative to the target cytidine is A, U, C, or G.
  • the preferred target base sequence is U>C>A ⁇ G, that is, when multiple arRNA sequences are compared, where all bases except the target base are the same, the target ArRNAs with U or C bases generally have higher editing efficiency. Therefore, preferably, in some embodiments, the targeting base is U or C.
  • the arRNA contains unpaired nucleotides at one or more positions corresponding to the upstream, downstream, or upstream and downstream of the target base of the target RNA, so as to form an upstream, downstream, upstream, downstream, or upstream from the target base. Or one or more nucleotide mismatches at upstream and downstream positions.
  • the 3' nearest neighbor base of the arRNA targeting base forms a mismatch with the target RNA.
  • the 3'nearest neighbor base of the target base forms a G-G mismatch with the target RNA.
  • the 5'nearest neighbor base of the target base when the arRNA hybridizes to the target RNA, does not form a mismatch with the target RNA. In some embodiments, the 5'nearest neighbor base of the targeted base does not form a mismatch with the target RNA, and wherein the 5'nearest neighbor base of the targeted base is U. In some embodiments, when the arRNA hybridizes to the target RNA, the preferred order of bases in the target RNA opposite to the 5'nearest neighbor base of the target base of the arRNA is G or C from high to low. , U or A.
  • the arRNA when the arRNA hybridizes complementary to the target RNA, the arRNA targets one or more bases other than the triplet to form a mismatch with the target RNA. And in some embodiments, the mismatch can further improve the efficiency of targeted editing based on the arRNA.
  • the target base triplet formed by the target base and its 5'and 3'adjacent bases only forms a mismatch at the target base, and
  • the target base in the arRNA has the same length from the 3'end and the 5'end.
  • the target base triplet is selected from: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU.
  • the target base triplet formed by the target base and its 5'and 3'adjacent bases only forms a mismatch at the target base, and , The target base in the arRNA is not equal in length from the 3'end and the 5'end.
  • the target base triplet is selected from: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU .
  • the length of the arRNA is >50nt, >55nt, >60nt, >65nt, >70nt, >75nt, >80nt, >85nt, >90nt, >95nt, >100nt, >105nt, >110nt,> 115n t, >120nt.
  • the arRNA is about 151-53 nt, 131-61 nt, 121-61 nt, 111-65, 101-71 nt, 91-71 nt, 81-71 nt in length.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • the target base in the arRNA has the same length from the 3'end and the 5'end. In some embodiments, the target bases in the arRNA have different lengths from the 3'end and the 5'end. In some embodiments, the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15nt, 24nt-11nt. In some specific embodiments, the length is selected from any positive integer within the length range defined in this item.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt, 55nt-45nt. In some specific embodiments, the length is selected from any positive integer within the length range defined in this item. In some embodiments, the length of the target base in the arRNA from the 5'end is greater than 80.
  • the arRNA is chemically synthesized. In some embodiments, the arRNA is an oligonucleotide. In some embodiments, the arRNA is chemically modified. In some embodiments, the chemical modification includes a 2'-O-methyl modification or an internucleotide 3'thio modification. In some embodiments, the chemical modification is selected from one or more of the following:
  • the first 3 and last 3 nucleotides of the sequence were modified by 2 ⁇ -OMe,
  • the first 3 and last 3 internucleotide linkages are all phosphorothioate linkages
  • the 3'nearest neighbor of the target base is 2'-OMe modified A
  • the 5'nearest neighbor of the targeted base is 2'-OMe modified C
  • the target base and its 3’ nearest neighbor base and 5’ nearest neighbor base are respectively connected by phosphorothioate bonds
  • the first 5 and last 5 nucleotides are modified by 2 ⁇ -OMe, and
  • the first 5 and last 5 nucleotide linkages are phosphorothioate linkages.
  • the arRNA is encoded by a construct and produced by transcription.
  • the construct is selected from linear nucleic acid strands, viral vectors, or plasmids.
  • This application provides a method for deaminating target cytosine in a target RNA in a cell-CUSPER (programmed C to U RNA editing), including introducing the following 1) and 2) into the cell:
  • the modified adenosine deaminase protein or its catalytic domain to the arRNA of the target RNA or a construct comprising the arRNA or the coding sequence thereof.
  • the adenosine deaminase protein or its catalytic domain has been modified to have the activity of catalyzing the deamination of cytidine
  • the arRNA comprises a complementary RNA sequence that hybridizes with the target RNA
  • the arRNA recruits the adenosine
  • the deaminase protein or its catalytic domain to the target RNA causes the target cytidine in the target RNA to be deaminated.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) that has cytidine deaminization activity by deleting, adding, or substituting one or more amino acids. protein.
  • the modified adenosine deaminase protein of the present application is an adenosine deaminase protein (such as ADAR2 protein) or its catalytic domain is cytidine deaminated by substituting one or more amino acids Active protein.
  • the modified adenosine deaminase protein of the present application includes the following mutation modification: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/ S495N/K418E/S661T, wherein the amino acid numbering modified by the mutation is consistent with the amino acid numbering in NP_001103.1.
  • the modified adenosine deaminase protein of the present application includes other ADAR2 proteins as reference sequences and has E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G /S582T/V440I/S495N/K418E/S661T corresponding mutant protein.
  • the construct for expressing the modified adenosine deaminase protein or its catalytic domain comprises the coding sequence of the modified adenosine deaminase protein or its catalytic domain.
  • the construct expressing the modified adenosine deaminase protein or its catalytic domain and the construct comprising the coding sequence of the arRNA are the same construct.
  • the construct expressing the coding sequence of the modified adenosine deaminase protein or its catalytic domain and the construct comprising the coding sequence of the arRNA are different constructs, that is, the modified The coding sequence of the adenosine deaminase protein or its catalytic domain and the coding sequence of the arRNA are respectively located on different constructs.
  • the different constructs are two or more than two.
  • the different constructs are introduced into the cell simultaneously or separately. It should be understood that the different constructs mentioned in this paragraph refer to different constructs, and it does not mean that the different constructs belong to different construct categories.
  • the construct expressing the modified adenosine deaminase protein or its catalytic domain is combined with the recruitment of the modified adenosine deaminase protein or its catalytic domain to
  • the arRNA of the target RNA or a construct containing the arRNA or a construct containing the coding sequence of the arRNA is introduced into the same cell.
  • the modified adenosine deaminase protein or its catalytic domain or a construct expressing the modified adenosine deaminase protein or its catalytic domain, and a variety of arRNA or comprising the The arRNA construct or the construct containing the coding sequence of the arRNA is introduced into the cell to realize high-throughput editing of the target RNA, wherein the multiple arRNAs are arRNAs targeting different target RNAs, or the same
  • the target RNA is arRNA at different target base (for example, C) sites.
  • this application also covers a method for high-throughput editing of target cytosine in target RNA in a cell, including introducing the following 1) and 2) into the cell:
  • the modified adenosine deaminase protein or its catalytic domain is recruited to multiple (e.g., 2 or more, 5 or more, 5 or more, 10 or more) arRNAs of the target RNA or comprise the arRNA or A construct comprising its coding sequence, wherein the adenosine deaminase protein or its catalytic domain has been modified to have the activity of catalyzing the deamination of cytidine, and the multiple types (e.g., 2 or more, 5 or more, 5
  • the above, 10 or more) arRNA includes complementary RNA sequences that hybridize with the target RNA, and the arRNA recruits the adenosine deaminase protein or its catalytic domain to the target RNA, causing the target cytidine in the target RNA to be depleted Amino.
  • the construct expressing the modified adenosine deaminase protein or its catalytic domain and the construct comprising the coding sequence of the arRNA are the same construct, or the expression modified adenosine
  • the construct of the glycoside deaminase protein or its catalytic domain and the construct containing the coding sequence of the arRNA are separate constructs, and the separate constructs are introduced into the cell simultaneously or separately.
  • the adenosine deaminase undergoes mutation modification at one or more sites so that it has the activity of deaminating cytidine to convert it into uridine.
  • the adenosine deaminase protein or its catalytic domain is ADAR2 protein or its homologous protein or its catalytic domain.
  • the mutation modification comprises: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T, wherein the mutation The modified amino acid numbering is consistent with the amino acid numbering in NP_001103.1.
  • the construct is selected from viral vectors, plasmids, and linear nucleic acids.
  • the modified adenosine deaminase protein or its catalytic domain or a construct expressing the modified adenosine deaminase protein or its catalytic domain and comprising the modified adenosine deaminase protein The adenosine deaminase protein or its catalytic domain is recruited to the oligonucleotide of the arRNA of the target RNA or the construct for transcribing the arRNA through viral infection, chemical transfection, electrotransfection, exosomal delivery, or Nanolipid particle delivery and other methods are introduced into the cell.
  • the adenosine deaminase is mutated and modified at one or more sites so as to have the activity of deaminating cytidine to convert it into uridine.
  • the adenosine deaminase protein or its catalytic domain is ADAR2 protein or its homologous protein or its catalytic domain.
  • the mutation modification comprises: E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T, wherein the mutation The modified amino acid numbering is consistent with the amino acid numbering in NP_001103.1.
  • the target base relative to the target cytidine is A, U, C, or G. In some embodiments, when the arRNA hybridizes to the target RNA, the target base relative to the target cytidine is preferably U or C.
  • the arRNA contains unpaired nucleotides at one or more positions corresponding to the upstream, downstream, or upstream and downstream of the target base of the target RNA, so as to form an upstream, downstream, upstream, downstream, or upstream from the target base. Or one or more nucleotide mismatches at upstream and downstream positions.
  • the 3' nearest neighbor base of the arRNA targeting base forms a mismatch with the target RNA.
  • the 3'nearest neighbor base of the target base forms a G-G mismatch with the target RNA.
  • the 5'nearest neighbor base of the target base does not form a mismatch with the target RNA.
  • the 5' nearest neighbor base of the targeting base is U.
  • the preferred order of bases in the target RNA opposite to the 5'nearest neighbor base of the target base of the arRNA is G or C from high to low. , U or A (G ⁇ C>U ⁇ A).
  • the base in the target RNA opposite to the 5'nearest base of the target base of the arRNA is preferably G or C.
  • the base in the target RNA opposite to the 5' nearest base of the target base of the arRNA is most preferably G.
  • the arRNA when the arRNA hybridizes complementary to the target RNA, the arRNA targets one or more bases other than the triplet to form a mismatch with the target RNA. And in some embodiments, the mismatch can further improve the efficiency of targeted editing based on the arRNA.
  • the target base triplet formed by the target base and its 5'and 3'adjacent bases only forms a mismatch at the target base, and
  • the target base in the arRNA has the same length from the 3'end and the 5'end.
  • the target base triplet is selected from: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU.
  • the target base triplet formed by the target base and its 5'and 3'adjacent bases only forms a mismatch at the target base, and , The target base in the arRNA is not equal in length from the 3'end and the 5'end.
  • the target base triplet is selected from: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU .
  • the length of the arRNA is >50nt, >55nt, >60nt, >65nt, >70nt, >75nt, >80nt, >85nt, >90nt, >95nt, >100nt, >105nt, >110nt,> 115nt, >120nt.
  • the arRNA is about 151-53 nt, 131-61 nt, 121-61 nt, 111-65, 101-71 nt, 91-71 nt, 81-71 nt in length.
  • the length of the arRNA is any positive integer within the length defined by this term.
  • the target base in the arRNA has the same length from the 3'end and the 5'end. In some embodiments, the target bases in the arRNA have different lengths from the 3'end and the 5'end. In some embodiments, the length of the target base in the arRNA from the 3'end is 45-5nt, 40-5nt, 35-10nt, 25nt-15nt, 24nt-11nt. In some specific embodiments, the length is any positive integer within the length range defined by this item.
  • the length of the target base in the arRNA from the 5'end is 80-30nt, 70-35nt, 60-40nt, 55nt-35nt, 55nt-45nt. In some specific embodiments, the length is any positive integer within the length range defined by this item. In some embodiments, the length of the target base in the arRNA from the 5'end is greater than 80.
  • the arRNA is or is contained in an oligonucleotide.
  • the oligonucleotide is chemically modified.
  • the chemical modification includes a 2'-O-methyl modification or an internucleotide 3'thio modification.
  • the chemical modification is selected from one or more of the following:
  • the first 3 and last 3 nucleotides of the sequence were modified by 2 ⁇ -OMe,
  • the first 3 and last 3 internucleotide linkages are all phosphorothioate linkages
  • the 3'nearest neighbor of the target base is 2'-OMe modified A
  • the 5'nearest neighbor of the targeted base is 2'-OMe modified C
  • the target base and its 3’ nearest neighbor base and 5’ nearest neighbor base are respectively connected by phosphorothioate bonds
  • the first 5 and last 5 nucleotides are modified by 2 ⁇ -OMe, and
  • the first 5 and last 5 nucleotide linkages are phosphorothioate linkages.
  • the arRNA is encoded by a construct and produced by transcription.
  • the construct is selected from linear nucleic acid strands, viral vectors, or plasmids.
  • This application provides a modified adenosine deaminase protein, wherein the adenosine deaminase protein is ADAR2, which contains the following amino acid mutations in ADAR2 corresponding to the Genebank accession number NP_001103.1: E488Q/V351G/ S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T, or the amino acid mutation in the corresponding position of the homologous ADAR2 protein of NP_001103.1, the ADAR2
  • the protein has the activity of catalyzing the deamination of cytidine by the mutation modification.
  • the application also provides the use of the modified adenosine deaminase protein to catalyze the deamination and conversion of cytidine into uridine.
  • the use of catalyzing the deamination of cytidine to uridine takes place intracellularly.
  • the use of catalyzing the deamination of cytidine to uridine occurs outside the cell.
  • This application provides a method for treating diseases caused by T to C mutations, which includes using the method of editing RNA as described above to deaminate the target base C in messenger RNA containing the T to C mutation transcription, To correct the mutation.
  • the method of treating a disease caused by a T to C mutation includes injecting an engineered composition or system as described above into a subject.
  • the treatment will include injection of the following 1) and 2) into the subject.
  • the adenosine deaminase protein is the aforementioned RNA editing-related enzyme protein.
  • the injection is intravenous injection, arterial infusion, intramuscular injection, subcutaneous injection, or intratumoral injection.
  • the diseases caused by T to C mutations include genetic diseases and cancer.
  • the application also provides a kit for catalyzing the deamination and conversion of cytidine to uridine.
  • the kit comprises an engineered composition or system as described previously.
  • the kit comprises encoding or expressing the modified adenosine deaminase protein or its catalytic domain in the engineered composition system and/or the modified adenosine deaminase Aminozyme protein or its catalytic domain is a construct of arRNA that recruits target RNA.
  • the new technology recruits editing proteins (such as adenosine deaminase (ADAR)), unlike RESCUE technology, it is necessary to design a guide RNA containing Cas13b recruitment backbone, and it does not require the overexpression of bacterial-derived Cas13b, which reduces
  • the length of the exogenously expressed protein makes it easy and diversified to be loaded by viral vectors and delivered in the human body.
  • it can also reduce the possibility of gene editing failure caused by the neutralization of the exogenously expressed nuclease, which makes it useful In the medical field, it has significant advantages over RESCUE technology.
  • the new system is not like LEAPER technology.
  • LEAPER technology can only realize the editing of RNA editing from A to I, while the new system broadens the editing to C to U, which makes a lot of T on the genome.
  • Genetic diseases with mutations in C and other applications that require the conversion of C to T/U can be treated with the technology of this application.
  • the technology of the present application has a wider application range, and is safer and more effective.
  • Example 1 Molecular construction of modified ADAR2 and BFP reporter system
  • RNA adenosine deaminase 2 RNA adenosine deaminase 2.
  • the mutation site is the same as r16 in the literature (dADAR2(E488Q/V351G/S486A/T375S/S370C/P462A/ N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T) r16, https://benchling.com/s/seq-19Ytwwh0i0vSI byXYZ95 ), wherein the amino acid number modified by the mutation is the same as NP_001103 The amino acid numbers in .1 are the same.
  • the ADAR2 coding sequence fragment containing the above-mentioned mutations was synthesized in vitro using conventional DNA synthesis techniques in the field, and inserted into the pLenti-ADAR2 plasmid vector (pLenti-ADAR2 plasmid backbone was gifted by Professor Wei Wensheng’s laboratory) with the ADAR2 XmaI restriction site and Between AscI restriction sites.
  • the plasmid constructed through the above steps was named pLenti-ADAR2-r16, and the ADAR2 gene containing the above-mentioned mutation was named ADAR2-r16.
  • the full-length cDNA sequence of ADAR2-r16 is: SEQ ID NO 1.
  • pCAG-VSVG is a gift from Arthur Nienhuis&Patrick Salmon (Addgene plasmid#35616; http://n2t.net/addgene:35616; RRID: Addgene_35616);
  • pCMVR8.74 is a gift from Didier Trono (Addgene plasmid #35616; http://n2t.net/addgene:35616; RRID: Addgene_35616) #22036; http://n2t.net/addgene:22036 ; RRID:Addgene_22036)) package pLenti-ADAR2-r16 into a lentivirus, and use the lentivirus to infect 293T cells and use 10ug/ml 48 hours later The final concentration of Blasicidin (Solarbio B9300) was screened for resistance. The cells that survive the screening are called ADAR2-r16-293T.
  • the BFP reporting system was constructed with reference to Reference 7. All BFP (blue fluorescent protein) cDNA sequences were synthesized in vitro, and the specific sequence is: SEQ ID NO 2.
  • the BFP cDNA sequence is cloned into the pCDH-CMV plasmid vector through the multiple cloning site behind the CMV promoter (the pCDH-CMV plasmid backbone is a gift from Kazuhiro Oka, Addgene Plasmid#72265; http://n2t.net/addgene:72265; RRID: Addgene_72265).
  • the target base to be edited in the reporting system is base C at position 199 of the BFP sequence, corresponding to histidine at position 66, see Figure 1.
  • the 198th, 199th, and 200th bases of this sequence are CCA, named BFP-CCA, and abbreviated as C*.
  • BFP-CCA When the base C at position 199 is edited to U by deamination at the RNA level, the BFP fluorescent protein will change from the original blue fluorescence to green fluorescence, which can be measured by the flow cytometer FITC (Fluorescein isothiocyanate, isothiocyanate). Acid fluorescein) channel detects signal. Since nucleotide 198 is mutated from C to A, T, G, the corresponding codons (bases 196, 197, 198) ACC, ACA, ACT, ACG all encode threonine, so this mutation is synonymous mutation.
  • a site-directed mutagenesis kit can be used accordingly ( Site-DirectedMutagenesis Kit, NEB E0554S) introduced mutations into base 198, so that the three bases at position 198,199,200 are: GCA, named BFP-GCA, abbreviated as G*; ACA, named BFP-ACA, abbreviated A*; TCA is named BFP-UCA, abbreviated as U*.
  • the arRNA used in this embodiment includes an antisense RNA complementary to the target mRNA, the target base is located in the middle of the arRNA, and the 5'upstream and 3'downstream extend to both sides with the same length. Due to the limitation of synthesis length, in this example, RNA with a length of 91 nt was selected for in vitro synthesis. As shown in Figure 3, when the 46th nucleotide of the arRNA is A, U, G, and C, they are abbreviated as A ⁇ , U ⁇ , G ⁇ , C ⁇ , respectively. The specific sequences of the four synthetic arRNAs are shown in Table 1 below.
  • the design of the four arRNAs in this batch of experiments is to determine the editing efficiency when the target base is mismatched with the target base on the arRNA.
  • Different target bases were tested, that is, the 46th arRNA with A, U, G, and C was tested.
  • the 5'nearest neighbor base at position 47 of arRNA (corresponding to position 198 of the reporter system) is designed according to the target triplet base of the BFP sequence before the mutation (ie CCA), that is, the arRNA target base
  • the 5'nearest neighbor (position 47) of is U complementary to A.
  • arRNA is consistent with LEAPER technology if and only when the reporting system for subsequent tests is BFP-CCA, that is: when arRNA hybridizes with target RNA, there is only a mismatch in the target base; and when When the reporting system is BFP-GCA, BFP-TCA, or BFP-ACA, the design of arRNA not only has mismatches in the target bases, but also has mismatches at the 3'adjacent bases of the arRNA target bases.
  • ADAR2-r16-293T was plated to a 6-well plate at a density of 300,000 cells/well, and transfected with Lipofectamine 3000 (Invitrogen L3000015) 24 hours after plating.
  • the transfection steps were carried out according to the instructions. According to the instructions, use different concentrations of Lipofectamine 3000 transfection reagent for two repeated experiments, repeat 1 use 3.75 ⁇ L Lipofectamine 3000 per well, and repeat 2 use 7.5 ⁇ L Lipofectamine 3000 per well.
  • BFP reporter plasmid selected from: BFP-GCA, abbreviated as G*; BFP-ACA, abbreviated as A*; BFP-TCA, abbreviated as T*; BFP-CCA, abbreviated as C*
  • MFI Mean Fluorescent Intensity
  • the mRNA row represents the BFP reporter plasmid added in the corresponding well
  • the arRNA row represents the arRNA added in the corresponding well.
  • the three bases 198,199,200 are CCA in the original sequence, and when the C at position 198 is changed to A, T or G, the corresponding amino acid at position 65 is threonine, so BFP-GCA, BFP- The 198-bit changes in the four different reporting systems of CCA, BFP-ACA, and BFP-TCA will not cause changes in the original protein function.
  • the GFP signal MFI is approximately 2.4 ⁇ 10 6 to 3.1 ⁇ 10 6 , which is about 100 times higher than the background value. Therefore, if all C at position 199 becomes U at the RNA level, it can lead to an approximately 100-fold increase in GFP MFI.
  • the system preference for the 5'-adjacent bases of the target base in the repeated experiments showed a pattern similar to that of the related tests in the second part of Example 2 shown in Figure 3, all being 5'-adjacent.
  • the base is G or C
  • the editing efficiency is better, and G is greater than C.
  • RNA single-base editing system whether it is A to I (Qu et al., 2019) or C to U (Abudayyeh et al., 2019), has the 5'adjacent base and 3'of the target base A or C. Adjacent bases have a greater impact on its editing efficiency.
  • Example 1.2 Referring to the steps in Example 1.2, according to the different DNA sequences at positions 198, 199, and 200 in the DNA sequence of BFP (SEQ ID NO 2), we constructed 16 different reporting systems, namely ACA, ACT (corresponding to mRNA: ACU ), ACC, ACG, TCA (corresponding to mRNA: UCA), TCT (corresponding to mRNA: UCU), TCC (corresponding to mRNA: UCC), TCG (corresponding to mRNA: UCG), CCA, CCT (corresponding to mRNA: CCU), CCC, CCG, GCA, GCT (corresponding mRNA: GCU), GCC, GCG. And referring to the lentivirus packaging and infection steps in Example 1.1, 293T was infected and stably integrated into 293T cells.
  • ACA ACT
  • ACT corresponding to mRNA: ACU
  • ACC ACG
  • TCA corresponding to mRNA: UCA
  • TCT corresponding to mRNA: UCU
  • the 3'and 5'nearest neighbors of the arRNA target base can be in the target RNA.
  • the 5'and 3'adjacent bases of the target base are complementary paired according to the Watson Creek base pairing principle.
  • the synthesized arRNA sequence is shown in Table 3, and the corresponding relationship between the target in mRNA and its 5'and 3'adjacent bases and the arRNA target base and its 5'and 3'nearest neighbor bases are shown in Figure 7.
  • Cell culture uses DMEM (Hyclone SH30243.01) containing 10% FBS (Vistech SE100-011).
  • the reporter system cells are transferred to a 12-well plate at 15,000 cells/well. At this time, it is recorded as 0 hours.
  • RNAi MAX Invitrogen, 13778150
  • the constructed sequencing library will be used for high-throughput sequencing through the NovaSeq6000 platform in PE150 mode.
  • the raw data obtained by high-throughput sequencing is quality controlled with fastp (v0.19.6) to filter out low-quality, linker sequences, and sequences containing polyG.
  • the high-quality sequencing data obtained was split into each sample according to the corresponding Barcode sequence using a self-developed split script, and the BWA (v0.7.17-r1188) software was used to compare with the amplified target region sequence, through SAMtools ( v1.9) Perform format conversion to generate BAM files, count and compare information, re-sort and build indexes.
  • the second-generation sequencing results are shown in Figure 8. It can be confirmed by second-generation sequencing that the CUSPER system can indeed achieve C to U single-base editing of mRNA. Compared with Example 2, it can be seen that if there is no mismatch between the 5'and 3'nearest neighbors of the target base and the target RNA, the editing efficiency is relatively low. When adjacent bases and target RNA form GG mismatches, editing efficiency can be improved. In addition, in addition to the CU mismatch of the target base, the 5'and 3'nearest neighbors of the target base are both the 3'and 5'neighbors of the target base according to the Watson-Crick principle. When paired, higher editing efficiency can be obtained when the target base triplet on the mRNA is the following sequence: ACG, ACC, UCC, UCG, CCC, CCG, UCA, UCU.
  • RNA editing for genetic restoration the relationship between the structure and deamination efficiency of carboxyvinyldeoxyurcle oligodeoxynucleides chemical ,87(4),583-593.

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Abstract

提供了一种使细胞中靶标RNA中的靶标胞嘧啶脱氨基的方法，该方法包括将经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA寡核苷酸或表达该arRNA的构建体导入该细胞中。还提供了一种用于RNA编辑的工程化组合物或系统，以及应用该工程化组合物或系统校正T到C突变治疗疾病的用途。

Description

一种靶向编辑RNA的新方法 技术领域
本申请属于基因编辑治疗领域，具体地，本申请创造了一种名为CUSPER(C to U Specific Programmable Editing of RNA)的靶向编辑RNA的方法，其包括利用CUSPER技术进行RNA上由C到U碱基的精准位点编辑，可用于治疗由于T到C突变导致的疾病。
背景技术
近年来，以CRISPR(Clustered regularly interspaced short palindromic repeats)为首的基因组编辑技术正在飞速发展，并对生物以及医学诸多领域产生了深远的影响。许多科研工作者和生物技术公司也在致力于将该技术推上临床。2019年9月，北京大学邓宏魁教授与其合作者发表文章11首次报道了利用CRISPR技术编辑干细胞并将其回输至患者，以治疗其艾滋病和白血病的临床试验结果，为CRISPR技术在基因治疗方向的转化做出了巨大的贡献。
尽管CRISPR技术存在极大的应用前景，但该技术也存在一系列缺陷，导致该技术从科研阶段向临床治疗应用的转化步履维艰。问题之一便是CRISPR技术用到的核心作用酶：Cas9。基于CRISPR的DNA编辑技术，必须外源表达Cas9或拥有相似功能的其它核酸酶，从而造成了以下几个问题。首先，需要外源表达的核酸酶通常具有较大的分子量，这使得通过病毒载体将其递送至体内的效率急剧下降。其次，由于核酸酶的外源表达，使这种方法存在潜在的核酸酶脱靶可能，这将导致其应用中的潜在致癌风险。最后，外源表达的Cas9等类似核酸酶是从细菌中发现的，而非人类或哺乳动物天然存在的，这使其可能引起患者体内的免疫反应，这一方面可能会对患者自身造成损伤，另一方面也可能会使外源表达的核酸酶被中和，从而失去应有的活性，影响治疗效果。
2017年，麻省理工学院张锋教授及其课题组报道了一种名为REPAIR(RNA Editing for Programmable A to I Replacement)的RNA编辑技术，通 过外源表达Cas13-ADAR融合蛋白及单向导RNA(single guide RNA,sgRNA)可以实现靶向目标RNA的A到I的编辑2，但是该方法同CRISPR技术一样，仍需要外源蛋白的表达。无法解决外源蛋白表达造成的问题。
2019年1月，Thorsten Stafforst课题组报道了名为RESTORE(recruiting endogenous ADAR to specific trans for oligonucleotide-mediated RNA editing,Merkle et al.,2019)的RNA单碱基编辑技术。RESTORE能够摆脱对外源蛋白的依赖，但RESTORE技术需要在IFN-γ存在的前提下才能有较高的编辑效率，而IFN-γ是决定自体免疫发展和严重程度的关键因子9，这使得该技术在医学领域的应用大打折扣。另一方面，RESTORE技术中同样也用到一段向导RNA，而其使用的向导RNA是化学合成的寡核苷酸，并且其合成的寡核苷酸需要人为引入大量的化学修饰以保证其稳定性。在这些化学修饰中，有一部分是非天然的修饰，使得该寡核苷酸可能存在毒性或免疫原性；还有一部分修饰会导致相同碱基链的不同构象，使得对于相同的RNA序列，可能有数十种不同的构象组合，这增加了其向细胞内递送的难度。
2019年7月，北京大学魏文胜教授课题组在Nature Biotechnology上发表的文章 4中，首次报道了一种核酸编辑技术：LEAPER(Leveraging Endogenous ADAR for Programmable Editing of RNA)。与CRISPR技术不同的是，一方面该技术从原理上摆脱了对外源核酸酶过表达的依赖，并且可以通过化学合成RNA完成，也可以通过腺相关病毒(AAV)、慢病毒等载体递送至患者发挥功能，这使得其递送手段的选择上更加灵活多变，使得该技术在向医学领域转化的过程中，具有更大的优势；另一方面该技术只能实现腺苷A到肌酐I(肌酐I在蛋白质翻译过程中会被识别为鸟酐G)的编辑，使得对其他突变，例如T到C的突变无能为力。此外，与CRISPR技术类似，该技术同样需要一段RNA作为向导，以便将内源的核酸酶招募至所需编辑的位点。该段向导RNA被命名为arRNA(adar-recruiting RNA)。
2019年7月，张锋教授课题组报道了一种名为RESCUE(RNA Editing for Specific C to U Exchange)的新技术 1。该技术基于2017年该课题组报道的Cas13-ADAR基本骨架，在负责反应的ADAR催化结构域上做了不同的突变尝试。最终将ADAR催化结构域的A到I编辑活性修改为C到U编辑活性，从而实现了特定位点RNA上C到U的编辑，进一步拓展了对碱基 的精准编辑范围。然而，该技术仍需要外源表达Cas13与ADAR突变后的融合蛋白，无法解决细菌源蛋白表达造成的问题。
发明内容
为了解决上述基因编辑技术中的问题，以便将基因编辑技术更好地应用于医学领域，迫切地需要找到一种易于递送且可高效精准纠正T至C突变的定向基因编辑技术。
本申请创造了一种新的RNA编辑技术CUSPER(C to U Specific Programmable Editing of RNA)，该技术无需依赖细菌来源的Cas13b的表达，并且将RNA编辑的应用范围从A到I的编辑拓展到C到U的编辑。
在本申请中，一方面由于无需依赖细菌来源的大分子蛋白的表达，因此减少了潜在的免疫系统风险，和因免疫系统对外源蛋白的攻击而对系统编辑效率带来的影响；另一方面，随着需要引入的蛋白分子量的显著缩减，其递送方式也变得更加灵活，可以包括化学转化及生物学递送，例如AAV递送等。因此，本申请提供的技术方案，不仅解决了C到U的单碱基编辑技术问题，而且提高了编辑系统的安全性、稳定性和使用的灵活性，更加利于体内应用，更具生物医药领域的应用前景。
具体地，本申请涉及：
1.一种用于RNA编辑的工程化组合物或系统，包含：
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，和
2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体；
其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
在一些实施方案中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为同一构建体。在一些实施方案中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包 含所述arRNA的编码序列的构建体为分开的构建体。
在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)经过缺失、添加或取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)或其催化结构域是经过取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含如下突变修饰：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含以其它ADAR2同源蛋白作为参考序列并具有E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T相应突变的蛋白。
在一些实施方案中，本申请用于RNA编辑的工程化组合物或系统包含上述经修饰的腺苷脱氨酶蛋白的催化结构域。在一些实施方案中，所述催化结构域为上述经修饰的腺苷脱氨酶蛋白的催化结构域。
2.项1的工程化组合物或系统，其中所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。
3.项2的工程化组合物或系统，其中所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。
4.项3中的工程化组合物或系统，其中所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
在一些实施方案中，所述经修饰的腺苷脱氨酶蛋白或其催化结构域为NP_001103.1蛋白的同源蛋白，具有与如下突变相应的突变：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T。
5.项1-4中任一项的工程化组合物或系统，其中所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。
6.项5中所述的工程化组合物或系统，其中所述靶向碱基为U或C。
7.项1-6中任一项的工程化组合物或系统，其中：
所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。
8.项7中的工程化组合物或系统，其中所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。
9.项8中所述的工程化组合物或系统，其中所述arRNA与靶标RNA杂交时，所述靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。
10.项1-9中任一项的工程化组合物或系统，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。
11.项10中的工程化组合物或系统，其中所述靶向碱基的5’最近邻碱基为U。
12.项9中的工程化组合物或系统，其中当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A(G≈C>U≈A)。
13.项1-6中任一项的工程化组合物或系统，其中当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
14.项1-13任一项的工程化组合物或系统，其中所述arRNA长度>50nt、>55nt、>60nt、>65nt、>70nt、>75nt、>80nt、>85nt、>90nt、>95nt、>100nt、>105nt、>110nt、>115nt、>120nt。在一些实施方案中，所述arRNA长约151-53nt、131-61nt、121-61nt、111-65、101-71nt、91-71nt、81-71nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
15.项1-14任一项的工程化组合物或系统，其中所述arRNA中靶向碱基距离3’端及5’端的长度相等。
16.项1-14中任一项的组合物，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。在一些具体实 施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
17.项1-14任一项的工程化组合物或系统，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
18.项1-17任一项的工程化组合物或系统，其中所述arRNA是化学修饰的。
19.项18的工程化组合物或系统，其中所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。
20.一种使细胞中靶标RNA中的靶标胞嘧啶脱氨基的方法，包括将如下1)和2)导入所述细胞：
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和
2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体，其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)经过缺失、添加或取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)或其催化结构域是经过取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含如下突变修饰：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含以其它ADAR2蛋白作为参考序列并具有E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418 E/S661T相应突变的蛋白。
在一些实施方案中，本申请用于RNA编辑的工程化组合物或系统包含上述经修饰的腺苷脱氨酶蛋白的催化结构域。
21.项20的方法，其中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为同一构建体，被同时导入所述细胞，或者所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为分开的构建体，所述分开的构建体被同时或分开地导入所述细胞。
22.项20或21的方法，其中所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。
23.项22的方法，其中所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。
24.项23中的方法，其中所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
在一些实施方案中，所述经修饰的腺苷脱氨酶蛋白或其催化结构域为NP_001103.1蛋白的同源蛋白，具有与如下突变相应的突变：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T。
25.项21-24中任一项方法，其中所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。
26.项25中所述的方法，其中所述靶向碱基为U或C。
27.项21-26中任一项的方法，其中：
所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。
28.项27中的方法，其中所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。
29.项28中所述的方法，其中所述arRNA与靶标RNA杂交时，所述 靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。
30.项21-29中任一项的方法，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。
31.项30中的方法，其中所述靶向碱基的5’最近邻碱基为U。
32.项29中的方法，其中当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A(G≈C>U≈A)。
33.项21-26中任一项的方法，其中当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
34.项21-33任一项的方法，其中所述arRNA长度>50nt、>55nt、>60nt、>65nt、>70nt、>75nt、>80nt、>85nt、>90nt、>95nt、>100nt、>105nt、>110nt、>115nt、>120nt。在一些实施方案中，所述arRNA长约151-53nt、1131-61nt、121-61nt、111-65、101-71nt、91-71nt、81-71nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
35.项21-34任一项的方法，其中所述arRNA中靶向碱基距离3’端及5’端的长度相等。
36.项21-34中任一项的方法，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
37.项21-34任一项的方法，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
38.项21-37任一项的方法，其中所述arRNA是化学修饰的。
39.项38的方法，其中所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。
40.项21-39中任一项的方法，其中所述细胞为哺乳动物细胞。
41.一种治疗由T到C突变引起的疾病的方法，包括使用如项21-39中任一项的方法使包含所述T到C突变转录形成的信使RNA中靶标碱基C 脱氨基，以校正所述突变。
42.一种经修饰的腺苷脱氨酶蛋白，其中所述腺苷脱氨酶蛋白为ADAR2，其包含E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T突变修饰，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致，所述ADAR2蛋白经过所述突变修饰而具有催化胞苷脱氨基的活性。
在一些实施方案中，所述经修饰的腺苷脱氨酶蛋白或其催化结构域为NP_001103.1蛋白的同源蛋白，具有与如下突变相应的突变：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T。
43.如项42中所述的经修饰的腺苷脱氨酶蛋白用于催化胞苷脱氨基转化为尿苷的用途。
附图说明
图1显示了BFP报告体系以及待进行C到U编辑的靶标碱基。
图2显示了当靶向为点为U时，CUSPER和RESCUE技术设计向导RNA时靶标碱基的选择以及靶向为点相邻碱基的设计原则。
图3显示了对CUSPER编辑体系的测试。图中“/”代表未添加对应质粒或arRNA，仅添加相同体积水。
图4显示了LEAPER技术对靶标碱基相邻上下游碱基的偏好性。图中箭头所示为与图3对应的当靶标碱基的3’相邻碱基为A时，LEAPER技术对5’相邻上游的偏好性。
图5显示了RESCUE技术对靶标碱基相邻上下游碱基的偏好性。图中箭头所示为与图3对应的当3’相邻碱基为A时，RESCUE技术对5’相邻碱基的偏好性。
图6显示了对CUSPER编辑体系的重复测试结果。图中“/”代表未添加对应质粒或arRNA而仅添加相同体积水。
图7显示靶标C对应U，及其相邻碱基无错配的情况下，mRNA与对应arRNA序列。
图8显示靶标C对应U，及其相邻碱基无错配的情况下全部16种3’和 5’相邻碱基组合的C到U RNA编辑效率
发明详述
为解决现有基因编辑技术中普遍依赖异种外源蛋白的困境，在更多种类的碱基上实现精准的单个碱基编辑，本申请创造了一种RNA编辑技术CUSPER(C to U Specific Programmable Editing of RNA)。CUSPER将RNA编辑的应用范围从A到I的编辑拓展到C到U的编辑，且由于使用了哺乳动物本身表达的酶蛋白进行编辑，避免了向细胞内引入异种外源蛋白，从而提高了安全性，并使整个基因编辑过程更加高效便利。
定义
如本文所用，“CUSPER技术”是本申请技术方案中的原创技术，其中的CUSPER是“C to U Specific Programmable Editing of RNA”的缩写，即“将胞苷C转变为尿苷U的特异性可编程RNA编辑”。该技术使用一段可与靶标RNA互补杂交的短RNA，将经修饰的腺苷脱氨酶蛋白或包含其催化结构域的蛋白募集到靶标RNA，以对靶标胞苷脱氨基，使其转变为尿苷。其中所述经修饰的腺苷脱氨酶蛋白或包含其催化结构域的蛋白经修饰而具有催化胞嘧啶脱氨基的活性。所述可与靶标RNA互补杂交的短RNA即arRNA。本申请中所使用的“arRNA(adar-recruiting RNA)”是指可以招募腺苷脱氨酶蛋白或包含其催化结构域至靶标RNA的单链RNA，该arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。如本文所用，核酸的“互补”是指一条核酸通过传统的Watson-Crick碱基配对与另一条核酸形成氢键的能力。百分比互补性表示核酸分子中可与另一核酸分子形成氢键(即，Watson-Crick碱基配对)的残基的百分比(例如，10个中的约5、6、7、8、9、10个分别为约50％，60％，70％，80％，90％和100％互补)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数量的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250或更多个核苷酸的区域内，至少约70％，75％，80％，85％，90％，95％，97％，98％，99％或100％中的任何一个的互补程度，或指在严格条件下杂交的两条核酸。对于单个碱基或单个核苷酸，按照 Watson-Crick碱基配对原则，A与T或U、C与G或I配对时，被称为互补或匹配，反之亦然；而除此以外的碱基配对都称为不互补或不匹配。
“杂交”是指其中一种或多种多核苷酸反应形成复合物的反应，所述复合物通过核苷酸残基的碱基之间的氢键稳定。所述氢键可以通过Watson Crick碱基配对，Hoogstein结合或以任何其他序列特异性的方式发生。能够与给定序列杂交的序列称为给定序列的“互补序列”。
如本文所用，术语“递送”是指将核酸、蛋白等生物大分子通过某些途径从细胞膜外引入细胞膜内。所述“递送”例如电转染、脂质体转染、脂质-纳米颗粒递送、病毒递送、外泌体递送等方式。
如本文所用，术语“靶标RNA”是指待编辑的目标RNA，其包含待编辑的胞苷。所述靶标RNA可以是成熟mRNA或mRNA前体。所述待编辑的胞苷被称为“靶标碱基”、“靶标胞苷”或“靶标C”。在靶标RNA的5’端与靶标胞苷相邻的碱基称为“5’相邻碱基”；在靶标RNA的3’端与靶标胞苷相邻的碱基称为“3’相邻碱基”；靶标碱基及其3’和5’相邻碱基组成的碱基三联体在本文中被称为“靶标碱基三联体”。当arRNA与靶标RNA杂交时，在arRNA上与所述靶标碱基相对的碱基称为“靶向碱基”，在arRNA的5’端与靶向碱基相邻的碱基称为“5’最近邻碱基”；在arRNA的3’端与靶向碱基相邻的碱基称为“3’最近邻碱基”；靶向碱基及其3’和5’最近邻碱基组成的碱基三联体在本文中被称为“靶向碱基三联体”。
在本文中，靶向碱基距离3’端的长度是指靶向碱基的3’最近邻碱基至3’最末端碱基的所有碱基个数；所述靶向碱基距离5’端的长度是指靶向碱基的5’最近邻碱基至5’最末端碱基的所有碱基个数。
如本文所用，术语“腺苷脱氨酶(Adenosine to inosine acting on RNA enzyme，ADAR)”是指是一类在真核生物(包括人等哺乳动物)各组织中广泛表达的腺苷脱氨酶，能够催化RNA分子中腺苷A到肌苷I的转换。
在本申请中，E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T突变是指发生于ADAR2蛋白的一系列突变，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致，即使用NP_001103.1作为参考序列。本领域技术人员应当知晓，对于不同的ADAR2蛋白的参考序列，所述突变中的氨基酸 编号可能发生变化。因此，如本申请所用，E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T突变包括不同ADAR2蛋白参考序列中与所述突变氨基酸位置对应的突变，所述ADAR2蛋白经过所述突变修饰而具有催化胞苷脱氨基的活性。相应的，本申请提供的经修饰的腺苷脱氨酶蛋白，包括以NP_001103.1作为参考序列并包含E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T突变修饰的ADAR2，也涵盖以不同ADAR2蛋白作为参考序列并包含相应突变修饰的ADAR2。
如本文所用，术语“构建体”是指包含某一核酸序列的核酸载体，所述核酸载体可以是线性核酸分子、质粒或病毒载体等。所述核酸分子可以是单链也可以是双链分子。所述特定的核酸序列可以是DNA序列也可以是RNA序列。在一些实施方案中，所述核酸序列不经过转录、翻译或表达而直接发挥其功能。在一些实施方案中，所述核酸序列为DNA序列，其经过转录形成RNA后以RNA分子形式发挥功能。在一些实施方案中，所述核酸序列为RNA，其经过翻译后以多肽或蛋白质的形式发挥作用。在一些实施方案中，所述核酸序列为DNA，其经过转录和翻译步骤形成蛋白质后以蛋白质的形式发挥功能。所述构建体可以通过包装为病毒、脂质纳米颗粒或外泌体等形式进入细胞，亦可以通过电转化、显微注射、化学转化等方式进入细胞。
本申请所用术语“修饰”是指通过化学方法例如基因工程方法改变核酸或蛋白的组成或结构，从而使所述核酸或蛋白的一项或多项特性或功能发生改变。例如，在本申请中，腺苷脱氨酶蛋白或其催化结构域经过修饰，例如添加、缺失和/或突变一个或多个氨基酸后具有催化胞苷脱氨基的作用。
工程化组合物或系统
本申请提供了一种用于RNA编辑的工程化组合物或系统，其包含：
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，和
2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到靶标RNA 的arRNA或包含该arRNA或包含其编码序列的构建体；
其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)经过缺失、添加或取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)或其催化结构域是经过取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含如下突变修饰：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含以其它ADAR2同源蛋白作为参考序列并具有E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T相应突变的蛋白。
在一些实施方案中，本申请用于RNA编辑的工程化组合物或系统包含上述经修饰的腺苷脱氨酶蛋白或其催化结构域。在一些实施方案中，所述催化结构域为上述经修饰的腺苷脱氨酶蛋白的催化结构域。
在一些实施方案中，所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。在一些实施方案中，所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或所述ADAR2蛋白的催化结构域或所述ADAR2蛋白同源蛋白的催化结构域。在一些实施方案中，所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
在一些实施方案中，所述经修饰的腺苷脱氨酶蛋白或其催化结构域通过构建体导入细胞进行表达，所述构建体选自线性核酸、质粒及病毒载体中的任意。在一些实施方案中，所述细胞为真核细胞。在一些实施方案中，所述细胞为哺乳动物细胞。在一些实施方案中，所述细胞为人细胞。
在一些实施方案中，所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。在一些实施方案中，优选的靶向碱基顺序为U＞C＞A≈G，即，当多条arRNA序列相比，其中除靶向碱基以外其他的所有碱基均相同时，靶向碱基为U或C的arRNA通常具有更高的编辑效率。因此优选地，在一些实施方案中，所述靶向碱基为U或C。
在一些实施方案中，所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。在一些实施方案中，所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。在一些实施方案中，所述arRNA与靶标RNA杂交时，所述靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。在一些实施方案中，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。在一些实施方案中，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配，且其中所述靶向碱基的5’最近邻碱基为U。在一些实施方案中，当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A。在一些实施方案中，当所述arRNA与靶标RNA互补杂交时，所述arRNA靶向三联体以外的一个或多个碱基与靶标RNA形成错配。并且在一些实施方案中，所述错配可以进一步提高基于所述arRNA的靶向编辑效率。
在一些实施方案中，当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，并且，所述arRNA中靶向碱基距离3’端及5’端的长度相等，此时，优选地其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。在一些实施方案中，当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，并且，所述arRNA中靶向碱基距离3’端及5’端的长度不相等，此时，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
在一些实施方案中，所述arRNA长度>50nt、>55nt、>60nt、>65nt、>70nt、>75nt、>80nt、>85nt、>90nt、>95nt、>100nt、>105nt、>110nt、>115n t、>120nt。在一些实施方案中，所述arRNA长约151-53nt、131-61nt、121-61nt、111-65、101-71nt、91-71nt、81-71nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
在一些实施方案中，所述arRNA中靶向碱基距离3’端及5’端的长度相等。在一些实施方案中，所述arRNA中靶向碱基距离3’端及5’端的长度不相等。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。在一些具体实施方案中，所述长度选自本项定义的长度范围内的任一正整数。
在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。在一些具体实施方案中，所述长度选自本项定义的长度范围内的任一正整数。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度大于80。
在一些实施方案中，所述arRNA由化学合成。在一些实施方案中，所述arRNA为寡核苷酸。在一些实施方案中，所述arRNA是化学修饰的。在一些实施方案中，所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。在一些实施方案中，所述化学修饰选自如下的一项或多项：
序列前3个和后3个核苷酸分别被2`-OMe修饰，
前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
序列中全部U均被2`-OMe修饰，
靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
前5个和后5个核苷酸分别被2`-OMe修饰，和
前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
在一些实施方案中，所述arRNA由一种构建体编码并转录生成。在一些实施方案中，所述构建体选自线性核酸链、病毒载体或质粒。
编辑RNA的方法
本申请提供了一种使细胞中靶标RNA中的靶标胞嘧啶脱氨基的方法——CUSPER(程序化的C到U的RNA编辑)，包括将如下1)和2)导入所 述细胞：
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和
2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体。其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
在一些实施方案中，所述细胞为真核细胞。在一些实施方案中，所述细胞为哺乳动物细胞。在一些实施方案中，所述细胞为人细胞。在一些实施方案中，所述细胞为小鼠细胞。
在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)经过缺失、添加或取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请的经修饰的腺苷脱氨酶蛋白是腺苷脱氨酶蛋白(例如ADAR2蛋白)或其催化结构域是经过取代一个或多个氨基酸而具有胞苷脱氨基的活性的蛋白质。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含如下突变修饰：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。在一些实施方案中，本申请经修饰的腺苷脱氨酶蛋白包含以其它ADAR2蛋白作为参考序列并具有E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T相应突变的蛋白。
本领域技术人员应当知晓，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体包含所述经修饰的腺苷脱氨酶蛋白或其催化结构域的编码序列。在一些实施方案中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体与包含所述arRNA的编码序列的构建体为同一构建体。在一些实施方案中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的编码序列的构建体与包含所述arRNA的编码序列的构建体为不同构建体，即所述经 修饰的腺苷脱氨酶蛋白或其催化结构域的编码序列与所述arRNA的编码序列分别位于不同构建体上。在一些实施方案中，所述不同构建体为两个或多于两个。在一些实施方案中，所述不同的构建体同时或分开地导入所述细胞。应当理解，本段所说的不同构建体指的是非同一个构建体，并不代表所述不同构建体分别属于不同的构建体类别。在一些实施方案中，将所述表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体与所述将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA或包含该arRNA的构建体或包含该arRNA的编码序列的构建体导入同一细胞。在一些实施方案中，将经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和多种arRNA或包含所述arRNA的构建体或包含所述arRNA的编码序列的构建体导入细胞，以实现对靶标RNA的高通量编辑，其中所述的多种arRNA为靶向不同靶标RNA的arRNA，或为靶向同一靶标RNA不同靶标碱基(例如C)位点的arRNA。
因此，本申请还涵盖一种高通量编辑细胞中靶标RNA中的靶标胞嘧啶的方法，包括将如下1)和2)导入所述细胞：
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和
将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的多种(例如2种以上、5种以上、5种以上、10种以上)arRNA或包含该arRNA或包含其编码序列的构建体，其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，所述多种(例如2种以上、5种以上、5种以上、10种以上)arRNA包含分别与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
在一些实施方案中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为同一构建体，或者所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为分开的构建体，所述分开的构建体被同时或分开地导入所述细胞。
在一些实施方案中，所述腺苷脱氨酶在一个或多个位点发生突变修饰从 而具有对胞苷脱氨使其转化成尿苷的活性。
在一些实施方案中，所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。
在一些实施方案中，所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。在一些实施方案中，所述构建体选自病毒载体、质粒和线性核酸。在一些实施方案中，所述经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体以及包含将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA的寡核苷酸或转录该arRNA的构建体通过病毒侵染、化学转染、电转染、外泌体递送、或纳米脂质颗粒递送等方式被导入所述细胞。
在一些实施方案中，所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。在一些实施方案中，所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。在一些实施方案中，所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
在一些实施方案中，所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。在一些实施方案中，所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基优选为U或C。
在一些实施方案中，所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。在一些实施方案中，所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。在一些实施方案中，所述arRNA与靶标RNA杂交时，所述靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。在一些实施方案中，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。在一些实施方案中，所述靶向碱基的5’最近邻碱基为U。在一些实施方案 中，当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A(G≈C>U≈A)。在一些实施方案中，当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选为G或C。在一些实施方案中，当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基最优选为G。在一些实施方案中，当所述arRNA与靶标RNA互补杂交时，所述arRNA靶向三联体以外的一个或多个碱基与靶标RNA形成错配。并且在一些实施方案中，所述错配可以进一步提高基于所述arRNA的靶向编辑效率。
在一些实施方案中，当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，并且，所述arRNA中靶向碱基距离3’端及5’端的长度相等，此时，优选地其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。在一些实施方案中，当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，并且，所述arRNA中靶向碱基距离3’端及5’端的长度不相等，此时，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
在一些实施方案中，所述arRNA长度>50nt、>55nt、>60nt、>65nt、>70nt、>75nt、>80nt、>85nt、>90nt、>95nt、>100nt、>105nt、>110nt、>115nt、>120nt。在一些实施方案中，所述arRNA长约151-53nt、131-61nt、121-61nt、111-65、101-71nt、91-71nt、81-71nt。在一些具体实施方案中，所述arRNA的长度是该项定义的长度范围内的任一正整数。
在一些实施方案中，所述arRNA中靶向碱基距离3’端及5’端的长度相等。在一些实施方案中，所述arRNA中靶向碱基距离3’端及5’端的长度不相等。在一些实施方案中，所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。在一些具体实施方案中，所述长度为本项定义的长度范围内的任一正整数。
在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。在一些具体实施方案中，所述 长度为本项定义的长度范围内的任一正整数。在一些实施方案中，所述arRNA中靶向碱基距离5’端的长度大于80。
在一些实施方案中，所述arRNA为寡核苷酸或包含于寡核苷酸。在一些实施方案中，所述寡核苷酸是化学修饰的。在一些实施方案中，所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。在一些实施方案中，所述化学修饰选自如下的一项或多项：
序列前3个和后3个核苷酸分别被2`-OMe修饰，
前3个和后3个核苷酸间连接均为硫代磷酸酯键连接，
序列中全部U均被2`-OMe修饰，
靶向碱基的3’最近邻碱基为2`-OMe修饰的A，
靶向碱基的5’最近邻碱基为2`-OMe修饰的C，
靶向碱基与其3’最近邻碱基和5’最近邻碱基分别以硫代磷酸酯键连接，
前5个和后5个核苷酸分别被2`-OMe修饰，和
前5个和后5个核苷酸间连接为硫代磷酸酯键连接。
在一些实施方案中，所述arRNA由一种构建体编码并转录生成。在一些实施方案中，所述构建体选自线性核酸链、病毒载体或质粒。
RNA编辑相关酶蛋白及其用途
本申请提供了一种经修饰的腺苷脱氨酶蛋白，其中所述腺苷脱氨酶蛋白为ADAR2，其包含对应于Genebank登记号为NP_001103.1的ADAR2中如下氨基酸突变：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，或所述NP_001103.1的同源ADAR2蛋白相应位置的氨基酸突变，所述ADAR2蛋白经过所述突变修饰而具有催化胞苷脱氨基的活性。
本申请同时还提供了所述经修饰的腺苷脱氨酶蛋白用于催化胞苷脱氨基转化为尿苷的用途。在一些实施方案中，所述催化胞苷脱氨基转化为尿苷的用途发生于细胞内。在一些实施方案中，所述催化胞苷脱氨基转化为尿苷的用途发生于细胞外。
疾病治疗方法
本申请提供了一种治疗由T到C突变引起的疾病的方法，包括使用如前所述的编辑RNA的方法使包含所述T到C突变转录形成的信使RNA中靶标碱基C脱氨基，以校正所述突变。
在一些实施方案中，所述治疗由T到C突变引起的疾病的方法包括将如前所述的工程化组合物或系统注射入受试者体内。在一些实施方案中，所述治疗将包括将如下1)和2)注射入受试者体内。
1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和
2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体。其中，所述腺苷脱氨酶蛋白为如前所述的RNA编辑相关酶蛋白。
在一些实施方案中，所述注射为静脉注射、动脉灌注、肌肉注射、皮下注射或瘤内注射。
在一些实施方案中，所述由T到C突变引起的疾病包括遗传性疾病和癌症。
试剂盒及制剂
本申请还提供了一种试剂盒，其用于催化胞苷脱氨基转化为尿苷。在一些实施方案中，所述试剂盒包含如前所述的工程化组合物或系统。在一些实施方案中，所述试剂盒包含编码或表达所述工程化组合物系统中所述经修饰的腺苷脱氨酶蛋白或其催化结构域和/或将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到靶标RNA的arRNA的构建体。
本申请提供的利用CUSPER技术对RNA进行靶向编辑的方法，具有以下优点：
一方面，新的技术招募编辑蛋白(例如腺苷脱氨酶(ADAR))时，不像RESCUE技术，需要设计含Cas13b招募骨架的向导RNA，也不需要细菌来源的Cas13b过表达，减小了外源表达蛋白的长度使得通过病毒载体进行装载及人体内递送容易、多样，同时，也可以减小外源表达的核酸酶被中和导致 的基因编辑失败的可能性，这使得其在应用至医疗领域时比RESCUE技术具有显著优势。
另一方面，新的体系不像LEAPER技术，LEAPER技术仅能实现RNA编辑的应用范围从A到I的编辑，而新的体系将该编辑拓宽至C到U，这使得很多在基因组上呈现T到C突变的遗传病，以及其它需要将C转变为T/U的应用，可以用本申请的技术治疗。
因此，本申请的技术相比于现有技术而言，应用范围更广，更加安全有效。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
下面结合具体实施例，对本发明的技术方案作进一步的详述，但本发明并不限于以下实施例。如未特别指出，以下所涉及的试剂均可通过商业途径购得。为简便起见，部分操作未详述操作的参数、步骤及所使用的仪器，应当理解，这些都是本领域技术人员所熟知并可重复再现的。
实施例
实施例1：经修饰的ADAR2及BFP报告体系的分子构建
1.突变型ADAR2-r16-293T的构建
参照参考文献1中报道的RESCUE技术对ADAR2(RNA腺苷脱氨酶2)催化结构域进行诱变，突变位点与文献中r16相同(dADAR2(E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T)r16, https://benchling.com/s/seq-19Ytwwh0i0vSI byXYZ95)，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。使用本领域常规DNA合成技术体外合成包含上述突变的ADAR2编码序列片段，并通过酶切连接插入pLenti-ADAR2质粒载体(pLenti-ADAR2质粒骨架由魏文胜教授试验室惠赠)的ADAR2 XmaI酶切位点及AscI酶切位点之间。通过以上步骤构建的质粒命名为pLenti-ADAR2-r16，其包含上述突变的ADAR2基因命名为ADAR2-r16。ADAR2-r16全长cDNA序列 为：SEQ ID NO 1。通过第二代慢病毒包装体系(pCAG-VSVG由Arthur Nienhuis&Patrick Salmon惠赠(Addgene plasmid#35616；http://n2t.net/addgene:35616；RRID:Addgene_35616)；pCMVR8.74由Didier Trono惠赠(Addgene plasmid#22036； http://n2t.net/addgene:22036；RRID:Addgene_22036))将pLenti-ADAR2-r16包装成慢病毒，并使用所述慢病毒侵染293T细胞并于48小时后使用10ug/ml终浓度的Blasticidin(Solarbio B9300)进行抗性筛选。筛选过后存活的细胞称为ADAR2-r16-293T。
SEQ ID NO 1：
Figure PCTCN2020142218-appb-000001
Figure PCTCN2020142218-appb-000002
2.BFP报告体系的构建
BFP报告体系参照参考文献7构建，全部BFP(蓝色荧光蛋白)cDNA序列在体外合成，具体序列为：SEQ ID NO 2。BFP cDNA序列通过CMV启动子后的多克隆位点克隆至pCDH-CMV质粒载体(pCDH-CMV质粒骨架由Kazuhiro Oka惠赠，Addgene plasmid#72265；http://n2t.net/addgene:72265；RRID:Addgene_72265)。报告体系中待编辑的靶标碱基为BFP序列第199位的碱基C，对应第66位组氨酸，参见图1。
SEQ ID NO 2：
Figure PCTCN2020142218-appb-000003
Figure PCTCN2020142218-appb-000004
该序列第198,199,200位的碱基依次为CCA，命名为BFP-CCA，缩略为C*。当199位的碱基C在RNA水平通过脱氨基化被编辑为U之后，BFP荧光蛋白会从原先的蓝色荧光变为绿色荧光，从而可以通过流式细胞仪FITC(Fluorescein isothiocyanate，异硫氰酸荧光素)通道检测到信号。由于198位核苷酸从C突变为A、T、G后，对应密码子(196、197、198位碱基)ACC、ACA、ACT、ACG均编码苏氨酸，因此该位突变为同义突变。这使得该报告体系可以同时测定和比较在mRNA上靶标碱基的5’上游相邻碱基不同时，C到U的编辑效率。可据此使用定点诱变试剂盒(
Figure PCTCN2020142218-appb-000005
Site-DirectedMutagenesis Kit,NEB E0554S)向198位置碱基中引入突变，从而198,199,200三个位置碱基分别为：GCA，命名为BFP-GCA，缩略为G*；ACA，命名为BFP-ACA，缩略为A*；TCA命名为BFP-UCA，缩略为U*。
实施例2：CUSPER体系的初步测试
1.arRNA的设计与合成
本实施例中使用的arRNA包含与靶标mRNA互补的反义RNA，靶标碱基位于arRNA的中间位置，5’上游以及3’下游按照相同长度向两边延伸。由于合成长度的限制，本实施例选取长度为91nt的RNA进行体外合成。如图3所示，当所述arRNA第46位核苷酸分别为A、U、G、C时，则分别缩写为A^、U^、G^、C^。四种合成的arRNA具体序列见下表1。如图2所示，与LEAPER技术设计方法不同的是，本批试验中四条arRNA的设计，由于试验目的是确定靶标碱基与arRNA上的靶向碱基错配时的编辑效率，因此使用了不同的靶向碱基进行了测试，即对第46位分别为A、U、G、C的arRNA进行了测试。而在arRNA第47位的5’最近邻碱基(对应报告体系第198位)则按照引入突变前的BFP序列的靶标三联体碱基(即CCA)进行设计，即所述arRNA靶向碱基的5’最近邻碱基(第47位)为与A互补的U。这意味着，当且仅当后续测试的报告体系为BFP-CCA的时候，arRNA的设计方才与LEAPER技术一致，即：当arRNA与靶标RNA杂交时，仅在靶标碱基存在错配；而当报告体系为BFP-GCA，BFP-TCA，BFP-ACA的时候，arR NA的设计不仅在靶标碱基存在错配，而且在arRNA靶标碱基的3’相邻碱基处也存在错配。
表1
Figure PCTCN2020142218-appb-000006
注：大写字母只为突出序列间差异，同一字母的大小写不同不代表碱基的差异。
2.C到U编辑测试
ADAR2-r16-293T以300000个细胞/孔的密度铺板至6孔板，铺板后24小时，用Lipofectamine 3000转染(Invitrogen L3000015)，转染步骤按照说明书进行。按照说明书采用不同浓度的Lipofectamine 3000转染试剂进行两次重复试验，重复1每孔使用3.75μL的Lipofectamine 3000，重复每孔2使用7.5μL的Lipofectamine 3000。每孔添加2.5μg BFP报告质粒(选自：BFP-GCA，缩略为G*；BFP-ACA，缩略为A*；BFP-TCA，缩略为T*；BFP-CCA，缩略为C*)以及25pmol化学合成的arRNA。转染后48h通过FACS检测FITC通道信号强度。阳性细胞平均荧光强度(Mean Fluorescent Intensity,MFI)统计结果如图3所示。
图3中mRNA行表示对应孔中添加的BFP报告体系质粒，arRNA行表示对应孔中添加的arRNA。BFP报告体系中，198,199,200三个碱基在原始序列中为CCA，而当198位C变为A、T或G的时候，对应的65位氨基酸均为苏氨酸，所以BFP-GCA、BFP-CCA、BFP-ACA、BFP-TCA四种不同报告体系198位的改变不会造成原本蛋白功能的改变。而当199位C被编辑变为U时，199、200、201形成的密码子从CAC变为UAC，对应的66位氨 基酸从组氨酸(Histidine,His,H)变为酪氨酸(Tyrosine，Tyr，Y)，从而实现BFP到GFP的荧光转变。如图3所示，当不加入任何arRNA时，报告体系本底GFP信号MFI约为5×10 4(mRNA行标注为U*，arRNA行标注为/的报告体系；以及，mRNA行标注为A*，arRNA标注为/的报告体系)。而当通过DNA水平的点突变，使199位C突变为T时，GFP信号MFI约为2.4×10 6～3.1×10 6，比本底值高约100倍。因此，如果199位的C在RNA水平全部变为U，则可导致GFP MFI的约100倍提升。
在DNA水平199位C不变的基础上，如图3所示，而当加入arRNA后，GFP的MFI最多能提升至超过5×10 5，荧光强度超过了DNA水平的199位C点突变成T后荧光强度的20％。这说明，本申请的技术可以在不改变DNA序列的前提下，通过在转录水平上将199位的C转变为U，从而改变最终的蛋白功能。
在LEAPER技术文献中(Qu et al.,2019，原图2f，对应于本申请图4)，当靶标碱基3’相邻碱基(如N 2位置所示)为A时，LEAPER技术对靶标碱基5’相邻碱基(如N1位置所示)的偏好(指在5’上游碱基为A、U、G或C时，相同arRNA所得到的编辑效率)是：U>C≈A>G。在RESCUE技术文献中(Abudayyeh et al.,2019，原图1c，对应于本申请图5)当靶标碱基3’相邻碱基为A时，RESCUE技术对靶标碱基5’相邻碱基的偏好是：U≈A>>C≈G。而在本申请的CUSPER技术中，我们意外地发现，该技术在当靶标碱基3’相邻碱基为A时，对靶标碱基5’上游碱基的偏好与LEAPER技术和RESCUE技术两者均不相同，如图3所示，如果固定arRNA为编辑效率较高的U^或者C^，可以看到，本申请的技术对5’上游碱基的偏好是：G>C>>U≈A。
3.对CUSPER碱基偏好性的进一步确定
为进一步确定CUSPER的编辑能力及其碱基偏好性，我们对实施例2第2部分做了进一步重复，如图6所示。相较于实施例2第2部分，本部分中补充了实施例2第2部分中未涉及的仅有BFP-GCA和BFP-CCA两个报告体系质粒而不添加任何arRNA情况下的对照试验。并且对图3相关测试 中MFI超过本底值2倍以上的条件做了重复。同时，试验中重复1和重复2对应两株不同批次制作的ADAR2-r16-293T。
如图6所示，重复试验中该体系对靶标碱基5’相邻碱基的偏好呈现出与图3显示的实施例2第2部分的相关测试类似的模式，均为在5’相邻碱基为G或C时有较好编辑效率，且G大于C。
以上结果表明，本申请的技术可以在不改变DNA序列的前提下，从转录水平影响最终蛋白功能。同时，该技术对靶标碱基5’相邻碱基的偏好可能与LERPER和RESCUE技术均不相同，具体参见表2。值得注意的是，本研究中对于BFP-GCA、BFP-TCA、BFP-ACA的测试中，arRNA的设计不仅在靶标碱基处存在一个错配，在arRNA的3’下游也存在另外一个错配(如图2所示)。因此，后续还需进一步测试当这些位置不存在错配时，该技术对5’上游碱基的偏好是否还存在类似的模式。
表2.三种RNA编辑技术靶标碱基相邻碱基偏好性对比
Figure PCTCN2020142218-appb-000007
实施例3：3’及5’相邻碱基均无错配的情况下，对CUSPER编辑体系的测试
通过对GFP信号的读取，我们可以快速粗略地判断不同arRNA的编辑效率，但如果需要进一步确认编辑效率，则需通过二代测序(Next Generation Sequencing,NGS)来最终确认mRNA中有多少比例的C被编辑成U。同时，RNA单碱基编辑体系无论是A到I(Qu et al.,2019)还是C到U(Abudayyeh et al.,2019)其靶标碱基A或者C的5’相邻碱基和3’相邻碱基对其编辑效率影响较大。由于BFP到GFP报告体系的限制，如果以BFP的DNA序列(SEQ ID NO 2)中第199位的C为靶标碱基，当5’相邻碱基(第198位)C变为A、T、G时均不影响对应的65位氨基酸；但当3’相邻碱基(第200位)的A变为T、C或G时，则会导致其完全失去GFP信号。因此，通过 报告体系读取GFP的试验无法对3’相邻碱基为T、C、G的体系进行测试。而如果通过二代测序则可直接读取编辑后其mRNA中U占A、U、C、G的百分比，从而较为方便地比较4种不同5’相邻碱基和4种不同3’相邻碱基共16种情况下，其编辑效率的高低。
参照实施例1.2的步骤，根据BFP的DNA序列(SEQ ID NO 2)中第198、199、200位的DNA序列不同，我们构建了16个不同的报告体系，即ACA、ACT(对应mRNA：ACU)、ACC、ACG、TCA(对应mRNA：UCA)、TCT(对应mRNA：UCU)、TCC(对应mRNA：UCC)、TCG(对应mRNA：UCG)、CCA、CCT(对应mRNA：CCU)、CCC、CCG、GCA、GCT(对应mRNA：GCU)、GCC、GCG。并且参照实施例1.1的慢病毒包装、侵染步骤，侵染293T，使其稳定整合入293T细胞中。
与以上16个不同的报告体系相对应，在mRNA靶标碱基C对应arRNA靶向碱基为U的基础上，使arRNA靶向碱基的3’及5’最近邻碱基可与靶标RNA中靶标碱基的5’及3’相邻碱基以沃森克里克碱基配对原则互补配对。所合成arRNA序列见表3，其中mRNA中靶标以及其5’和3’相邻碱基与arRNA靶向碱基以及其5’和3’最近邻碱基的对应关系表见图7。
按照以下步骤，向16个不同的报告体系细胞中，转染入对应的arRNA，并进行RNA提取和二代测序。
1.细胞培养采用含有10％FBS(Vistech SE100-011)的DMEM(Hyclone SH30243.01)。报告体系细胞以15000个细胞/孔，传至12孔板。此时记为0小时。
2.细胞传代后24小时，用RNAi MAX(Invitrogen,13778150)试剂将12.5pmol的arRNA转入每个孔。转染步骤参照供应商说明书。
3.细胞传代后72小时，用胰酶(Invitrogen 25300054)消化整孔细胞，用800μL TRIzol(Invitrogen,15596018)收样，采用Direct-zol RNA Miniprep试剂盒(Zymo Resaerch,R2052)进行RNA提取，每个样品取1000ng提取好的总RNA用
Figure PCTCN2020142218-appb-000008
One-Step gDNA Removal and cDNA Synthesis SuperMix试剂盒(全式金，AT311)进行反转录合成cDNA，取1μL反转录产物，用序列为ggagtgagtacggtgtgcCTACGGCAAGCTGACCCTGAAGTT(SEQ ID NO：7)和gagttggatgctggatggGTAGTTGCCGTCGTCCTTGAAGAAG (SEQ ID NO：8)的两个引物以及Q5热启动酶(NEB,M0494L)进行PCR。PCR产物用Hi-TOM试剂盒(诺禾致源，REF PT045)进行建库并按照以下步骤完成二代测序和数据分析。
i.Illumina测序
将构建好的测序文库，通过NovaSeq6000平台以PE150方式进行高通量测序。
ii.测序数据处理
高通量测序得到的原始数据以fastp(v0.19.6)进行质控，过滤掉低质量、带有接头序列、及含有polyG等的序列。将得到的高质量测序数据以自主开发的拆分脚本按照相应的Barcode序列拆分到每个样本，使用BWA(v0.7.17-r1188)软件与扩增的目标区域序列进行比对，通过SAMtools(v1.9)进行格式转换以生成BAM文件、统计比对信息并重新排序和建立索引。
iii.编辑效率分析
使用JACUSA(v1.3.0)软件检测所有的mRNA靶标碱基，所用参数为：call-1 -a B,R,D,I,Y,M:4 -C ACGT -c 2 -p 1 -P UNSTRANDED -R -u Dir Mult-CE。过滤掉同时在对照和处理样本中出现的高频突变之后，以C->U突变之外的平均突变频率的三倍作为阈值，将靶标碱基C到U的突变频率在阈值之上的部分作为真实的靶标C突变为U的频率。
二代测序结果如图8显示。通过二代测序可以证实，CUSPER体系确实可以实现mRNA的C到U单碱基编辑。相较于实施例2可以看出，如果靶向碱基的5’及3’最近邻碱基与靶标RNA均无错配，则编辑效率相对较低，而当靶向碱基的5’最近邻碱基与靶标RNA形成G-G错配时，便可以提高编辑效率。此外，当除靶标碱基的C-U错配以外，靶向碱基的5’及3’最近邻碱基均与靶标碱基的3’及5’相邻碱基以沃森-克里克原则配对时，当mRNA上靶标碱基三联体为下述序列时可以获得较高的编辑效率：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
表3. 3’及5’相邻碱基均无错配的arRNA序列
Figure PCTCN2020142218-appb-000009
Figure PCTCN2020142218-appb-000010
Figure PCTCN2020142218-appb-000011
注：大小写字母无区别，大写字母只为突出序列间差异。其中arRNA，以其靶向的靶标碱基三联体命名。
参考文献
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4.Merkle,T.,Merz,S.,Reautschnig,P.,Blaha,A.,Li,Q.,Vogel,P.,...&Stafforst,T.(2019).Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.Nature biotechnology,37(2),133.
5.Pollard,K.M.,Cauvi,D.M.,Toomey,C.B.,Morris,K.V.,&Kono,D.H.(2013).Interferon-γand systemic autoimmunity.Discovery medicine,16(87),123.
6.Qu,L.,Yi,Z.,Zhu,S.,Wang,C.,Cao,Z.,Zhou,Z.,...&Bao,Y.(2019).Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.Nature biotechnology,37(9),1059-1069.
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Claims (43)

  1. 一种用于RNA编辑的工程化组合物或系统，包含：
    1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，其中，所述腺苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，和
    2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体；
    其中所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
  2. 权利要求1的工程化组合物或系统，其中所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。
  3. 权利要求2的工程化组合物或系统，其中所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。
  4. 权利要求3中的工程化组合物或系统，其中所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
  5. 权利要求1-4中任一项的工程化组合物或系统，其中所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。
  6. 权利要求5中所述的工程化组合物或系统，其中所述靶向碱基为U或C。
  7. 权利要求1-6中任一项的工程化组合物或系统，其中：
    所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。
  8. 权利要求7中的工程化组合物或系统，其中所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。
  9. 权利要求8中所述的工程化组合物或系统，其中所述arRNA与靶标RNA杂交时，所述靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。
  10. 权利要求1-9中任一项的工程化组合物或系统，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。
  11. 权利要求10中的工程化组合物或系统，其中所述靶向碱基的5’最近邻碱基为U。
  12. 权利要求9中的工程化组合物或系统，其中当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A。
  13. 权利要求1-6中任一项的工程化组合物或系统，其中当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
  14. 权利要求1-13任一项的工程化组合物或系统，其中所述arRNA的长度>50nt。
  15. 权利要求1-14任一项的工程化组合物或系统，其中所述arRNA中靶向碱基距离3’端及5’端的长度相等。
  16. 权利要求1-14中任一项的组合物，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。
  17. 权利要求1-14任一项的工程化组合物或系统，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。
  18. 权利要求1-17任一项的工程化组合物或系统，其中所述arRNA是化学修饰的。
  19. 权利要求18的工程化组合物或系统，其中所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。
  20. 一种使细胞中靶标RNA中的靶标胞嘧啶脱氨基的方法，包括将如下1)和2)导入所述细胞：
    1)经修饰的腺苷脱氨酶蛋白或其催化结构域或表达该经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体，和
    2)将所述经修饰的腺苷脱氨酶蛋白或其催化结构域募集到所述靶标RNA的arRNA或包含该arRNA或包含其编码序列的构建体，其中，所述腺 苷脱氨酶蛋白或其催化结构域经过修饰具有催化胞苷脱氨基的活性，所述arRNA包含与所述靶标RNA杂交的互补RNA序列，并且所述arRNA募集所述腺苷脱氨酶蛋白或其催化结构域至靶标RNA致使靶标RNA中的靶标胞苷脱氨基。
  21. 权利要求20的方法，其中，所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为同一构建体，或者所述表达经修饰的腺苷脱氨酶蛋白或其催化结构域的构建体和包含所述arRNA的编码序列的构建体为分开的构建体，所述分开的构建体被同时或分开地导入所述细胞。
  22. 权利要求20或21的方法，其中所述腺苷脱氨酶在一个或多个位点发生突变修饰从而具有对胞苷脱氨使其转化成尿苷的活性。
  23. 权利要求22的方法，其中所述腺苷脱氨酶蛋白或其催化结构域为ADAR2蛋白或其同源蛋白或其催化结构域。
  24. 权利要求23中的方法，其中所述突变修饰包含：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，其中所述突变修饰的氨基酸编号与NP_001103.1中的氨基酸编号一致。
  25. 权利要求21-24中任一项方法，其中所述arRNA与靶标RNA杂交时，其与靶标胞苷相对的靶向碱基为A、U、C、或G。
  26. 权利要求25中所述的方法，其中所述靶向碱基为U或C。
  27. 权利要求21-26中任一项的方法，其中：
    所述arRNA在对应于靶标RNA的靶标碱基的上游、下游、或上游和下游的一个或多个位置包含非配对核苷酸，以形成和靶标碱基上游、下游、或上游和下游的一个或多个位置的核苷酸错配。
  28. 权利要求27中的方法，其中所述arRNA靶向碱基的3’最近邻碱基与靶标RNA形成错配。
  29. 权利要求28中所述的方法，其中所述arRNA与靶标RNA杂交时，所述靶向碱基的3’最近邻碱基与靶标RNA形成G-G错配。
  30. 权利要求21-29中任一项的方法，所述arRNA与靶标RNA杂交时，所述靶向碱基的5’最近邻碱基与靶标RNA不形成错配。
  31. 权利要求30中的方法，其中所述靶向碱基的5’最近邻碱基为U。
  32. 权利要求29中的方法，其中当所述arRNA与靶标RNA杂交时，与所述arRNA的靶向碱基的5’最近邻碱基相对的靶标RNA中的碱基优选次序从高到低为G或C、U或A。
  33. 权利要求21-26中任一项的方法，其中当所述arRNA与靶标RNA杂交时，所述靶标碱基及其5’和3’相邻碱基形成的靶标碱基三联体仅在靶标碱基处形成错配，其中所述靶标碱基三联体选自：ACG、ACC、UCC、UCG、CCC、CCG、UCA、UCU。
  34. 权利要求21-33任一项的方法，其中所述arRNA长度>50nt。
  35. 权利要求21-34任一项的方法，其中所述arRNA中靶向碱基距离3’端及5’端的长度相等。
  36. 权利要求21-34中任一项的方法，其中所述arRNA中靶向碱基距离3’端的长度为45-5nt，40-5nt，35-10nt，25nt-15nt，24nt-11nt。
  37. 权利要求21-34任一项的方法，其中所述arRNA中靶向碱基距离5’端的长度为80-30nt，70-35nt，60-40nt，55nt-35nt，55nt-45nt。
  38. 权利要求21-37任一项的方法，其中所述arRNA是化学修饰的。
  39. 权利要求38的方法，其中所述化学修饰包括2’-O-甲基修饰或核苷酸间3’硫代修饰。
  40. 权利要求21-39中任一项的方法，其中所述细胞为哺乳动物细胞。
  41. 一种治疗由T到C突变引起的疾病的方法，包括使用如权利要求21-39中任一项的方法使包含所述T到C突变转录形成的信使RNA中靶标碱基C脱氨基，以校正所述突变。
  42. 一种经修饰的腺苷脱氨酶蛋白，其中所述腺苷脱氨酶蛋白为ADAR2，其包含对应于Genebank登记号为NP_001103.1的ADAR2中如下氨基酸突变：E488Q/V351G/S486A/T375S/S370C/P462A/N597I/L332I/I398V/K350I/M383L/D619G/S582T/V440I/S495N/K418E/S661T，或所述NP_001103.1的同源ADAR2蛋白相应位置的氨基酸突变，所述ADAR2蛋白经过所述突变修饰而具有催化胞苷脱氨基的活性。
  43. 如权利要求42中所述的经修饰的腺苷脱氨酶蛋白用于催化胞苷脱氨基转化为尿苷的用途。
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