Methods and Compositions for Editing RNA

Abstract

The present invention provides a method for editing RNA by introducing a deaminase-recruiting RNA into a host cell to deaminate adenosine in a target RNA. The present application also provides a deaminase-recruiting RNA for use in the RNA editing method, and a composition comprising the same.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to an international application with international application number PCT / CN2019 / 082713 filed on April 15, 2019, and an international application with international application number CN2019 / 129952 filed on December 30, 2019. Their contents are hereby incorporated by reference in their entirety into this application.

[0002] All of the foregoing applications and all documents cited therein, all documents cited during the application process ("cited documents") and all documents cited by the cited documents, as well as all documents cited or referenced in this specification ("documents cited in the text"), and all documents cited or referenced by the documents cited herein, and any product mentioned herein or any manufacturer's description, specifications, product standards, and product lists incorporated into this specification by reference, are hereby incorporated by reference into this specification and can be used in the practice of the present invention. More specifically, all references are incorporated by reference such that each individual document is specifically and individually incorporated by reference.

[0003] (Submission of Sequence Listing in ASCII Text File) The content submitted in the ASCII text file is hereby incorporated by reference in its entirety into this application: the computer - readable form (CRF) of the sequence listing (file name: FD0020PCT - sequence listing.TXT, recording date: April 13, 2020, size: 133KB).

[0004] The present invention relates to methods and compositions for editing RNA using engineered RNAs that can recruit adenosine deaminase to deaminate one or more adenosines in a target RNA.

Background Art

[0005] Genome editing is a powerful tool for biomedical research and the development of disease therapies. So far, the most popular gene editing technology is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system developed from the adaptive immune systems of bacteria and archaea. CRISPR-Cas can accurately target and cleave genomic DNA to generate double-strand DNA breaks (DSBs). DSBs can be repaired via the non-homologous end joining (NHEJ) pathway, causing insertions or deletions (Indels), which in most cases inactivate genes. Alternatively, the homology-directed repair (HDR) pathway can repair the DSBs using homologous template dsDNA or ssDNA, enabling precise genome editing.

[0006] Recently, new tools for RNA editing have been developed using deaminase proteins (such as adenosine deaminase acting on RNA (ADAR)). Mammalian cells have three types of ADAR proteins: ADAR1 (two isoforms, p110 and p150), ADAR2, and ADAR3 (catalytically inactive). The catalytic substrate of the ADAR protein is double-stranded RNA, which can remove the -NH2 group from the adenosine (A) nucleobase and change A to inosine (I). This is recognized as guanosine (G) and pairs with cytidine (C) during subsequent cell transcription and translation processes. The researchers constructed a λN-ADARDD system by fusing the λN peptide to the human ADAR1 or ADAR2 deaminase domain. This system can be induced by a fusion RNA consisting of a BoxB stem-loop and an antisense RNA to bind to a specific RNA target. In this method, the target A can be edited to I (by introducing an A-C mismatch at the base of the target A). As a result, the RNA base from A to G is edited. Other methods of RNA editing include methods of editing target RNA by fusing antisense RNA to an R / G motif (ADAR-recruiting RNA scaffold) and overexpressing ADAR1 or ADAR2 proteins in mammalian cells, and methods of accurately targeting and editing RNA using dCas13-ADAR. The PCT / EP2017 / 071912 application disclosed a method of RNA editing that does not require a recruiting structural region on an exogenous protein or nucleic acid. A synthetic RNA containing a sequence complementary to the target RNA was used to induce base editing from A to G. The RNA used in this method is short (less than 54 nt) and must be specially modified to increase the editing efficiency.

Prior Art Documents

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Summary of the Invention

[0008] Nucleic acid editing holds great promise for biological research and the development of therapeutic methods. Current tools for DNA or RNA editing rely on introducing exogenous proteins into organisms. This can be affected by potential risks or technical barriers due to possible abnormal effector activities, delivery limitations, and immunogenicity. Some other tools require complex chemical modifications, yet still have low editing efficiency. In some aspects, the present application provides a programmable approach that uses short RNAs to utilize deaminases for targeted RNA editing. In some embodiments, the deaminase is an ADAR (adenosine deaminase acting on RNA) protein. In some embodiments, the ADAR is an endogenous ADAR protein. In some aspects, the present application provides engineered RNAs that are partially complementary to the target transcript and recruit native ADAR1 or ADAR2 to change adenosine to inosine at specific sites of the target RNA. The methods described herein are collectively referred to as "LEAPER" (programmable editing of RNA using endogenous ADAR), and the ADAR-recruiting RNAs are alternatively referred to as "dRNA" or "arRNA".

[0009] In one aspect, the present application provides a method for editing a target RNA in a host cell, comprising introducing into the host cell a deaminase-recruiting RNA (dRNA) or a construct encoding the deaminase-recruiting RNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA is capable of recruiting a deaminase to deaminate a target nucleotide. In some embodiments, the adenosine deaminase acting on RNA (ADAR) deaminates a target adenosine (A) in the target RNA. In certain embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a mouse cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a primary cell. In some embodiments, the host cell is a T cell.

[0010] In certain embodiments, the ADAR is naturally or endogenously present in the host cell, e.g., naturally or endogenously present in eukaryotic cells. In some embodiments, the ADAR is endogenously expressed by the host cell. In certain embodiments, the ADAR is exogenous to the host cell. In some embodiments, the ADAR is encoded by a nucleic acid (e.g., DNA or RNA). In some embodiments, the method includes introducing an ADAR or a construct encoding the ADAR into the host cell. In some embodiments, the method does not include introducing a protein into the host cell. In certain embodiments, the ADAR is ADAR1 and / or ADAR2. In some embodiments, the ADAR is one or more ADARs selected from the group consisting of hADAR1, hADAR2, mouse ADAR1, and mouse ADAR2.

[0011] In certain embodiments, the Cas (CRISPR-associated protein) does not recognize the dRNA. In some embodiments, the dRNA does not include a crRNA, tracrRNA, or gRNA used in the CRISPR / Cas system. In some embodiments, the method does not include introducing a Cas or a Cas fusion protein into the host cell.

[0012] In certain embodiments, the deamination of target A in the target RNA causes a missense mutation, premature stop codon, abnormal splicing, or alternative splicing in the target RNA. In some embodiments, the target RNA encodes a protein, and the deamination of target A in the target RNA causes a point mutation, cleavage, elongation, and / or misfolding of the protein. In some embodiments, the deamination of target A in the target RNA causes the recovery of a missense mutation, premature stop codon, abnormal splicing, or alternative splicing in the target RNA. In some embodiments where the target RNA encodes a cleaved, extended, mutated, or misfolded protein, the deamination of the target adenosine in the target RNA causes a functional, full-length, correctly folded, and / or wild-type protein by the recovery of a missense mutation, premature stop codon, abnormal splicing, or alternative splicing in the target RNA. In some embodiments, the target RNA is a regulatory RNA, and the deamination of target A changes the expression of downstream molecules regulated by the target RNA. In certain embodiments, the method edits on the target RNA using an endogenous adenosine deaminase, causing a point mutation and / or misfolding of the protein encoded by the target RNA, and / or generating a premature stop codon, abnormal splicing site, or alternative splicing site in the target RNA.

[0013] In certain embodiments, provided is a method for editing a plurality of target RNAs in a host cell, comprising introducing into the host cell a plurality of deaminase-recruiting RNAs (dRNAs) or constructs encoding the plurality of dRNAs, wherein each of the plurality of deaminase-recruiting RNAs comprises a complementary RNA sequence that hybridizes to a corresponding target RNA among the plurality of target RNAs, and each dRNA is capable of recruiting an adenosine deaminase (ADAR) that acts on the RNA to deaminate a target adenosine (A) in the corresponding target RNA.

[0014] In some embodiments, an edited RNA or a host cell having the edited RNA generated by any one of the above RNA editing methods is provided.

[0015] In one aspect, the present application provides a method for treating or preventing a disease or disorder in an individual, comprising editing a target RNA associated with the disease or disorder in the cells of the individual according to any one of the above RNA editing methods. In some embodiments, the method comprises editing the target RNA in the cells ex vivo. In some embodiments, the method comprises administering the edited cells to the individual. In some embodiments, the method comprises administering to the individual an effective amount of dRNA or a construct encoding dRNA. In some embodiments, the method further comprises introducing into the cells an ADAR or a construct encoding ADAR (e.g., a viral vector). In some embodiments, the method further comprises administering to the individual an ADAR or a construct encoding ADAR (e.g., a viral vector). In some embodiments, the disease or disorder is a genetic disease. In some embodiments, the disease or disorder is associated with one or more acquired gene mutations (e.g., drug resistance).

[0016] One aspect of the present application provides a dRNA for deaminating a target adenosine in a target RNA by mobilizing a deaminase, comprising a complementary RNA sequence that hybridizes to the target RNA. In some embodiments, the deaminase deaminates the target adenosine in the target RNA by acting on an adenosine deaminase acting on RNA (ADAR).

[0017] In some embodiments by any one of the methods or dRNAs described herein, the dRNA comprises an RNA sequence comprising a cytidine (C), adenosine (A), or uridine (U) opposite the target adenosine to be edited in the target RNA when binding to the target RNA. The cytidine (C), adenosine (A), and uridine (U) opposite the target adenosine to be edited in the target RNA are collectively referred to as "target nucleotides", or are referred to as "target C", "target A", and "target U", respectively. In certain embodiments, the RNA sequence further comprises one or more guanosines opposite each of the non-target adenosines in the target RNA. In certain embodiments, the neighbor closest to the 5' of the target A in the target RNA is a nucleotide selected from U, C, A, and G, with the priority being U > C ≒ A > G, and the neighbor closest to the 3' of the target A in the target RNA is a nucleotide selected from G, C, A, and U, with the priority being G > C > A ≒ U. In certain embodiments, the target A in the target RNA is located in a three-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU. In certain embodiments, the three-base motif is UAG, and the dRNA comprises an A opposite the U of the three-base motif, a C opposite the target A, and a C, G, or U opposite the G of the three-base motif. In certain embodiments, the three-base motif is UAG in the target RNA, and the dRNA comprises ACC, ACG, or ACU opposite the UAG of the target RNA.

[0018] In some embodiments by any one of the methods or dRNAs described herein, the deaminase recruiting RNA comprises more than 40, 45, 50, 55, 60, 65, 70, 75 or 80 nucleotides. In certain embodiments, the deaminase recruiting RNA has a length of 40 - 260, 45 - 250, 50 - 240, 60 - 230, 65 - 220, 70 - 210, 70 - 200, 70 - 190, 70 - 180, 70 - 170, 70 - 160, 70 - 150, 70 - 140, 70 - 130, 70 - 120, 70 - 110, 70 - 100, 70 - 90, 70 - 80, 75 - 200, 80 - 190, 85 - 180, 90 - 170, 95 - 160, 100 - 150 or 105 - 140 nucleotides. In some embodiments, the length of the dRNA is about 60 - 200 nucleotides (e.g., about 60 - 150, 65 - 140, 68 - 130, or 70 - 120 nucleotides).

[0019] In some embodiments by any of the methods or dRNAs described herein, the dRNA described herein comprises, from the 5' end to the 3' end: a 5' portion, a cytidine mismatch opposite the target A in the target RNA, and a 3' portion. In some embodiments, the length of the 3' portion is about 7 nt or more (e.g., 8 nt or more, 9 nt or more, and 10 nt or more) nucleotides. In some embodiments, the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 8 nt to 25 nt, 9 nt to 25 nt, 10 nt to 25 nt, 11 nt to 25 nt, 12 nt to 25 nt, 13 nt to 25 nt, 14 nt to 25 nt, 15 nt to 25 nt, 16 nt to 25 nt, 17 nt to 25 nt, 18 nt to 25 nt, 19 nt to 25 nt, 20 nt to 25 nt, 21 nt to 25 nt, 22 nt to 25 nt, 23 nt to 25 nt, 24 nt to 25 nt, e.g., 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the length of the 5' portion is about 25 nt or more (e.g., about 30 nt or more, about 35 nt or more, about 40 nt or more, and about 45 nt or more) nucleotides. In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides). In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides), and the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the 5' portion is longer than the 3' portion. In some embodiments, the 5' portion is about 55 nucleotides in length and the 3' portion is about 15 nucleotides in length.In some embodiments, the position of the cytidine mismatch in the dRNA is due to any of the dRNAs described in the examples herein, and the dRNA is, for example, in the form of Xnt-c-Ynt [where X represents the length of the 5' portion and Y represents the length of the 3' portion], 55nt-c-35nt, 55nt-c-25nt, 55nt-c-24nt, 55nt-c-23nt, 55nt-c-22nt, 55nt-c-21nt, 55nt-c-20nt, 55nt-c-19nt, 55nt-c-18nt, 55nt-c-17nt, 55nt-c-16nt, 55nt-c-15nt, 55nt-c-14nt, 55nt-c-13nt, 55nt-c-12nt, 55nt-c-11nt, 55nt-c-10nt, 55nt-c-9nt, 55nt-c-8nt, 55nt-c-7nt, 55nt-n-20nt, 50nt-n-20nt, 45nt-n-20nt, 55nt-n-15nt, 50nt-n-15nt, 45nt-c-45nt, 45nt-c-55nt, 54nt-c-12nt, 53nt-c-13nt, 52nt-c-14nt, 51nt-c-15nt, 50nt-c-16nt, 49nt-c-17nt, 48nt-c-18nt, 47nt-c-19nt, 46nt-c-20nt, 45nt-c-21nt, 44nt-c-22nt, 43nt-c-23nt, 54nt-c-15nt, 53nt-c-16nt, 52nt-c-17nt, 51nt-c-18nt, 50nt-c-19nt, 49nt-c-20nt, 48nt-c-21nt, 47nt-c-22nt, 46nt-c-23nt, 54nt-c-17nt, 53nt-n-18nt, 52nt-n-19nt, 51nt-n-20nt, 50nt-n-21nt, 49nt-n-22nt, and 48nt-c-23.

[0020] In certain embodiments, the target RNA is an RNA selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA, and small RNAs (e.g., miRNA).

[0021] In some embodiments by either the method or the dRNA described in this specification, the dRNA is single-stranded RNA. In some embodiments, the complementary RNA sequence is single-stranded, where the dRNA further comprises one or more double-stranded regions.

[0022] In some embodiments, the dRNA comprises one or more modifications such as 2'-O-methylation and / or phosphorothioation. In some embodiments, the dRNA is about 60 to 200 nucleotides in length and comprises one or more modifications (e.g., 2'-O-methylation and / or phosphorothioation). In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides and / or phosphorothioation at the internucleotide linkages between the first and last three nucleotides. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides, phosphorothioation at the internucleotide linkages between the first and last three nucleotides, and 2'-O-methylation at one or more uridines, e.g., all uridines. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides, phosphorothioation at the internucleotide linkages between the first and last three nucleotides, 2'-O-methylation at single or multiple or all uridines, and modifications at the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine. In certain embodiments, the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is 2'-O-methylation. In certain embodiments, the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is a phosphorothioate linkage, such as a 3'-phosphorothioate linkage for example. In certain embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides, phosphorothioation at the internucleotide linkages between the first and last three nucleotides, 2'-O-methylation at all uridines, and further comprises 2'-O-methylation at the nucleotide most adjacent to the 3' or 5' of the nucleotide opposite the target adenosine.In certain embodiments, the dRNA comprises 2'-O-methylation at each of the first and last 3 nucleotides, phosphorothioation at the internucleotide linkages between the first and last 3 nucleotides, 2'-O-methylation at all uridines, and 3'-phosphorothioation at the nucleotide opposite the target adenosine and / or the nucleotides most adjacent to it at its 5' and / or 3'. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last 5 nucleotides and phosphorothioation at the internucleotide linkages between the first and last 5 nucleotides.

[0023] In certain embodiments according to any one of the methods described herein, the editing efficiency on the target RNA is at least about 30%, for example, any one of at least about 32%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%.

[0024] In some embodiments, constructs (such as viral vectors or plasmids) encoding any one of the above dRNAs are provided. In some embodiments, the construct comprises a promoter operably linked to the sequence encoding the dRNA. In some embodiments, the construct is a DNA construct.

[0025] In some embodiments, libraries are provided that contain multiple dRNAs according to any one of the above dRNAs or multiple constructs according to any one of the above constructs.

[0026] Also provided are compositions, host cells, kits, and products comprising any one of the dRNAs described herein, any one of the constructs described herein, or any one of the libraries described herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0028] The present application provides an RNA editing method (referred to herein as the "LEAPER" method) and a specially designed RNA (referred to herein as deaminase-recruiting RNA ("dRNA") or ADAR-recruiting RNA ("arRNA")) for editing a target RNA in a host cell. Without being bound by theory or hypothesis, the dRNA hybridizes sequence-specifically to its target RNA to form a double-stranded RNA, recruits adenosine deaminase acting on RNA (ADAR) that acts on RNA, and deaminates the target adenosine in the target RNA. Thus, in some embodiments, efficient RNA editing can be achieved without ectopic or overexpression of the ADAR protein in the host cell. The present invention further provides methods and compositions for treating or preventing a disease or disorder of an individual using the RNA editing method.

[0029] The RNA editing method described in this specification does not use a fusion protein comprising ADAR and a protein (such as Cas) that specifically binds to a guide nucleic acid. The deaminase-recruiting RNA (dRNA) described in this specification does not include crRNA, tracrRNA, or gRNA used in the CRISPR / Cas system. In some embodiments, the dRNA does not include an ADAR recruitment domain or chemical modifications. In some embodiments, the dRNA can be expressed from a plasmid or viral vector or synthesized as an oligonucleotide, which can achieve a desired editing efficiency. Without being bound by theory or fundamental mechanisms, it has been discovered that certain dRNAs with specific lengths, mismatch positions, and / or modification patterns exhibit higher efficiency in RNA editing. Accordingly, this application further provides an RNA editing method that is superior to those previously reported.

[0030] The LEAPER method described herein has a manageable off-target rate for the target transcript and rare global off-targets. The inventors used the LEAPER method to restore p53 function by repairing specific cancer-related point mutations. The LEAPER method described in this specification can also be applied to a wide range of cell types, including multiple human primary cells, and can be used to restore α-L-iduronidase catalytic activity in primary fibroblasts from Hurler syndrome patients without inducing a natural immune response. In some embodiments, the LEAPER method comprises a single molecule (i.e., dRNA) system. The LEAPER method as referred to herein enables accurate and efficient RNA editing and has the potential to revolutionize basic research and therapies.

[0031] Definitions The present invention will be described with reference to specific drawings using specific embodiments, but the present invention is not limited thereto and is limited only by the scope of the claims. The reference symbols in the claims should not be construed as limiting the scope. When the term "comprising" is used in this specification and the claims, it does not exclude other elements or steps. When referring to a singular noun, for example, when an indefinite article or a definite article such as "one" or "a kind" or "the" is used, the plural form of that noun is also included unless otherwise specified. Regarding the description of the numerical range of nucleotides in this specification, each intervening numerical value is clearly considered. For example, for the range of 40 to 260 nucleotides, in addition to the numbers of 40 nucleotides and 260 nucleotides, any integer number of nucleotides between 40 and 260 nucleotides is also considered.

[0032] The following terms or definitions are provided only to assist in the understanding of the present invention. Unless specifically defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art of the present invention. For definitions and terms in this field, one of ordinary skill in the art can refer, in particular, to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbour Press, Plainsview, New York (1989), Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999). The definitions provided herein should not be construed as being narrower than would be understood by one of ordinary skill in the art.

[0033] The terms "deaminase-recruiting RNA", "dRNA", "ADAR-recruiting RNA" and "arRNA" can be used interchangeably herein and refer to engineered RNAs that can recruit ADAR to deaminate target adenosines in RNA.

[0034] The terms "polynucleotide", "nucleotide sequence" and "nucleic acid" may be used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or any analogs thereof. Two nucleotides are joined by a phosphodiester bond, and multiple nucleotides are joined by phosphodiester bonds to form a polynucleotide or nucleic acid. The bond between nucleotides can be phosphorothioated, referred to as a "phosphorothioate bond" or "phosphorothiolated bond".

[0035] As used herein, the terms "adenine", "guanine", "cytosine", "thymine", "uracil" and "hypoxanthine" refer to the nucleobases themselves. The terms "adenosine", "guanosine", "cytidine", "thymidine", "uridine" and "inosine" refer to nucleobases attached to a ribose or deoxyribose sugar moiety. The term "nucleoside" refers to a nucleobase attached to ribose or deoxyribose. The term "nucleotide" refers to the respective nucleobase-ribosyl-phosphate ester or nucleobase-deoxyribosyl-phosphate ester. Adenosine and adenine (abbreviated "A"), guanosine and guanine (abbreviated "G"), cytosine and cytidine (abbreviated "C"), uracil and uridine (abbreviated "U"), thymine and thymidine (abbreviated "T"), inosine and hypoxanthine (abbreviated "I") refer to the corresponding nucleobases, nucleosides or nucleotides and are used interchangeably. Unless the context clearly dictates otherwise, the terms nucleobase, nucleoside and nucleotide may be used interchangeably.

[0036] In the context of the present application, "target RNA" is designed such that the deaminase-recruiting RNA sequence has perfect or substantial complementarity, and hybridization between the target sequence and the dRNA forms a double-stranded RNA (dsRNA) region containing the target adenosine, and its recruitment acts on RNA adenosine deaminase (ADAR) to deaminate the target adenosine. In some embodiments, the ADAR naturally exists in a host cell such as a eukaryotic cell (preferably, a mammalian cell, more preferably, a human cell). In some embodiments, the ADAR is introduced into the host cell.

[0037] As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid by conventional Watson-Crick base pairing. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., out of 10, about 5, 6, 7, 8, 9, 10 are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Completely complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100, any one of which, over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0038] As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly to the target sequence and not substantially to non-target sequences. Stringent conditions generally depend on the sequence and vary with many factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes Part I, Second Chapter “Principles of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N, Y.

[0039] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or other sequence-specific methods. A sequence that can hybridize to a given sequence is called the "complementary sequence" of the given sequence.

[0040] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. It should be understood that due to either intentional or inadvertent mutations, the DNA content of all progeny is not necessarily exactly the same. The present invention includes progeny of mutants having the same function or biological activity as the original cell.

[0041] Methods of RNA editing In the present invention, the dRNA used herein includes an RNA sequence containing a cytidine (C), adenosine (A), or uridine (U) opposite a target adenosine that is edited by the target RNA when binding to the target RNA. The cytidine (C), adenosine (A), and uridine (U) opposite the target adenosine are collectively referred to as "target nucleotides", or are referred to as "target C", "target A", and "target U", respectively. The target nucleotide and the two nucleotides directly adjacent to the target nucleotide form a triplet called a "target triplet" herein.

[0042] In some embodiments, the present application provides a method for editing a target RNA in a host cell (e.g., a eukaryotic cell) that includes introducing into the host cell a deaminase mobilizing RNA (dRNA) or a construct encoding the dRNA, wherein the dRNA includes a complementary RNA sequence that hybridizes to the target RNA, and wherein the dRNA is capable of mobilizing an adenosine deaminase (ADAR) that acts on the RNA to deaminate a target adenosine (A) in the target RNA.

[0043] In some embodiments, the present application provides a method for editing a target RNA in a host cell (e.g., a eukaryotic cell) that includes introducing into the host cell a dRNA or a construct encoding the dRNA, wherein the dRNA includes a complementary RNA sequence that hybridizes to the target RNA, and wherein the dRNA mobilizes an endogenously expressed ADAR in the host cell to deaminate a target A in the target RNA. In some embodiments, the method does not include introducing into the host cell any protein or construct encoding a protein (e.g., Cas, ADAR, or a fusion protein of ADAR and Cas).

[0044] In some embodiments, provided is a method for editing a target RNA in a host cell (e.g., a eukaryotic cell) comprising introducing into the host cell (a) a dRNA or a construct encoding a dRNA, and (b) a construct encoding an ADAR or an ADAR, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA recruits ADAR to deaminate a target A in the target RNA. In some embodiments, the ADAR is an endogenously encoded ADAR of the host cell, and introducing the ADAR comprises overexpressing the ADAR in the host cell. In some embodiments, the ADAR is exogenous to the host cell. In some embodiments, the construct encoding the ADAR is a vector such as a plasmid, or a viral vector (e.g., a lentiviral vector).

[0045] In some embodiments, provided is a method for editing a plurality (e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100 or more) of target RNAs in a host cell (e.g., a eukaryotic cell) comprising introducing into the host cell a plurality of dRNAs or constructs encoding a plurality of dRNAs, wherein each dRNA comprises a complementary RNA sequence that hybridizes to a corresponding target RNA among the plurality of target RNAs, and each dRNA is capable of recruiting ADAR to deaminate a target A of the corresponding target RNA.

[0046] In some embodiments, provided is a method for editing a plurality (e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100 or more) of target RNAs in a host cell (e.g., a eukaryotic cell) comprising introducing into the host cell a plurality of dRNAs or constructs encoding a plurality of dRNAs, wherein each dRNA comprises a complementary RNA sequence that hybridizes to a corresponding target RNA among the plurality of target RNAs, and each dRNA recruits an endogenously expressed ADAR to deaminate a target A of the corresponding target RNA.

[0047] In some embodiments, provided is a method for editing a plurality (e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100, 1000 or more) of target RNAs in a host cell (e.g., a eukaryotic cell), comprising introducing into the host cell (a) a plurality of dRNAs or a construct encoding a plurality of dRNAs, and (b) a construct encoding ADAR or ADAR, wherein each dRNA comprises a complementary RNA sequence that hybridizes to a corresponding target RNA among the plurality of target RNAs, and each dRNA recruits ADAR to deaminate a target A in the corresponding target RNA.

[0048] In one aspect, the present application provides a method for editing a plurality of RNAs in a host cell by introducing into the host cell a plurality of deaminase-recruiting RNAs, one or more constructs encoding deaminase-recruiting RNAs, or a library described herein.

[0049] In certain embodiments, a method for editing a target RNA comprises introducing into a host cell a plurality of deaminase-recruiting RNAs or one or more constructs comprising a plurality of deaminase-recruiting RNAs to effect a reaction of recruiting and deaminating adenosine deaminase (ADAR) that acts on the RNA (to one or more target adenosines in one or more target RNAs), wherein each deaminase-recruiting RNA comprises an RNA sequence complementary to the corresponding target RNA.

[0050] In one aspect, the present application provides a method for generating in a host cell (e.g., a eukaryotic cell) one or more modifications of a target RNA and / or a protein encoded by the target RNA, comprising introducing into the host cell a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the one or more modifications are selected from the group consisting of a point mutation of the protein encoded by the target RNA, misfolding of the protein encoded by the target RNA, an early stop codon in the target RNA, an abnormal splicing site in the target RNA, and an alternative splicing site in the target RNA.

[0051] In certain embodiments, a method for generating one or more modifications of a plurality of target RNAs and / or proteins encoded by the target RNAs in a host cell (e.g., a eukaryotic cell) involves introducing into the host cell a plurality of deaminase-recruiting RNAs or constructs encoding a plurality of deaminase-recruiting RNAs. Here, each dRNA includes a complementary RNA sequence that hybridizes to a corresponding target RNA among the plurality of target RNAs, and each dRNA can recruit ADAR to deaminate the target A of the corresponding target RNA.

[0052] In one aspect, the present application provides the use of a deaminase-recruiting RNA by any one of the dRNAs described herein for editing a target RNA in a host cell. In certain embodiments, the deaminase-recruiting RNA includes a complementary RNA sequence that hybridizes to the target RNA to be edited.

[0053] In one aspect, the present application provides the use of a deaminase-recruiting RNA by any one of the dRNAs described herein for generating one or more modifications to a target RNA and / or a protein encoded by the target RNA. The one or more modifications are selected from the group consisting of a point mutation of a protein encoded by the target RNA, misfolding of a protein encoded by the target RNA, an early termination codon in the target RNA, an abnormal splicing site in the target RNA, and an alternative splicing site in the target RNA. In certain embodiments, the deaminase-recruiting RNA includes a complementary RNA sequence that hybridizes to the target RNA to be edited.

[0054] The present invention also relates to a method for editing a target RNA in a eukaryotic cell using endogenous adenosine deaminase, which includes introducing into the eukaryotic cell the dRNA or a construct encoding the dRNA described herein to recruit natural endogenous adenosine deaminase (ADAR) and act on the RNA to perform a deamination reaction on the target adenosine in the target RNA sequence.

[0055] In certain embodiments according to any one of the methods or uses described herein, the dRNA comprises any one of nucleotides greater than about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250. In certain embodiments, the dRNA is any one of about 40 - 260, 45 - 250, 50 - 240, 60 - 230, 65 - 220, 70 - 220, 70 - 210, 70 - 200, 70 - 190, 70 - 180, 70 - 170, 70 - 160, 70 - 150, 70 - 140, 70 - 130, 70 - 120, 70 - 110, 70 - 100, 70 - 90, 70 - 80, 75 - 200, 80 - 190, 85 - 180, 90 - 170, 95 - 160, 100 - 200, 100 - 150, 100 - 175, 110 - 200, 110 - 175, 110 - 150, or 105 - 140 nucleotides. In some embodiments, the dRNA is about 60 - 200 nucleotides in length, for example, 60 - 150, 65 - 140, 68 - 130 or 70 - 120 nucleotides in length. In some embodiments, the dRNA is about 71 nucleotides in length. In some embodiments, the dRNA is about 111 nucleotides in length.

[0056] In certain embodiments according to any one of the methods or uses described herein, the dRNA does not contain an ADAR recruitment domain. An "ADAR recruitment domain" can be a nucleotide sequence or structure that binds to ADAR with high affinity, or a nucleotide sequence that binds to a binding partner fused to ADAR in an engineered ADAR construct. Exemplary ADAR recruitment regions include, but are not limited to, the GluR-2, GluR-B(R / G), GluR-B(Q / R), GluR-6(R / G), 5HT2C, and FlnA(Q / R) domains; for example, Wahlstedt, Helene, and Marie, “Site-selective versus promiscuous A-to-I editing”, Wiley Interdisciplinary Reviews: RNA 2.6 (2011): 761-771, which is hereby incorporated by reference in its entirety. In some embodiments, the dRNA does not contain a double-stranded portion. In some embodiments, the dRNA does not contain a hairpin such as an MS2 stem-loop. In some embodiments, the dRNA is single-stranded. In some embodiments, the ADAR does not contain a DSB binding domain. In some embodiments, the dRNA consists of (or consists essentially of) complementary RNA sequences.

[0057] In certain embodiments according to any one of the methods or uses described herein, the dRNA does not contain chemical modifications. In some embodiments, the dRNA does not contain chemically modified nucleotides such as 2'-O-methyl nucleotides or nucleotides with phosphorothioate linkages. In some embodiments, the dRNA contains 2'-O-methylation and phosphorothioate linkages only at the first three and last three residues. In some embodiments, the dRNA is not an antisense oligonucleotide (ASO).

[0058] In certain embodiments according to any one of the methods or uses described herein, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell. In some embodiments, the host cell is a mouse cell. In some embodiments, the host cell is a plant cell or a fungal cell.

[0059] In some embodiments according to any one of the methods or uses described herein, the host cell is a cell line such as HEK293T, HT29, A549, HepG2, RD, SF268, SW13, and HeLa cells. In some embodiments, the host cell is a primary cell such as a fibroblast, an epithelial cell, or an immune cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a post-mitotic cell. In some embodiments, the host cell is a brain cell, e.g., a cell of the central nervous system (CNS) such as a cerebellar cell.

[0060] In some embodiments, a method of editing a target RNA in a primary host cell (e.g., a T cell or a CNS cell) is provided, comprising introducing a dRNA or a construct encoding the dRNA into the host cell, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA recruits an endogenously expressed ADAR in the host cell to deaminate a target A in the target RNA.

[0061] In certain embodiments according to any one of the methods or uses described herein, the ADAR is endogenous to the host cell. In some embodiments, the adenosine deaminase acting on RNA (ADAR) is naturally or endogenously present in the host cell, for example, naturally or endogenously present in a eukaryotic cell. In some embodiments, the ADAR is endogenously expressed by the host cell. In certain embodiments, the ADAR is exogenously introduced into the host cell. In some embodiments, the ADAR is ADAR1 and / or ADAR2. In certain embodiments, the ADAR is one or more ADARs selected from the group consisting of hADAR1, hADAR2, mouse ADAR1, and ADAR2. In some embodiments, the ADAR is the p110 isoform of ADAR1 ("ADAR1 p110 ") and / or the p150 isoform of ADAR1 ("ADAR1 p150 "), such as ADAR1. In some embodiments, the ADAR is ADAR2. In some embodiments, the ADAR is ADAR2 expressed by the host cell, for example, ADAR2 expressed by cerebellar cells.

[0062] In some embodiments, the ADAR is an ADAR that is exogenous to the host cell. In some embodiments, the ADAR is a gain-of-function mutant of a naturally occurring ADAR. In some embodiments, the ADAR is ADAR1 comprising the E1008Q mutation. In some embodiments, the ADAR is not a fusion protein comprising a binding domain. In some embodiments, the ADAR does not comprise an engineered double-stranded nucleic acid binding domain. In some embodiments, the ADAR does not comprise an MCP domain that binds to an MS2 hairpin fused to a complementary RNA sequence of dRNA. In some embodiments, the ADAR does not comprise a DSB.

[0063] In some embodiments according to any one of the methods or uses described herein, the host cell is ADAR1 (ADAR1 p110 and / or ADAR1 p150a high expression level, such as at least about 10%, 20%, 50%, 100%, 2x, 3x, 5x or more of the protein expression level of β-tubulin. In some embodiments, the host cell has a high expression level of ADAR2, such as at least about 10%, 20%, 50%, 100%, 2x, 3x, 5x or more of the protein expression level of β-tubulin. In some embodiments, the host cell has a low expression level of ADAR3, such as not exceeding any one of about 5x, 3x, 2x, 100%, 50%, 20% or less of the protein expression level of β-tubulin.

[0064] In certain embodiments according to any one of the methods or uses described herein, the complementary RNA sequence comprises a cytidine, adenosine, or uridine opposite the target A of the target RNA. In some embodiments, the complementary RNA sequence comprises a cytidine mismatch opposite the target A of the target RNA. In some embodiments, the cytidine mismatch is located at least 5 nucleotides, e.g., at least 10, 15, 20, 25, 30, or more nucleotides away from the 5' end of the complementary RNA sequence. In some embodiments, the cytidine mismatch is located at least 20 nucleotides, e.g., at least 25, 30, 35, or more nucleotides away from the 3' end of the complementary RNA sequence. In some embodiments, the cytidine mismatch is not located within 20 (e.g., 15, 10, 5 or less) nucleotides from the 3' end of the complementary RNA sequence. In some embodiments, the cytidine mismatch is located at least 20 nucleotides (e.g., at least 25, 30, 35, or more nucleotides) away from the 3' end and at least 5 nucleotides (e.g., at least 10, 15, 20, 25, 30 or more nucleotides) away from the 5' end of the complementary RNA sequence. In some embodiments, the cytidine mismatch is located at the center of the complementary RNA sequence. In some embodiments, the cytidine mismatch is located within 20 nucleotides (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide) from the center of the complementary sequence of the dRNA. In certain embodiments according to any one of the methods or uses described herein, the complementary RNA sequence further comprises one or more guanosines, such as one, two, three, four, five, six, or more Gs, each opposite a non-target adenosine of the target RNA. In some embodiments, the complementary RNA sequence comprises two or more consecutive mismatched nucleotides (e.g., 2, 3, 4, 5, or more mismatched nucleotides) opposite a non-target adenosine of the target RNA. In some embodiments, the target RNA comprises about 20 or fewer non-target As, such as any one of 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-target A or less.G and consecutive mismatched nucleotides opposite to non-target A can reduce the off-target editing effect by ADAR.

[0065] In some embodiments by any of the methods or dRNAs described herein, the dRNA described herein can be characterized as comprising, from the 5'-end to the 3'-end: a 5' portion, a cytidine mismatch opposite the target A in the target RNA, and a 3' portion. In some embodiments, the length of the 3' portion is about 7 nt or more (e.g., 8 nt or more, 9 nt or more, and 10 nt or more) nucleotides. In some embodiments, the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 8 nt to 25 nt, 9 nt to 25 nt, 10 nt to 25 nt, 11 nt to 25 nt, 12 nt to 25 nt, 13 nt to 25 nt, 14 nt to 25 nt, 15 nt to 25 nt, 16 nt to 25 nt, 17 nt to 25 nt, 18 nt to 25 nt, 19 nt to 25 nt, 20 nt to 25 nt, 21 nt to 25 nt, 22 nt to 25 nt, 23 nt to 25 nt, 24 nt to 25 nt, e.g., 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the length of the 5' portion is about 25 nt or more (e.g., about 30 nt or more, about 35 nt or more, about 40 nt or more, and about 45 nt or more) nucleotides. In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides). In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides), and the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the 5' portion is longer than the 3' portion. In some embodiments, the 5' portion is about 55 nucleotides in length and the 3' portion is about 15 nucleotides in length.

[0066] In some embodiments, the position of the cytidine mismatch in the dRNA is due to any of the dRNAs described in the examples herein, and the dRNA can be, for example, in the form of Xnt-c-Ynt. Here, X represents the length of the 5' portion and Y represents the length of the 3' portion: 55nt-c-35nt, 55nt-c-25nt, 55nt-c-24nt, 55nt-c-23nt, 55nt-c-22nt, 55nt-c-21nt, 55nt-c-20nt, 55nt-c-19nt, 55nt-c-18nt, 55nt-c-17nt, 55nt-c-16nt, 55nt-c-15nt, 55nt-c-14nt, 55nt-c-13nt, 55nt-c-12nt, 55nt-c-11nt, 55nt-c-10nt, 55nt-c-9nt, 55nt-c-8nt, 55nt-c-7nt, 55nt-n-20nt, 50nt-n-20nt, 45nt-n-20nt, 55nt-n-15nt, 50nt-n-15nt, 45nt-c-45nt, 45nt-c-55nt, 54nt-c-12nt, 53nt-c-13nt, 52nt-c-14nt, 51nt-c-15nt, 50nt-c-16nt, 49nt-c-17nt, 48nt-c-18nt, 47nt-c-19nt, 46nt-c-20nt, 45nt-c-21nt, 44nt-c-22nt, 43nt-c-23nt, 54nt-c-15nt, 53nt-c-16nt, 52nt-c-17nt, 51nt-c-18nt, 50nt-c-19nt, 49nt-c-20nt, 48nt-c-21nt, 47nt-c-22nt, 46nt-c-23nt, 54nt-c-17nt, 53nt-n-18nt, 52nt-n-19nt, 51nt-n-20nt, 50nt-n-21nt, 49nt-n-22nt, and 48nt-c-23.

[0067] In certain embodiments according to any one of the methods or uses described herein, the complementary RNA sequence further comprises one or more guanosines (G), such as 1, 2, 3, 4, 5, 6 or more Gs, each of which is opposite a non-target adenosine in the target RNA. In some embodiments, the complementary RNA sequence comprises two or more consecutive mismatched nucleotides (e.g., 2, 3, 4, 5 or more mismatched nucleosides) opposite non-target adenosines in the target RNA. In some embodiments, the target RNA comprises about 20 or fewer non-target As, such as, for example, about 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer non-target As. The G opposite the non-target A and the consecutive mismatched nucleotides can reduce the off-target editing effect of ADAR.

[0068] In certain embodiments according to any one of the methods or uses described herein, the neighbor closest to the 5' of the target A is a nucleotide selected from U, C, A, and G, with the priority U > C ≒ A > G, and the neighbor closest to the 3' of the target A is a nucleotide selected from G, C, A, and U, with the priority G > C > A ≒ U. In certain embodiments, the target A in the target RNA is a 3-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU. In certain embodiments, the 3-base motif is UAG, and the dRNA comprises the A opposite the U of the 3-base motif, the C opposite the target A, and the C, G, or U opposite the G of the 3-base motif. In certain embodiments, the 3-base motif is UAG in the target RNA, and the dRNA comprises ACC, ACG, or ACU opposite the UAG of the target RNA. In certain embodiments, the 3-base motif is UAG in the target RNA, and the dRNA comprises ACC opposite the UAG of the target RNA.

[0069] In some embodiments, the dRNA comprises one or more modifications. Exemplary modifications to the dRNA include, but are not limited to, phosphorothioate backbone modifications, 2'-substitutions in the ribose (such as 2'-O-methylation and 2'-fluoro substitution), LNA, and L-RNA. In some embodiments, the dRNA comprises one or more modifications such as 2'-O-methylation and / or phosphorothiolation. In some embodiments, the dRNA is about 60 to 200 (this range covers any consecutive positive integers between 60 and 200, e.g., 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, etc.) nucleotides in length and further comprises one or more modifications (such as 2'-O-methylation and / or 3'-phosphorothiolation). In some embodiments, the dRNA is about 60 to 200 nucleotides in length and comprises one or more modifications. In some embodiments, the dRNA is about 60 to 200 nucleotides in length and comprises modifications of 2'-O-methylation and / or phosphorothiolation. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides and / or phosphorothiolation at the internucleotide linkages between the first and last three nucleotides. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides, phosphorothiolation at the internucleotide linkages between the first and last three nucleotides, and 2'-O-methylation in one or more uridines, e.g., all uridines. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last three nucleotides, phosphorothiolation at the internucleotide linkages between the first and last three nucleotides, 2'-O-methylation in single or multiple or all uridines, and modifications in the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine. In certain embodiments, the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is 2'-O-methylation.In certain embodiments, the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine are phosphorothioate linkages, such as 3'-phosphorothioate linkages. In certain embodiments, the dRNA includes 2'-O-methylation at each of the first and last three nucleotides, includes phosphorothiolation at the internucleotide linkages between the first and last three nucleotides, includes 2'-O-methylation at all uridines, and further includes 2'-O-methylation at the nucleotide most adjacent to the 3' or 5' of the nucleotide opposite the target adenosine. In certain embodiments, the dRNA includes 2'-O-methylation at each of the first and last three nucleotides, includes phosphorothiolation at the internucleotide linkages between the first and last three nucleotides, includes 2'-O-methylation at single, multiple, or all uridines, and further includes 3'-phosphorothioate linkages at the nucleotide opposite the target adenosine and / or the nucleotide most adjacent to its 5' and / or 3'. In some embodiments, the dRNA includes 2'-O-methylation at each of the first and last five nucleotides and includes phosphorothiolation at the internucleotide linkages between the first and last five nucleotides.

[0070] In certain embodiments according to any one of the methods or uses described herein, the target RNA is any one selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA, and small RNAs (e.g., miRNA). In some embodiments, the target RNA is pre-messenger RNA. In some embodiments, the target RNA is messenger RNA.

[0071] In certain embodiments according to any one of the methods or uses described herein, the method further comprises introducing an inhibitor of ADAR3 into a host cell. In some embodiments, the inhibitor of ADAR3 is RNAi against ADAR3, such as shRNA against ADAR3 or siRNA against ADAR3. In some embodiments, the method further comprises introducing a stimulator of interferon into the host cell. In some embodiments, ADAR is inducible by interferon, for example, ADAR is ADAR p150 . In some embodiments, the stimulator of interferon is IFNα. In some embodiments, the inhibitor of ADAR3 and / or the stimulator of interferon are encoded by the same construct (e.g., vector) encoding dRNA.

[0072] In certain embodiments according to any one of the methods or uses described herein, the efficiency of editing of the target RNA is at least about 20%, such as at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or any one of more than that. In some embodiments, the efficiency of editing is determined by Sanger sequencing. In some embodiments, the efficiency of editing is determined by next-generation sequencing.

[0073] In certain embodiments according to any one of the methods or uses described herein, the method has a low off-target editing rate. In some embodiments, the method has an editing efficiency of less than about 1% (such as any one of 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or less) for non-target A in the target RNA. In some embodiments, the method does not edit non-target A in the target RNA. In some embodiments, the method has an editing efficiency of less than about 0.1% (such as any one of 0.05%, 0.01%, 0.005%, 0.001%, 0.0001% or less) for A in non-target RNA.

[0074] In certain embodiments according to any one of the methods or uses described herein, the method does not induce an immune response, such as a natural immune response. In some embodiments, the method does not induce the expression of interferon and / or interleukin in host cells. In some embodiments, the method does not induce IFN-β and / or IL-6 expression in host cells.

[0075] The present invention further provides a host cell having the edited RNA or the edited RNA produced by any one of the methods described herein. In some embodiments, the edited RNA contains inosine. In some embodiments, the host cell contains RNA having a missense mutation, an early termination codon, an alternative splicing site, or an abnormal splicing site. In some embodiments, the host cell contains a mutated, truncated, or misfolded protein.

[0076] As used herein, "host cell" refers to any cell type that can be used as a host cell, provided that it can be modified as described herein. For example, the host cell can be a host cell that endogenously expresses adenosine deaminase acting on RNA (ADAR), or a host cell into which adenosine deaminase acting on RNA (ADAR) is introduced by methods known in the art. For example, the host cell can be a prokaryotic cell, a eukaryotic cell, or a plant cell. In some embodiments, the host cell is derived from a pre-established cell line, such as a mammalian cell line including a human cell line or a non-human cell line. In some embodiments, the host cell is derived from an individual, such as a human individual.

[0077] As used herein, "introducing" or "introduction" means delivering to the host cell one or more polynucleotides, such as dRNA, or one or more constructs comprising a vector described herein, and one or more transcripts thereof. The present invention is used as a basic platform for RNA to enable targeted editing of, for example, pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA, and small RNAs (such as miRNA). The methods of the present application can achieve introduction of the dRNA or construct described herein into a host cell using many delivery systems including, but not limited to, viruses, liposomes, electroporation, microinjection, and conjugation. Nucleic acids can be introduced into mammalian cells or target tissues using conventional virus- and non-virus-based gene transfer methods. Using such methods, nucleic acids encoding the dRNA of the present application can be administered into cells in culture or within a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (such as transcripts of the constructs described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. The viral vector delivery systems include DNA and RNA viruses having either episomal or integrated genomes for delivery to the host cell.

[0078] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistic, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugate, electroporation, nanoparticle, exosome, microvesicle, or gene gun, naked DNA, and artificial virion.

[0079] The use of RNA- or DNA virus-based systems for delivery of nucleic acids has high efficiency in targeting viruses to specific cells and transporting the viral payload to the cell nucleus.

[0080] In certain embodiments according to any one of the methods or uses described herein, the method comprises introducing into a host cell a viral vector (such as a lentiviral vector) encoding dRNA. In some embodiments, the method comprises introducing into a host cell a plasmid encoding dRNA. In some embodiments, the method comprises introducing (e.g., by electroporation) dRNA (e.g., synthetic dRNA) into a host cell. In some embodiments, the method comprises transfection of dRNA into a host cell.

[0081] After deamination, the modification of the target RNA and / or the protein encoded by the target RNA can be determined using different methods depending on the position of the target adenosine in the target RNA. For example, to confirm whether "A" has been edited to "I" in the target RNA, RNA sequencing methods known in the art can be used to detect modifications in the RNA sequence. When the target adenosine is in the coding region of the mRNA, RNA editing can change the amino acid sequence encoded by the mRNA. For example, point mutations can be introduced into the mRNA, or congenital or acquired point mutations in the mRNA can be reverted to yield a wild-type gene product. This is because it has been converted from "A" to "I". Amino acid sequencing by methods known in the art can be used to find changes in amino acid residues in the encoded protein. Modification of the stop codon can be determined by assessing the presence of functional, extended, truncated, full-length and / or wild-type proteins. For example, when the target adenosine is located in the UGA, UAG, or UAA stop codon, modification of the target A (UGA or UAG) or multiple As (UAA) can create read-through mutations and / or extended proteins. Alternatively, truncated proteins encoded by the target RNA can be restored to create functional full-length and / or wild-type proteins. Editing of the target RNA can also generate abnormal splicing sites and / or alternative splicing sites in the target RNA, and thus, by restoring extended, truncated, or misfolded proteins, or abnormal or alternative splicing sites encoded by the target RNA, functional and correctly folded full-length and / or wild-type proteins can be created. In some embodiments, the present application contemplates editing of both congenital and acquired genetic changes, such as missense mutations, premature stop codons, abnormal splicing or alternative splicing sites encoded by the target RNA. Using known methods to evaluate the function of the protein encoded by the target RNA can confirm whether the RNA editing has achieved the desired effect.The deamination of adenosine (A) to inosine (I) can correct mutated A at the target position of mutant RNA encoding a protein. Therefore, the identification of deamination to inosine can provide an evaluation of whether a functional protein exists, or whether the disease or drug resistance-related RNA caused by the presence of the mutated adenosine has been restored or partially restored. Similarly, since the deamination of adenosine (A) to inosine (I) may introduce point mutations into the resulting protein, the identification of deamination to inosine can find functional indicators for identifying the cause of a disease or disease-related factors.

[0082] If the presence of the target adenosine causes abnormal splicing, the readout may be an evaluation of the occurrence and frequency of abnormal splicing. On the other hand, when deamination of the desired target adenosine is introduced into the splicing site, a similar method can be used to check whether the required type of splicing occurs. Exemplary suitable methods for identifying the presence of inosine after deamination of the target adenosine using methods well known to those skilled in the art are RT-PCR and sequencing.

[0083] The effects of deamination of the target adenosine include, for example, point mutations, premature stop codons, abnormal splicing sites, alternative splicing sites, and misfolding of the resulting protein. These effects can induce structural and functional changes in disease-related RNA and / or proteins, or structural and functional changes in RNA and / or proteins related to the development of drug resistance, regardless of whether they are genetically inherited or caused by acquired gene mutations. Therefore, by changing the structure and / or function of disease-related RNA and / or proteins, dRNA, constructs encoding dRNA, and the RNA editing methods of the present application can be used for the prevention or treatment of genetic diseases or disorders, or diseases or disorders related to acquired gene mutations.

[0084] In some embodiments, the target RNA is a regulatory RNA. In some embodiments, the target RNA to be edited is ribosomal RNA, transfer RNA, long non-coding RNA or small RNA (e.g., miRNA, pri-miRNA, pre-miRNA, piRNA, siRNA, snoRNA, snRNA, exRNA or scaRNA). The effects of deamination of the target adenosine include, for example, loss or gain of function and / or three-dimensional structure of ribosomal RNA, transfer RNA, long non-coding RNA or small RNA (e.g., miRNA). In some embodiments, deamination of the target A in the target RNA changes the expression levels of one or more downstream molecules (e.g., proteins, RNAs and / or metabolites) of the target RNA. The change in the expression level of the downstream molecule may be an increase or decrease in the expression level.

[0085] Some embodiments of the present application relate to multiplex editing of target RNA in host cells, which can be used to screen different variants of a target gene or different genes in a host cell. In some embodiments where the method involves introducing multiple dRNAs into the host cell, at least two of the multiple dRNAs have different sequences and / or have different target RNAs. In some embodiments, each dRNA has a different sequence and / or a different target RNA. In some embodiments, the method generates multiple (e.g., at least 2, 3, 5, 10, 50, 100, 1000 or more) modifications to a single target RNA in the host cell. In some embodiments, the method generates modifications to multiple (e.g., at least 2, 3, 5, 10, 50, 100, 1000 or more) target RNAs in the host cell. In some embodiments, the method involves editing multiple target RNAs in multiple host cell populations. In some embodiments, each host cell population receives a different dRNA or a dRNA having a target RNA different from other host cell populations.

[0086] Deaminase mobilizing RNAs, constructs, and libraries In one aspect, the present application provides a deaminase-recruiting RNA that can be used in any one of the methods described herein. Any one of the dRNAs described in this section can be used in the RNA editing and therapies described herein. It is intended that any of the features and parameters described herein for dRNA can be combined with each other as if all combinations were individually described. The dRNAs described herein do not include tracrRNA, crRNA, or gRNA used in the CRISPR / Cas system.

[0087] In some embodiments, a deaminase-recruiting RNA (dRNA) is provided for deaminating a target adenosine in a target RNA by recruiting an ADAR that includes a complementary RNA sequence that hybridizes to the target RNA.

[0088] In one aspect, the present invention provides a construct comprising any one of the deaminase-recruiting RNAs described herein. In certain embodiments, the construct is a viral vector (preferably a lentiviral vector) or a plasmid. In some embodiments, the construct encodes a single dRNA. In some embodiments, the construct encodes multiple (e.g., about 1, 2, 3, 4, 5, 10, 20 or more) dRNAs.

[0089] In one aspect, the present application provides a library comprising multiple deaminase-recruiting RNAs or multiple constructs described herein.

[0090] In one aspect, the present application provides a composition or host cell comprising the deaminase-recruiting RNA or the construct described herein. In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell.

[0091] In certain embodiments by any one of the dRNAs, constructs, libraries or compositions described herein, the complementary RNA sequence comprises a cytidine, adenosine or uridine opposite the target adenosine (to be edited in the target RNA). In certain embodiments, the complementary RNA sequence further comprises one or more guanosines each opposite a non-target adenosine in the target RNA. In certain embodiments, the nucleotide closest to the 5' of the target A is a nucleotide selected from U, C, A, and G, with the priority U > C ≒ A > G, and the nucleotide closest to the 3' of the target A is a nucleotide selected from G, C, A, and U, with the priority G > C > A ≒. In some embodiments, the nucleotide closest to the 5' of the target A is U. In some embodiments, the nucleotide closest to the 5' of the target A is C or A. In some embodiments, the nucleotide closest to the 3' of the target A is G. In some embodiments, the nucleotide closest to the 3' of the target A is C.

[0092] In certain embodiments by any one of the dRNAs, constructs, libraries or compositions described herein, the target A in the target RNA is a 3-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU. In certain embodiments, the 3-base motif is UAG, and the dRNA comprises an A opposite the U of the 3-base motif, a C opposite the target A, and a C, G, or U opposite the G of the 3-base motif. In certain embodiments, the 3-base motif is UAG of the target RNA, and the dRNA comprises ACC, ACG, or ACU opposite the UAG of the target RNA.

[0093] In some embodiments, the dRNA comprises a cytosine mismatch opposite the target A in the target RNA. In some embodiments, the cytosine mismatch is close to the center of the complementary RNA sequence, such as within 20, 15, 10, 5, 4, 3, 2, or 1 nucleotide from the center of the complementary RNA sequence. In some embodiments, the cytosine mismatch is at least 5 nucleotides away from the 5' end of the complementary RNA sequence. In some embodiments, the cytosine mismatch is at least 20 nucleotides away from the 3' end of the complementary RNA sequence.

[0094] In certain embodiments by any one of the dRNAs, constructs, libraries, or compositions described herein, the dRNA comprises any one of more than about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nucleotides. In certain embodiments, the length of the dRNA is any one of 40 - 260, 45 - 250, 50 - 240, 60 - 230, 65 - 220, 70 - 220, 70 - 210, 70 - 200, 70 - 190, 70 - 180, 70 - 170, 70 - 160, 70 - 150, 70 - 140, 70 - 130, 70 - 120, 70 - 110, 70 - 100, 70 - 90, 70 - 80, 75 - 200, 80 - 190, 85 - 180, 90 - 170, 95 - 160, 100 - 150, or 105 - 140 nucleotides.

[0095] In some embodiments, the dRNA is about 60 - 200 (e.g., about 60 - 150, 65 - 140, 68 - 130, or 70 - 120) nucleotides in length. The dRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 25 - 44, 142 - 205, 341 - 342.

[0096] The dRNA of the present application contains a complementary RNA sequence that hybridizes to the target RNA. The complementary RNA sequence is fully or substantially complementary to the target RNA, enabling hybridization of the complementary RNA sequence to the target RNA. In some embodiments, the complementary RNA sequence has 100% sequence complementarity with the target RNA. In some embodiments, the complementary RNA sequence has at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more complementarity (over at least about 20, 40, 60, 80, 100, 150, 200, or more consecutive nucleotides in the target RNA). In some embodiments, the dsRNA formed by hybridization between the complementary RNA sequence and the target RNA has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-Watson-Crick base pairs (i.e., mismatches).

[0097] ADARs, such as human ADAR enzymes, edit double-stranded RNA (dsRNA) structures with different specificities depending on a number of factors. One important factor is the degree of complementarity between the two strands that make up the dsRNA sequence. Complete complementarity between the dRNA and the target RNA usually results in the catalytic domain of ADAR deaminating adenosine indiscriminately. The specificity and efficiency of ADAR can be modified by introducing mismatches into the dsRNA region. For example, an A-C mismatch is preferably recommended to enhance the specificity and efficiency of deamination of the adenosine to be edited. Conversely, at A (adenosine) positions other than the target A (i.e., "non-target A"), off-target editing can be reduced by a G-A mismatch. Substantial complementarity in the hybridization of the dsRNA between the dRNA and the target RNA and in the generation of the dsRNA is required, but complete complementarity is not necessarily required for dsRNA formation between the dRNA and its target RNA. In some embodiments, the dRNA sequence or its single-stranded RNA region has complementarity (upon optimal alignment) with the target RNA of at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner).

[0098] The nucleotides adjacent to the target adenosine also affect the specificity and efficiency of deamination. For example, the 5'-proximal nucleotide of the target adenosine edited in the target RNA sequence has a preference of U > C ≒ A > G, and the 3'-proximal nucleotide of the target adenosine edited in the target RNA sequence has a preference of G > C > A ≒ U from the viewpoints of the specificity and efficiency of adenosine deamination. In some embodiments, when the target adenosine in the target RNA can be a 3-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU, the specificity and efficiency of the deamination of said adenosine are higher than those of adenosines of other 3-base motifs. In some embodiments, when the target adenosine to be edited is in the 3-base motifs UAG, UAC, UAA, UAU, CAG, CAC, AAG, AAC, or AAA, the deamination efficiency of said adenosine is higher than that of adenosines of other motifs. For the same 3-base motif, different designs of dRNA may also lead to different deamination efficiencies. Taking the 3-base motif UAG as an example, in some embodiments, when the dRNA contains a cytidine (C) opposite the target adenosine to be edited, said adenosine (A) is opposite uridine, and cytidine (C), guanosine (G), or uridine (U) is opposite guanosine, and the deamination efficiency of the target adenosine is higher than that of deamination using other dRNA sequences. In some embodiments, when the dRNA contains ACC, ACG, or ACU opposite UAG of the target RNA, the editing efficiency of A at UAG of said target RNA can reach about 25% - 30%.

[0099] In addition to the target adenosine, there may be one or more adenosines in the target RNA, and it is not desirable to edit these. With respect to these adenosines, it is preferable to reduce their editing efficiency as much as possible. According to the present invention, it has been found that when guanosine is opposite an adenosine of the target RNA, the deamination efficiency is significantly reduced. Thus, to reduce off-target deamination, the dRNA can be designed to include one or more guanosines opposite one or more adenosines other than the target adenosine to be edited in the target RNA.

[0100] The desired levels of specificity and efficiency in editing the target RNA sequence vary depending on the various applications. In accordance with the description of this patent application, one of ordinary skill in the art can design a dRNA having a sequence complementary or substantially complementary to the target RNA sequence as needed, and can obtain the desired results by repeating trial and error. As used herein, the term "mismatch" refers to opposing nucleotides in double-stranded RNA (dsRNA) that do not form a complete base pair according to the Watson-Crick base pairing rules. Mismatched base pairs include, for example, G-A, C-A, U-C, A-A, G-G, C-C, U-U base pairs. Taking the A-C match as an example, when the target A is edited in the target RNA, the dRNA is designed to include a C opposite the A to be edited, generating an A-C mismatch in the dsRNA formed by hybridization between the target RNA and the dRNA.

[0101] In some embodiments, the dsRNA formed by hybridization between the dRNA and the target RNA does not contain mismatches. In some embodiments, the dsRNA formed by hybridization between the dRNA and the target RNA contains one or more, for example, 1, 2, 3, 4, 5, 6, 7 or more mismatches (e.g., the same type of mismatch in different types of mismatches). In some embodiments, the dsRNA formed by hybridization between the dRNA and the target RNA contains one or more types of mismatches, for example, 1, 2, 3, 4, 5, 6, 7 types of mismatches selected from the group consisting of G-A, C-A, U-C, A-A, G-G, C-C, and U-U.

[0102] The mismatched nucleotides of the dsRNA formed by hybridization between the dRNA and the target RNA can form bulges, which can promote the efficiency of target RNA editing. There may be one or more bulges (formed only at the target adenosine) formed by the mismatches. The mismatches that induce additional bulges can be upstream and / or downstream of the target adenosine. The bulge may be a single-mismatch bulge (caused by a base pair of one mismatch) or a multi-mismatch bulge (caused by a base pair of multiple consecutive mismatches, preferably 2 or 3 consecutive base pairs of mismatches).

[0103] The complementary RNA sequence in the dRNA is single-stranded. The dRNA can be completely single-stranded or can have one or more (e.g., 1, 2, 3, or more) double-stranded regions and / or one or more stem-loop regions. In some embodiments, the complementary RNA sequence is any one of at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides. In certain embodiments, the complementary RNA sequence has a length of 40 - 260, 45 - 250, 50 - 240, 60 - 230, 65 - 220, 70 - 220, 70 - 210, 70 - 200, 70 - 190, 70 - 180, 70 - 170, 70 - 160, 70 - 150, 70 - 140, 70 - 130, 70 - 120, 70 - 110, 70 - 100, 70 - 90, 70 - 80, 75 - 200, 80 - 190, 85 - 180, 90 - 170, 95 - 160, 100 - 200, 100 - 150, 100 - 175, 110 - 200, 110 - 175, 110 - 150, or 105 - 140 nucleotides. In some embodiments, the dRNA is about 60 - 200 (e.g., about 60 - 150, 65 - 140, 68 - 130, or 70 - 120) nucleotides in length. In some embodiments, the complementary RNA sequence is about 71 nucleotides in length. In some embodiments, the complementary RNA sequence is about 111 nucleotides in length.

[0104] In some embodiments, in addition to the complementary RNA sequence, the dRNA can further include a region for stabilizing the dRNA, e.g., one or more double-stranded regions and / or stem-loop regions. In some embodiments, the double-stranded region or stem-loop region of the dRNA can include any one of about 200, 150, 100, 50, 40, 30, 20, 10, or fewer base pairs. In some embodiments, the dRNA does not include a stem-loop or double-stranded region. In some embodiments, the dRNA includes an ADAR recruitment domain. In some embodiments, the dRNA does not include an ADAR recruitment domain.

[0105] The dRNA may include one or more modifications. In some embodiments, the dRNA has modified nucleotides that include one or more nucleobase modifications and / or backbone modifications. In some embodiments, the dRNA is about 60 to 200 nucleotides in length and includes one or more modifications (e.g., 2'-O-methylation and / or phosphorothioation). In some embodiments, the modified dRNA includes, from the 5' end to the 3' end: a 5' portion, a cytosine mismatch opposite the target A in the target RNA, and a 3' portion, and the length of the 3' portion is about 7 nt or more (e.g., 8 nt or more, 9 nt or more, and 10 nt or more) nucleotides. In some embodiments, the length of the 5' portion is about 25 nt or more (e.g., about 30 nt or more, about 35 nt or more, about 40 nt or more, and about 45 nt or more) nucleotides. In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides). In some embodiments, the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the length of the 5' portion is about 25 nt to 85 nt nucleotides (e.g., about 25 nt to 80 nt, 25 nt to 75 nt, 25 nt to 70 nt, 25 nt to 65 nt, 25 nt to 60 nt, 30 nt to 55 nt, 40 nt to 55 nt, or 45 nt to 55 nt nucleotides), and the length of the 3' portion is about 7 nt to 25 nt nucleotides (e.g., about 10 nt to 15 nt or 21 nt to 25 nt nucleotides). In some embodiments, the 5' portion is longer than the 3' portion. In some embodiments, the 5' portion is about 55 nucleotides in length and the 3' portion is about 15 nucleotides in length.In some embodiments, the position of the cytosine mismatch in the dRNA is by any of the dRNAs described in the examples herein, and the dRNA is, for example, in the form of Xnt-c-Ynt [where X represents the length of the 5' portion and Y represents the length of the 3' portion]: 55nt-c-35nt, 55nt-c-25nt, 55nt-c-24nt, 55nt-c-23nt, 55nt-c-22nt, 55nt-c-21nt, 55nt-c-20nt, 55nt-c-19nt, 55nt-c-18nt, 55nt-c-17nt, 55nt-c-16nt, 55nt-c-15nt, 55nt-c-14nt, 55nt-c-13nt, 55nt-c-12nt, 55nt-c-11nt, 55nt-c-10nt, 55nt-c-9nt, 55nt-c-8nt, 55nt-c-7nt, 55nt-n-20nt, 50nt-n-20nt, 45nt-n-20nt, 55nt-n-15nt, 50nt-n-15nt, 45nt-c-45nt, 45nt-c-55nt, 54nt-c-12nt, 53nt-c-13nt, 52nt-c-14nt, 51nt-c-15nt, 50nt-c-16nt, 49nt-c-17nt, 48nt-c-18nt, 47nt-c-19nt, 46nt-c-20nt, 45nt-c-21nt, 44nt-c-22nt, 43nt-c-23nt, 54nt-c-15nt, 53nt-c-16nt, 52nt-c-17nt, 51nt-c-18nt, 50nt-c-19nt, 49nt-c-20nt, 48nt-c-21nt, 47nt-c-22nt, 46nt-c-23nt, 54nt-c-17nt, 53nt-n-18nt, 52nt-n-19nt, 51nt-n-20nt, 50nt-n-21nt, 49nt-n-22nt, and 48nt-c-23.

[0106] In some embodiments, the dRNA is about 60 to 200 nucleotides in length and contains one or more modifications (e.g., 2'-O-methylation and / or phosphorothioation). In some embodiments, the dRNA contains 2'-O-methylation at each of the first and last 3 nucleotides and / or phosphorothioation at the internucleotide linkages between the first and last 3 nucleotides. In some embodiments, the dRNA contains 2'-O-methylation at each of the first and last 3 nucleotides, contains phosphorothioation at the internucleotide linkages between the first and last 3 nucleotides, and contains 2'-O-methylation at one or more uridines, e.g., all uridines. In some embodiments, the dRNA contains 2'-O-methylation at each of the first and last 3 nucleotides, contains phosphorothioation at the internucleotide linkages between the first and last 3 nucleotides, contains 2'-O-methylation at single, multiple, or all uridines, and contains modifications at the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine. In certain embodiments, the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is 2'-O-methylation. In certain embodiments, the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is a phosphorothioate linkage, such as a 3'-phosphorothioate linkage. In certain embodiments, the dRNA contains 2'-O-methylation at each of the first and last 3 nucleotides, contains phosphorothioation at the internucleotide linkages between the first and last 3 nucleotides, contains 2'-O-methylation at all uridines, and moreover contains 2'-O-methylation at the nucleotide most adjacent to the 3' or 5' of the nucleotide opposite the target adenosine.In certain embodiments, the dRNA comprises 2'-O-methylation at each of the first and last 3 nucleotides, phosphorothioation at the internucleotide linkages of each of the first and last 3 nucleotides, 2'-O-methylation at all uridines, and 3'-phosphorothioation at the nucleotide opposite the target adenosine and / or the nucleotides most adjacent to it at its 5' and / or 3'. In some embodiments, the dRNA comprises 2'-O-methylation at each of the first and last 5 nucleotides and phosphorothioation at the internucleotide linkages of each of the first and last 5 nucleotides. This application also contemplates constructs comprising the dRNAs described herein. As used herein, the term "construct" refers to a DNA or RNA molecule that includes a coding nucleotide sequence that can be transcribed into RNA or expressed as a protein. In some embodiments, the construct comprises one or more regulatory elements operably linked to a nucleotide sequence encoding an RNA or protein. When the construct is introduced into a host cell, under appropriate conditions, the coding nucleotide sequence in the construct can be transcribed or expressed.

[0107] In some embodiments, the construct comprises a promoter operably or spatially linked to a coding nucleotide sequence such that the promoter controls the transcription or expression of the coding nucleotide sequence. The promoter can be located 5' (upstream) of the coding nucleotide sequence under its control. The distance between the promoter and the coding sequence may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function. In some embodiments, the construct comprises a 5' UTR and / or a 3' UTR that regulate the transcription or expression of the coding nucleotide sequence.

[0108] In some embodiments, the construct is a vector encoding any one of the dRNAs disclosed in the present application. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. The vector includes, but is not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that include one or more free ends and nucleic acid molecules that do not include free ends (e.g., circular); nucleic acid molecules that include DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors can direct the transcription or expression of the coding nucleotide sequences to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0109] The recombinant expression vector can include the nucleic acid of the present invention in a form suitable for transcription or expression of the nucleic acid in a host cell. In some embodiments, the recombinant expression vector includes one or more regulatory elements that can be selected based on the host cell used for transcription or expression and that are operably linked to the nucleic acid sequence to be transcribed or expressed. In the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element in a manner that enables expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0110] In some embodiments, constructs (e.g., vectors such as viral vectors) comprising nucleotide sequences encoding dRNA are provided. In some embodiments, constructs (e.g., vectors such as viral vectors) comprising nucleotide sequences encoding the ADAR are provided. In some embodiments, constructs comprising a first nucleotide sequence encoding the dRNA and a second nucleotide sequence encoding the ADAR are provided. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to the same promoter. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters. In some embodiments, the promoter is inducible. In some embodiments, the construct does not encode an ADAR. In some embodiments, the vector further comprises a nucleic acid sequence encoding an inhibitor of ADAR3 (e.g., ADAR3 shRNA or siRNA) and / or a stimulator of interferon (e.g., IFN-α).

[0111] Treatment method The RNA editing methods and compositions described herein can be used to treat or prevent a disease or disorder of an individual, including but not limited to, hereditary genetic diseases and drug resistance.

[0112] In some embodiments, a method for editing a target RNA in a cell of an individual (e.g., a human individual) ex vivo is provided, comprising editing the target RNA using any one of the RNA editing methods described herein.

[0113] In some embodiments, a method of editing a target RNA in a cell of an individual (e.g., a human individual) ex vivo is provided, which includes introducing a dRNA or a construct encoding the dRNA into the cell of the individual, wherein the dRNA is a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate a target A in the target RNA. In some embodiments, the target RNA is associated with a disease or disorder of the individual. In some embodiments, the disease or disorder is a genetic disease, or a disease or disorder associated with one or more acquired gene mutations (e.g., drug resistance). In some embodiments, this method further includes obtaining cells from the individual.

[0114] In some embodiments, a method of treating or preventing a disease or disorder in an individual (e.g., a human individual) is provided, which includes editing a target RNA associated with the disease or disorder in a cell of the individual using any one of the RNA editing methods described herein.

[0115] In some embodiments, a method of treating or preventing a disease or disorder in an individual (e.g., a human individual) is provided, which includes introducing a dRNA or a construct encoding the dRNA into an isolated cell of the individual ex vivo, wherein the dRNA includes a complementary RNA sequence that hybridizes to a target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate a target A in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cell. In some embodiments, this method includes introducing an ADAR or a construct encoding the ADAR into the isolated cell. In some embodiments, this method further includes culturing the cells having the edited RNA. In some embodiments, this method further includes administering the cells having the edited RNA to the individual. In some embodiments, the disease or disorder is a genetic disease, or a disease or disorder associated with one or more acquired gene mutations (e.g., drug resistance).

[0116] In some embodiments, provided is a method of treating or preventing a disease or disorder in an individual (e.g., a human individual) comprising introducing into isolated cells of the individual in ex vivo dRNA or a construct encoding dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to a target RNA associated with the disease or disorder, and wherein the dRNA is capable of recruiting an endogenously expressed ADAR in the host cell to deaminate a target A in the target RNA. In some embodiments, the method further comprises culturing the cells having the edited RNA. In some embodiments, the method further comprises administering the cells having the edited RNA to an individual. In some embodiments, the disease or disorder is a genetic disease, or a disease or disorder associated with one or more acquired genetic mutations (e.g., drug resistance).

[0117] In some embodiments, provided is a method of treating or preventing a disease or disorder in an individual (e.g., a human individual) comprising administering to the individual an effective amount of dRNA or a construct encoding dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to a target RNA associated with the disease or disorder, and wherein the dRNA is capable of recruiting ADAR to deaminate a target A in the target RNA. In some embodiments, the ADAR is an ADAR endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding ADAR. In some embodiments, the disease or disorder is a genetic disease, or a disease or disorder associated with one or more acquired genetic mutations (e.g., drug resistance).

[0118] Diseases and disorders suitable for treatment using the methods of the present application include diseases associated with mutations such as missense mutations, premature stop codons, abnormal splicing, or G to A mutations that cause alternative splicing in RNA transcripts. Examples of disease-related mutations that can be restored by the methods of the present application include TP53 associated with cancer W53X(e.g., 158G>A), IDUA associated with mucopolysaccharidosis type I (MPS I) W402X (e.g., TGG>TAG mutation in exon 9), COL3A1 associated with Ehlers-Danlos syndrome W1278X (e.g., 3833G>A mutation), BMPR2 associated with primary pulmonary hypertension W298X (e.g., 893G>A), AHI1 associated with Joubert syndrome W725X (e.g., 2174G>A) syndrome, FANCC associated with Fanconi anemia W506X (e.g., 1517G>A), MYBPC3 associated with hypertrophic cardiomyopathy, familial, primary W1098X (e.g., 3293G>A), and IL2RG associated with X-linked severe combined immunodeficiency W237X (e.g., 710G>A) is included, but not limited to these. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is a single-gene disease. In some embodiments, the disease or disorder is a polygenic disease.

[0119] In some embodiments, provided is a method of treating cancer associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing a dRNA or a construct encoding the dRNA into isolated cells of the individual ex vivo, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA recruits ADAR to deaminate a target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing an ADAR or a construct encoding the ADAR into the isolated cells. In some embodiments, the target RNA is TP53 W53X (e.g., 158G>A). In some embodiments, the dRNA comprises a nucleic acid sequence of SEQ ID NO: 195, 196, or 197.

[0120] In some embodiments, provided is a method of treating or preventing cancer using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA. Here, the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is TP53 W53X (e.g., 158G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 195, 196, or 197.

[0121] In some embodiments, provided is a method of treating MPS I (e.g., Hurler syndrome or Scheie syndrome) associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into isolated cells of the individual, ex vivo, a dRNA or a construct encoding the dRNA. Here, the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is IDUA W402X (e.g., the TGG>TAG mutation in exon 9). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 204 or 205.

[0122] In some embodiments, provided is a method of treating or preventing MPS I (e.g., Hurler syndrome or Scheie syndrome) using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is IDUA W402X (e.g., a TGG>TAG mutation in exon 9). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 204 or 205.

[0123] In some embodiments, provided is a method of treating or preventing Ehlers-Danlos syndrome associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into isolated cells of the individual in ex vivo an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is COL3A1 W1278X (e.g., a 3833G>A mutation). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 198.

[0124] In some embodiments, provided is a method of treating or preventing Ehlers-Danlos syndrome using a target RNA having a mutation (e.g., a G>A mutation) in an individual, the method comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA is capable of recruiting ADAR to deaminate a target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is COL3A1 W1278X (e.g., the 3833G>A mutation). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 198.

[0125] In some embodiments, provided is a method of treating idiopathic pulmonary hypertension associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, the method comprising introducing into cells isolated from the individual ex vivo a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA is capable of recruiting ADAR to deaminate a target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is BMPR2 W298X (e.g., 893G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 199.

[0126] In some embodiments, provided is a method of treating or preventing primary pulmonary hypertension using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is BMPR2 W298X (e.g., 893G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 199.

[0127] In some embodiments, provided is a method of treating or preventing Joubert syndrome associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into cells isolated from the individual ex vivo a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is AH11 W725X (e.g., 2174G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 200.

[0128] In some embodiments, provided is a method of treating or preventing Joubert syndrome using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is AH11 W725X (e.g., 2174G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 200.

[0129] In some embodiments, provided is a method of treating Fanconi anemia associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into cells isolated from the individual ex vivo a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is FANCC W506X (e.g., 1517G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 201.

[0130] In some embodiments, provided is a method of treating or preventing Fanconi anemia using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is FANCC W506X (e.g., 1517G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 201.

[0131] In some embodiments, provided is a method of treating primary familial hypertrophic cardiomyopathy associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into cells isolated from the individual ex vivo a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is MYBPC3 W1098X (e.g., 3293G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 202.

[0132] In some embodiments, provided is a method of treating or preventing primary familial hypertrophic cardiomyopathy using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA associated with the disease or disorder, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is MYBPC3 W1098X (e.g., 3293G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 202.

[0133] In some embodiments, provided is a method of treating X-linked severe combined immunodeficiency associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising introducing into cells isolated from the individual ex vivo a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the dRNA can recruit ADAR to deaminate the target A in the target RNA, thereby rescuing the mutation of the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the isolated cells. In some embodiments, the method comprises introducing into the isolated cells an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is IL2RG W237X (e.g., 710G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 203.

[0134] In some embodiments, provided is a method of treating or preventing X-linked severe combined immunodeficiency using a target RNA having a mutation (e.g., a G>A mutation) in an individual, comprising administering to the individual an effective amount of a dRNA or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to a target RNA associated with a disease or disorder, and the dRNA is capable of recruiting ADAR to deaminate a target A in the target RNA, thereby rescuing the mutation in the target RNA. In some embodiments, the ADAR is an ADAR that is endogenously expressed in the cells of the individual. In some embodiments, the method comprises administering to the individual an ADAR or a construct encoding the ADAR. In some embodiments, the target RNA is IL2RG W237X (e.g., 710G>A). In some embodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO: 203.

[0135] As used herein, "treatment" or "treating" is an approach for obtaining a beneficial or desirable result, including clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, a decrease in one or more other symptoms resulting from the disease, a decrease in the extent of the disease, stabilization of the disease (e.g., prevention of or delay in the worsening of the disease), prevention or delay of the spread of the disease (e.g., metastasis), prevention or delay of the occurrence or recurrence of the disease, delay or slowing of the progression of the disease, improvement of the medical condition, provision of remission of the disease (whether partial or total), a decrease in the dosage of one or more other agents necessary to treat the disease, delay of the progression of the disease, improvement in the quality of life and / or prolongation of the survival period. "Treatment" also includes alleviation of the pathological consequences of a disease or disorder. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0136] The terms "subject", "individual", and "patient" are used interchangeably herein to describe mammals, including humans. Individuals include, but are not limited to, humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human. In some embodiments, the individual has a disease or disorder such as drug resistance. In some embodiments, the individual is in need of treatment.

[0137] As understood in the art, an "effective amount" refers to an amount of a composition (e.g., a dRNA or a construct encoding a dRNA) sufficient to produce a desired therapeutic result (e.g., a reduction in the severity or duration of one or more symptoms of a disease or disorder, stabilization of the severity of one or more symptoms of a disease or disorder, or elimination of one or more symptoms of a disease or disorder). In the case of therapeutic use, beneficial or desired results include, for example, a decrease in one or more symptoms (biochemical, histological, and / or behavioral) resulting from a disease, including its complications and intermediate pathological phenotypes presented during the onset of the disease, which includes an improvement in the quality of life of an individual having such a disease or disorder, a reduction in the dosage of other medications required to treat the disease, an enhancement of the effect of another medication, a delay in the progression of the disease, and / or an extension of the patient's survival.

[0138] In general, the dosage, schedule, and route of administration of a composition (e.g., a dRNA or a construct encoding a dRNA) can be determined according to the size and condition of the individual and according to standard pharmaceutical practice. Exemplary routes of administration include intravenous, intraarterial, intraperitoneal, intralung, intracystic, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, or transdermal.

[0139] The RNA editing methods of the present application can be used not only in animal cells, such as mammalian cells, but also for the modification of plant or fungal RNA, such as plant or fungal RNA that endogenously expresses ADAR. The methods described herein can be used to generate genetically engineered plants and fungi having improved properties.

[0140] Compositions, Kits, and Products This specification further provides a composition (such as a pharmaceutical composition) comprising any one of the dRNAs, constructs, libraries, or RNAs edited as described herein, or any one of the host cells.

[0141] In some embodiments, a pharmaceutical composition is provided that comprises any one of the dRNAs or constructs encoding dRNAs described herein, and a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m 3 -cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or TWEEN TM , PLURONICS TMIt contains a nonionic surfactant such as polyethylene glycol (PEG). In some embodiments, a lyophilized formulation is provided. The pharmaceutical composition for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through a sterile filtration membrane.

[0142] There is further provided a kit or product that can be used in any one of the RNA editing methods or treatment methods described herein, including any one of the dRNAs, constructs, compositions, libraries, or edited host cells described herein.

[0143] In some embodiments, there is provided a kit for editing a target RNA in a host cell, comprising a dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and wherein the dRNA can recruit ADAR to deaminate A in the target RNA. In some embodiments, the kit further comprises ADAR or a construct encoding ADAR. In some embodiments, the kit further comprises an inhibitor of ADAR3 or its construct. In some embodiments, the kit further comprises a stimulator of interferon or its construct. In some embodiments, the kit further comprises instructions for performing any one of the RNA editing methods described herein.

[0144] The kit of the present application is contained in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (such as sealed polyester films or plastic bags). The kit may optionally provide additional components such as transfection or transduction reagents, cell culture media, buffers, and interpretive information.

[0145] Accordingly, the present application also provides a product. The product can include a container and a label or protocol on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, and the like. In some embodiments, the container may contain a pharmaceutical composition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle). The container containing the pharmaceutical composition may be a reusable vial that allows for repeated administration (for example, 2 to 6 administrations) of the reconstituted formulation. The protocol refers to the instructions typically included in the commercial package of a therapeutic product and contains information such as indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such a product. Further, the product may further include a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0146] The kit or product may include a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in an amount sufficient for storage and use in pharmacies (such as hospital pharmacies and dispensing pharmacies).

[0147] Exemplary embodiments The embodiments provided herein are as follows. 1. A method for editing a target RNA in a host cell, comprising introducing into the host cell a deaminase mobilizing RNA (dRNA) or a construct encoding the dRNA, wherein the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the deaminase mobilizing RNA is capable of mobilizing an adenosine deaminase (ADAR) that acts on RNA to deaminate a target adenosine in the target RNA. 2. The method according to embodiment 1, wherein the RNA sequence comprises a cytidine, adenosine, or uridine that is directly opposite the target adenosine in the target RNA. 3. The method according to embodiment 2, wherein the RNA sequence comprises a cytidine mismatch that is directly opposite the target adenosine in the target RNA. 4. The method according to embodiment 3, wherein the cytidine mismatch is located at least 20 nucleotides away from the 3' end of the complementary sequence and at least 5 nucleotides away from the 5' end of the complementary sequence in the dRNA. 5. The method according to embodiment 4, wherein the cytidine mismatch is located within 10 nucleotides from the center (e.g., at the center) of the complementary sequence in the dRNA. 6. The method according to any one of embodiments 1 to 5, wherein the RNA sequence further comprises one or more guanosines that are opposite each of the non-target adenosines in the target RNA. 7. The method according to any one of embodiments 1 to 6, wherein the complementary sequence comprises two or more consecutive mismatched nucleotides that are opposite the non-target adenosines in the target RNA. 8. The nearest neighbor to the 5' of the target adenosine in the target RNA is a nucleotide selected from U, C, A, and G, with the priority being U > C ≒ A > G, and the nearest neighbor to the 3' of the target adenosine in the target RNA is a nucleotide selected from G, C, A, and U, with the priority being G > C > A ≒ U. The method according to any one of embodiments 1 to 7. 9. The method according to any one of embodiments 1 to 8, wherein the target adenosine in the target RNA is located in a 3-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU. 10. The 3-base motif is UAG, and the deaminase-recruiting RNA comprises an A opposite the uridine of the 3-base motif, a cytidine opposite the target adenosine, and a cytidine, guanosine, or uridine opposite the guanosine of the 3-base motif. The method according to embodiment 9.

[0148] 11. The method according to any one of Embodiments 1 to 10, wherein the deaminase-recruiting RNA is about 40 to 260 nucleotides in length. 12. The method according to Embodiment 11, wherein the deaminase-recruiting RNA is about 60 to 230 nucleotides in length. 13. The method according to Embodiment 11 or 12, wherein the dRNA has a length exceeding about 60 nucleotides. 14. The method according to any one of Embodiments 11 to 13, wherein the dRNA is about 100 to about 150 (e.g., about 110 - 150) nucleotides in length. 15. The method according to any one of Embodiments 1 to 14, wherein the target RNA is an RNA selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA, and small RNA. 16. The method according to Embodiment 15, wherein the target RNA is pre-messenger RNA. 17. The method according to any one of Embodiments 1 to 16, wherein the ADAR is endogenously expressed by the host cell. 18. The method according to any one of Embodiments 1 to 16, wherein the ADAR is exogenous to the host cell. 19. The method according to Embodiment 18, further comprising introducing the ADAR into the host cell. 20. The method according to Embodiment 18 or 19, wherein the ADAR contains the E1008 mutation.

[0149] 21. The method according to any one of Embodiments 1 to 20, wherein the deaminase-recruiting RNA is single-stranded RNA. 22. The method according to any one of Embodiments 1 to 20, wherein the complementary RNA sequence is single-stranded and the deaminase-recruiting RNA further comprises one or more double-stranded regions. 23. The method according to any one of Embodiments 1 to 22, wherein the dRNA does not contain an ADAR-recruiting domain (e.g., DSB-binding domain, GluR2 domain, or MS2 domain). The method according to any one of embodiments 1 to 23, wherein the 24.dRNA does not contain chemically modified nucleotides (for example, 2'-O-methylation or phosphorothioation). 25. The method according to embodiment 24, wherein the deamination of the target adenosine in the target RNA causes point mutations, cleavage, elongation and / or misfolding of the protein encoded by the target RNA, or, by restoring missense mutations, premature termination codons, abnormal splicing or alternative splicing in the target RNA, results in a functional, full-length, correctly folded and / or wild-type protein. 26. The method according to any one of embodiments 1 to 25, wherein the host cell is a eukaryotic cell. 27. The method according to embodiment 26, wherein the host cell is a mammalian cell. 28. The method according to embodiment 27, wherein the host cell is a human or mouse cell. 29. The method according to embodiment 27 or 28, wherein the ADAR is ADAR1 and / or ADAR2. 30. The method according to any one of embodiments 1 to 29, wherein the host cell is a primary cell.

[0150] 31. The method according to embodiment 30, wherein the host cell is a T cell. 32. The method according to embodiment 30, wherein the host cell is a post-mitotic cell. 33. The method according to any one of embodiments 1 to 32, further comprising introducing an inhibitor of ADAR3 into the host cell. 34. The method according to any one of embodiments 1 to 33, further comprising introducing a stimulator of interferon into the host cell. 35. The method according to any one of embodiments 1 to 34, comprising introducing a plurality of dRNAs targeting different target RNAs. 36. The method according to any one of embodiments 1 to 35, wherein the efficiency of editing the target RNA is at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more). 37. The method according to any one of embodiments 1 to 36, wherein the dRNA does not induce an immune response. 38. An edited RNA generated by the method according to any one of embodiments 1 to 37 or a host cell having the edited RNA. 39. A method for treating or preventing a disease or disorder in an individual, comprising editing a target RNA associated with the disease or disorder in the cells of the individual according to the method according to any one of embodiments 1 to 37. 40. The method according to embodiment 39, wherein the disease or disorder is a hereditary genetic disease or a disease or disorder associated with one or more acquired gene mutations.

[0151] 41. The method according to embodiment 39 or 40, wherein the target RNA has a mutation from G to A. 42. The method according to any one of embodiments 39 to 41, wherein the disease or disorder is a single-gene disease or disorder. 43. The method according to any one of embodiments 39 to 42, wherein the disease or disorder is a multi-gene disease or disorder. 44. (i) The target RNA is TP53 and the disease or disorder is cancer; (ii) The target RNA is IDUA and the disease or disorder is mucopolysaccharidosis type I (MPS I); (iii) The target RNA is COL3A1 and the disease or disorder is Ehlers-Danlos syndrome; (iv) The target RNA is BMPR2 and the disease or disorder is Joubert syndrome; (v) The target RNA is FANCC and the disease or disorder is Fanconi anemia; (vi) The target RNA is MYBPC3 and the disease or disorder is primary familial hypertrophic cardiomyopathy; or (vii) where the target RNA is IL2RG and the disease or disorder is X-linked severe combined immunodeficiency The method according to any one of Embodiments 39 to 43. 45. A deaminase mobilizing RNA (dRNA) for deaminating a target adenosine in the target RNA by mobilizing an adenosine deaminase (ADAR) acting on RNA, which comprises a complementary RNA sequence hybridizing to the target RNA. 46. The deaminase mobilizing RNA according to Embodiment 47, wherein the RNA sequence contains a cytidine, adenosine, or U directly opposite the target adenosine in the target RNA. 47. The dRNA according to Embodiment 48, wherein the RNA sequence contains a cytidine mismatch directly opposite the target adenosine in the target RNA. 48. The dRNA according to Embodiment 49, wherein the cytidine mismatch is located at least 20 nucleotides away from the 3' end of the complementary sequence and at least 5 nucleotides away from the 5' end of the complementary sequence in the dRNA. 49. The dRNA according to Embodiment 50, wherein the cytidine mismatch is located within 10 nucleotides from the center (e.g., in the center) of the complementary sequence in the dRNA. 50. The deaminase mobilizing RNA according to any one of Embodiments 47 to 51, wherein the RNA sequence further contains one or more guanosines opposite each of the non-target adenosines in the target RNA.

[0152] 51. The dRNA according to any one of Embodiments 47 to 51, wherein the complementary sequence contains two or more consecutive mismatched nucleotides opposite the non-target adenosine in the target RNA. 52. The deaminase mobilizing RNA according to any one of Embodiments 47 to 53, wherein the target adenosine in the target RNA is located in a 3-base motif selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU. 53. The deaminase-recruiting RNA according to embodiment 54, wherein the three-base motif is UAG, and the dRNA comprises adenosine opposite to the uridine of the three-base motif, cytidine opposite to the target adenosine, and cytidine, guanosine, or uridine opposite to the guanosine of the three-base motif. 54. The deaminase-recruiting RNA according to embodiment 55, wherein the three-base motif is UAG in the target RNA, and the deaminase-recruiting RNA comprises ACC, ACG, or ACU opposite to the UAG of the target RNA. 55. The deaminase-recruiting RNA according to any one of embodiments 47 to 56, wherein the deaminase-recruiting RNA is about 40 to 260 nucleotides in length. 56. The dRNA according to embodiment 57, wherein the dRNA has a length of about 70 nucleotides. 57. The dRNA according to embodiment 57 or 58, wherein the dRNA is about 100 to about 150 (e.g., about 110 - 150) nucleotides in length. 58. The dRNA according to any one of embodiments 47 to 59, wherein the dRNA does not contain an ADAR-recruiting domain (e.g., DSB-binding domain, GluR2 domain, or MS2 domain). 59. The dRNA according to any one of embodiments 47 to 60, wherein the dRNA does not contain chemically modified nucleotides (e.g., 2'-O-methylation or phosphorothioation). 60. A construct encoding the deaminase-recruiting RNA according to any one of embodiments 47 to 61.

[0153] 61. The construct according to embodiment 62, wherein the construct is a viral vector (e.g., a lentiviral vector) or a plasmid. 62. A library comprising the plurality of deaminase-recruiting RNAs according to any one of embodiments 47 to 61 or the construct according to embodiment 62 or 63. 63. A composition comprising the deaminase-recruiting RNA according to any one of embodiments 47 to 61, the construct according to embodiment 62 or 63, or the library according to embodiment 64. 64. A host cell comprising the deaminase-recruiting RNA according to any one of Embodiments 47 to 61 or the construct according to Embodiment 62 or 63. 65. The host cell according to Embodiment 66, wherein the host cell is a eukaryotic cell. 66. The host cell according to Embodiment 66 or 67, wherein the host cell is a primary cell. 67. A kit for editing a target RNA in a host cell, comprising a deaminase-recruiting RNA, wherein the deaminase-recruiting RNA comprises a complementary RNA sequence that hybridizes to the target RNA, and the deaminase-recruiting RNA can recruit ADAR to deaminate a target adenosine in the target RNA. 68. A deaminase-recruiting RNA (dRNA) of 60 to 200 nucleotides, a) the dRNA comprises a complementary RNA sequence that hybridizes to the target RNA, b) the dRNA can recruit a deaminase or a construct comprising a deaminase or a catalytic domain of a deaminase to deaminate a target adenosine in the target RNA, c) the dRNA comprises one or more chemical modifications, the dRNA. 69. The dRNA according to Embodiment 68, wherein the dRNA is longer than any of 60 nt, 65 nt, 70 nt, 80 nt, 90 nt, 100 nt, or 110 nt. 70. The dRNA according to Embodiment 1 or 69, comprising one or more mismatches, wobbles, and / or bulges with a complementary target RNA region.

[0154] 71. The dRNA according to any one of Embodiments 68 to 70, wherein the complementary RNA sequence comprises a cytidine, adenosine, or uridine opposite the target adenosine in the target RNA. 72. The cytidine, adenosine, or uridine opposite the target adenosine is located at least about 7 nucleotides from the 3' end, for example, at least about 8, 9, 10 or more nucleotides from the 3' end, or located about 7-25 nt from the 3' end, the dRNA according to embodiment 71. 73. The cytidine, adenosine, or uridine opposite the target adenosine is located at least about 25 nucleotides from the 5' end, for example, at least about 30, 35, 40, 45, 50 or 55 nucleotides from the 5' end, or located about 45-55 nt from the 5' end, the dRNA according to embodiment 71 or 72. 74. The lengths of the 5' and 3' sequences adjacent to the cytidine, adenosine, or uridine opposite the target adenosine are not equal, the dRNA according to any one of embodiments 71-73. 75. The length of the 5' sequence adjacent to the cytidine, adenosine, or uridine opposite the target adenosine is longer than the length of the 3' sequence, the dRNA according to any one of embodiments 71-74. 76. The dRNA according to any one of embodiments 68-75, which contains cytidine opposite the target adenosine in the target RNA. 77. The dRNA according to any one of embodiments 68-76, wherein the complementary RNA sequence further contains one or more guanosines opposite each of the non-target adenosines in the target RNA. 78. The dRNA according to any one of embodiments 68-77, wherein the complementary sequence contains two or more consecutive mismatched nucleotides opposite the non-target adenosine in the target RNA. 79. The nearest neighbor to the 5' of the target adenosine in the target RNA is a nucleotide selected from U, C, A, and G, with the priority U>C≒A>G, and the nearest neighbor to the 3' of the target adenosine in the target RNA is a nucleotide selected from G, C, A, and U, with the priority G>C>A≒U, the dRNA according to any one of embodiments 68-78. 80. The dRNA according to any one of embodiments 68 to 79, wherein the target adenosine is located in a 3-base motif in the target RNA selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU.

[0155] 81. The dRNA according to embodiment 80, wherein the 3-base motif is UAG, and the dRNA contains an A opposite the uridine of the 3-base motif, a cytidine opposite the target adenosine, and a cytidine, guanosine, or uridine opposite the guanosine of the 3-base motif. 82. The dRNA according to embodiment 81, which contains 5'-CCA-3' opposite the 3-base motif of UAG. 83. The dRNA according to any one of embodiments 68 to 82, wherein the chemical modification is methylation and / or phosphorothioation, for example, 2'-O-methylation and / or internucleotide phosphorothioate bond. 84. The dRNA according to embodiment 83, wherein the chemical modification includes 2'-O-methylation of each of the first and last 1 to 5, 2 to 5, 3 to 5, 4 to 5 nucleotides and / or phosphorothioation of the bonds between each of the first and last 1 to 5, 2 to 5, 3 to 5, 4 to 5 nucleotides. 85. The dRNA according to embodiment 83 or embodiment 84, wherein the chemical modification includes 2'-O-methylation and / or 3'-phosphorothioation in the nucleotide opposite the target adenosine and / or the nucleotides most adjacent to its 5' and / or 3'. 86. The chemical modification is 1) 2'-O-methylation of each of the first and last 3 nucleotides and / or phosphorothioation of the bonds between each of the first and last 3 nucleotides; 2) 2'-O-methylation of each of the first and last 3 nucleotides and / or phosphorothioation of the bonds between each of the first and last 3 nucleotides, and 2'-O-methylation of single or multiple uridines (for example, all uridines); 3) 2'-O-methylation of each of the first and last 3 nucleotides, phosphorothioation of the bond between each of the first and last 3 nucleotides, 2'-O-methylation of single, multiple, or all uridines, and modification of the nucleotide opposite the target adenosine and / or the nucleotides most adjacent to its 5' and / or 3'; 4) 2'-O-methylation of each of the first and last 3 nucleotides, phosphorothioation of the bond between each of the first and last 3 nucleotides, 2'-O-methylation of all uridines, and 2'-O-methylation of the nucleotides most adjacent to the 3' and / or 5' of the nucleotide opposite the target adenosine; 5) 2'-O-methylation of each of the first and last 3 nucleotides, phosphorothioation of the bond between each of the first and last 3 nucleotides, 2'-O-methylation of all uridines, and phosphorothioation of the nucleotide opposite the target adenosine and / or the nucleotides most adjacent to its 5' and / or 3'; 6) 2'-O-methylation of each of the first and last 5 nucleotides and / or phosphorothioation of the bond between each of the first and last 5 nucleotides The dRNA according to any one of Embodiments 1 to 85, selected from 87. The dRNA according to Embodiment 86, wherein the modification of the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine is 2'-O-methylation or a phosphorothioate bond, for example, a 3'-phosphorothioate bond. 88. The dRNA according to any one of Embodiments 68 to 87, which does not contain an ADAR recruitment domain capable of forming an intramolecular stem-loop structure for binding to an ADAR enzyme. 89. A construct comprising or encoding the dRNA according to any one of Embodiments 68 to 88. 90. A method for editing a target RNA in a host cell, comprising introducing the dRNA according to any one of Embodiments 68 to 89 into a host cell including, but not limited to, eukaryotic cells, primary cells, T cells, mammalian cells, human cells, murine cells, etc. by infection, electroporation, lipofection, endocytosis, liposome or lipid nanoparticle delivery, etc.

[0156] 91. The method according to Embodiment 90, further comprising introducing an inhibitor of ADAR3 into the host cell. 92. The method according to Embodiment 90 or 91, further comprising introducing a stimulator of interferon into the host cell. 93. The me...

Claims

1. A deaminase-recruiting RNA (dRNA) of 60 to 81 nucleotides, comprising: a) the dRNA does not contain an ADAR-recruiting domain capable of forming an intramolecular stem-loop structure for binding to the ADAR enzyme and contains a complementary RNA sequence that hybridizes to the target RNA; b) the dRNA is capable of recruiting a deaminase or a construct containing a deaminase or a construct containing a catalytic domain of a deaminase to deaminate a target adenosine in the target RNA; c) the dRNA contains one or more chemical modifications, the complementary RNA sequence contains a cytidine, adenosine, or uridine opposite the target adenosine in the target RNA, the cytidine, adenosine, or uridine opposite the target adenosine is located 9 to 25 nucleotides away from the 3'-end, and the cytidine, adenosine, or uridine opposite the target adenosine is located 45 to 55 nucleotides away from the 5'-end; dRNA.

2. The dRNA according to claim 1, comprising one or more mismatches, wobbles, and / or bulges with a complementary target RNA region.

3. The dRNA according to claim 1 or 2, comprising a cytidine opposite the target adenosine in the target RNA and / or the complementary RNA sequence contains one or more guanosines opposite each of the non-target adenosines in the target RNA.

4. The dRNA according to claim 1 or 2, wherein the complementary RNA sequence contains two or more consecutive mismatched nucleotides opposite non-target adenosines in the target RNA.

5. The dRNA according to claim 1 or 2, wherein the chemical modification comprises methylation and / or phosphorothiolation.

6. The dRNA according to claim 5, wherein the chemical modification comprises 2'-O-methylation and / or internucleotide phosphorothioate linkages.

7. The dRNA according to claim 5, wherein the chemical modification comprises 2'-O-methylation and / or 3'-phosphorothiolation at the nucleotide opposite the target adenosine and / or the nucleotide(s) most adjacent to its 5' and / or 3'-end.

8. The dRNA according to claim 7, wherein the nucleotide opposite the target adenosine and / or one or two nucleotides most adjacent to the nucleotide opposite the target adenosine are 2'-O-methylation and / or phosphorothioate bond.

9. A construct comprising or encoding the dRNA according to any one of claims 1 to 8.

10. A method for editing a target RNA in a host cell in vitro, comprising introducing the dRNA according to any one of claims 1 to 8 into the host cell.

11. The method according to claim 10, comprising introducing a plurality of dRNAs targeting different target RNAs, and optionally, the method further comprises introducing an inhibitor of ADAR3 and / or a stimulator of interferon into the host cell.

12. A construct, composition, cell, library, or kit comprising the dRNA according to any one of claims 1 to 8.

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